# Patent

## Title
Essential fungal polypeptide, CaYDR341c, and methods of use thereof

## Abstract

The present invention provides essential fungal polynucleotides and their encoded polypeptides, homologues thereof and their uses. Additionally, the invention provides methods for the identification of essential polynucleotides and fungal strains which may be used for drug screening.

## Background
This application is a divisional application of non-provisional application U.S. Ser. No. 10/424,324, filed Apr. 25, 2003, U.S. Pat. No. 7,465,568, issued Dec. 16, 2008, which claims benefit to provisional application U.S. Ser. No. 60/376,022 filed Apr. 26, 2002, under 35 U.S.C. 119(e). The entire teachings of the referenced application are incorporated herein by reference.


FIELD OF THE INVENTION
The present invention provides essential fungal polynucleotides and their encoded polypeptides, homologues thereof and uses thereof. Additionally, the invention provides methods for the identification of essential polynucleotides and fungal strains which may be used for drug screening.
BACKGROUND OF THE INVENTION
As the number of severe fungal infections continues to rise, the need for a broad spectrum antifungal agent becomes more urgent. The rise in fungal infections is primarily due to the increasing number of immuno-compromised patients as a result of medical advances (transplantation and chemotherapy) and as a result of the increasing population of AIDS patients.
Although fluconazole has been an effective drug against fungal pathogens for a number of years, resistance is increasing. Alternatives such as amphotericin B have serious drawbacks, including such side effects as nephrotoxicity and severe discomfort. Few new antifungals are on the horizon, and more knowledge about the pathogenicity of fungi as well as about their general biology is crucial if new drug targets are to be identified.
More than 80% of fungal infections in immuno-compromised patients are caused by Candida species. Cryptococcosis is the second most prevalent fungal infection in AIDS patients following candidiasis. Aspergillosis is responsible for at least 30% of the infections in cancer and organ transplant patients and has a high mortality rate.
In order to discover new drugs to combat fungal infections, compounds are often tested for their effects on particular, suitable polynucleotides and polynucleotide products. Suitable polynucleotides are generally those which are found to be essential to the viability of the pathogen. This determination of essentiality presents substantial obstacles to the identification of appropriate targets for drug screening. These obstacles are especially pronounced in diploid organisms, such as Candida albicans. 
A central technique used to investigate the role of a Candida albicans polynucleotide is to study the phenotype of a cell in which both copies of the polynucleotide have been deleted. Two popular polynucleotide deletion protocols have been reported. The first and most often used method, the ‘urablaster’ method, requires construction of a disruption cassette consisting of a selectable marker (URA and hisG flanking sequences) and sequences of the polynucleotide to be disrupted that are positioned at the 5′ and 3′ ends of the hisG-URA3-hisG cassette. A ura3− strain is then transformed and grown on minimal medium lacking uridine. ura3+ clones are isolated and transferred to a medium containing 5-fluoroortic acid, which selects for strains that have a ura3− genotype. These arise spontaneously as a subpopulation of the original transformed cells which will have undergone a recombinational event that retains one of the hisG sequences in the disrupted allele. The heterozygote ura3− strains can then be used in a second transformation event using the same cassette to disrupt the second allele resulting in a homozygous deletion strain.
A second type of popular method utilizes a PCR-based polynucleotide disruption strategy and multiple markers to construct homozygous mutants (Wilson et al J. Bacteriol. 181: 1868-74 (1999)). Although more rapid than the urablaster method, both methods have limitations. Since a homozygous deletion strain which lacks both essential polynucleotide copies would not be viable, such results are not an unequivocal explanation establishing the essential nature of the target polynucleotide because alternative explanations, including poor growth of a viable mutant strain, may be as likely a reason as essentiality for the negative results obtained.
Essential polynucleotides may also be identified using inducible promoter-regulated constructs to modify expression of the second polynucleotide copy, rather than completely inactivating it. With these methods, one polynucleotide copy is disrupted and the second copy is only expressed under certain conditions. The essentiality of the polynucleotide can be investigated since the fungal strain will only be viable under conditions in which the promoter is switched on (See, for example Nakayama et al. (Infection and Immunity 68: 6712-6719, (2000), and WO 01/60975).
One reportedly effective technique involves the use of the C. albicans MET3 promoter (Care et al., Molecular Microbiology 34 792-798 (1999)). The activity of the promoter is inhibited by methionine and/or cysteine and completely inactivated with both amino acids. Although the MET3 promoter is not the only regulated promoter to be characterized in C. albicans, one advantage of this promoter is that it is controlled by the addition of amino acids to the growth medium rather than a switch in carbon source. Switching carbon sources is likely to cause a bigger disturbance to cell physiology than adding amino acids (Care et at). However, the method developed for use of the MET3 promoter by Care et al. is cumbersome since it requires subcloning a portion of the polynucleotide under evaluation.
Additional methodologies enabling the identification of essential polynucleotides for drug screening which are both easy to use and give rapid results are needed, particularly for C. albicans. The C. albicans genome-sequencing project has recently begun and novel polynucleotides are being identified. There should be increasing demand to assess essential polynucleotide function and methodologies particularly amenable to high throughput screening will be useful.
Furthermore, although the identification of novel polynucleotides as essential in C. albicans is of value, the determination of essentiality for known polynucleotide sequences in C. albicans or other fungal genomes is also highly desirable since such polynucleotides will add to the set of drug targets. Furthermore, the use of C. albicans essential polynucleotides for drug screening for which orthologs in other pathogenic fungi are identified may result in the discovery of drugs effective in fighting infections from a variety of pathogens.
SUMMARY OF THE INVENTION
The invention provides a nucleic acid molecule including nucleotide sequences that hybridize under stringent conditions to a second nucleic acid molecule having a nucleotide sequence that encodes an essential polypeptide having an amino acid sequence selected from the group consisting of one of SEQ ID NO: 12 to 22.
The invention also provides a substantially pure oligonucleotide, said oligonucleotide comprising a region of nucleotide sequence capable of hybridizing under highly stringent conditions to at least about 12 consecutive nucleotides of one of SEQ ID NO: 1 to SEQ ID NO: 11.
The invention also provides a polynucleotide comprising the nucleotide sequence of a member of the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11, or the nucleotide sequence included in the deposited clone.
The invention also provides a recombinant DNA molecule comprising the isolated nucleic acid molecule comprising a nucleotide sequence of a member of the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11, or the nucleotide sequence included in the deposited clone.
The invention also provides a recombinant DNA molecule comprising a nucleotide sequence of a member of the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11, or the nucleotide sequence included in the deposited clone, wherein said polynucleotide is operably linked to one or more regulatory sequences.
The invention also provides an expression vector comprising an isolated polynucleotide comprising a nucleotide sequence of a member of the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11.
The invention also provides a host cell transformed to contain an expression vector comprising an isolated polynucleotide comprising a nucleotide sequence of a member of the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11, wherein said host cell is either a prokaryote or a eukaryote.
The invention also provides a substantially pure oligonucleotide, said oligonucleotide comprising a region of nucleotide sequence capable of hybridizing under highly stringent conditions to at least about 12 consecutive nucleotides of one of SEQ ID NO: 1 to SEQ ID NO: 11.
The invention also provides a substantially pure oligonucleotide, said oligonucleotide comprising a region of nucleotide sequence capable of hybridizing under highly stringent conditions to at least about 12 consecutive nucleotides of one of SEQ ID NO: 1 to SEQ ID NO: 11, wherein said oligonucleotide further comprises a detectable label attached thereto.
The invention also provides an isolated nucleic acid molecule obtained from an organism other than Candida albicans or Saccromyces cervisiae comprising a nucleotide sequence having at least 30% identity to a sequence selected from the group consisting of SEQ ID NO: 1 to 11.
The invention also provides a method for producing a polypeptide comprising the step of culturing a host cell transformed with the nucleic acid molecule comprising a nucleotide sequence of a member of the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11 under conditions in which the protein encoded by said nucleic acid molecule is expressed.
The invention also provides a polynucleotide that encodes a full length protein of a member of the group consisting of SEQ ID NO: 12 to 22, or the encoded sequence included in the deposited clone.
The invention also provides an isolated nucleic acid molecule obtained from an organism other than Candida albicans or Saccromyces cervisiae including a nucleotide sequence having at least 30% identity to a sequence selected from the group consisting of SEQ ID NO: 1 to 11, wherein said identity is determined using the CLUSTALW algorithm with default parameters.
The invention also provides an isolated nucleic acid molecule obtained from an organism other than Candida albicans or Saccromyces cervisiae including a nucleotide sequence having at least 30% identity to a sequence selected from the group consisting of SEQ ID NO: 1 to 11, wherein said identity is determined using the CLUSTALW algorithm with default parameters, wherein said organism is selected from the group consisting of Absidia corymbigera, Aspergillus flavis, Aspergillus fumigatus, Aspergillus niger, Botrytis cinerea, Candida dublinensis, Candida glabrata, Candida krusei, Candia parapsilopsis, Candia tropicalis, Coccidioides immitis, Cryptococcus neoformans, Erysiphe graminis, Exophalia dermatiditis, Fusarium osysproum, Histoplasma capsulatum, Magnaporthe grisea, Mucor rouxii, Pneumocystis carinii, Puccinia graminis, Puccinia recodita, Rhizomucor pusillus, Puccinia striiformis, Rhizopus arrhizus, Septoria avenae, Septoria nodorum, Septoria triticii, Tilletia controversa, Tilletia tritici, Trichospoon beigelii and Ustilago maydis. 
The invention also provides an isolated nucleic acid molecule obtained from an organism other than Candida albicans or Saccromyces cervisiae including a nucleotide sequence having at least 30% identity to a sequence selected from the group consisting of SEQ ID NO: 1 to 11, wherein said identity is determined using the CLUSTALW algorithm with default parameters, wherein said organism is Aspergillus fumigatus. 
Additionally provided is an isolated nucleic acid molecule having a fragment of at least 10 consecutive nucleotides of one of SEQ ID NO 1 to 11.
The invention provides a substantially purified polypeptide including an amino acid sequence selected from the group consisting of one of SEQ ID NO: 12 to SEQ ID NO: 22 or one of SEQ ID NO:48 to one of SEQ ID NO: 73.
Furthermore, the invention provides a strain of Candida albicans wherein a first copy of a polynucleotide having a nucleotide sequence selected from the group consisting of one of SEQ ID NO 1 to 11 is inactive and a second copy of the polynucleotide is under the control of a regulatable promoter.
Furthermore, the invention provides a strain of Candida albicans wherein a first copy of a polynucleotide having a nucleotide sequence selected from the group consisting of one of SEQ ID NO 1 to 11 is inactive and a second copy of the polynucleotide is under the control of a regulatable promoter, wherein said regulatable promoter is MET3.
The invention also provides a strain of Candida albicans comprising a nucleic acid molecule having a nucleotide sequence selected from one of SEQ ID NO: 1 to 11 under the control of a regulatable promoter.
Additionally provided herein is an isolated nucleic acid molecule having a nucleotide sequence encoding a polypeptide required for growth of Candida albicans, wherein said polypeptide includes an amino acid sequence of one of SEQ ID NO: 12 to 22.
The invention also provides a method for identifying essential polynucleotides in diploid fungal cells, said method having the steps of:
(a) inactivating a first copy of a polynucleotide in diploid fungal cells by recombination using a PCR-based polynucleotide disruption cassette, thereby providing heterozygous diploid fungal cells;
(b) modifying a second copy of the polynucleotide in the heterozygous diploid fungal cells by recombination using a PCR-based promoter swapping cassette including a nucleotide sequence encoding a regulatable promoter, such that expression of the second copy of the polynucleotide is regulated by said promoter;
(c) culturing said cells with a promoter suppressor; and
(d) assessing growth of said cultured cells in comparison to control cells.
The invention also provides a method for identifying essential polynucleotides in diploid fungal cells, said method having the steps of:
(a) inactivating a first copy of a polynucleotide in diploid fungal cells by recombination using a PCR-based polynucleotide disruption cassette, thereby providing heterozygous diploid fungal cells;
(b) modifying a second copy of the polynucleotide in the heterozygous diploid fungal cells by recombination using a PCR-based promoter swapping cassette including a nucleotide sequence encoding a regulatable promoter, such that expression of the second copy of the polynucleotide is regulated by said promoter;
(c) culturing said cells with a promoter suppressor; and
(d) assessing growth of said cultured cells in comparison to control cells, wherein said diploid fungal cells are Candida albicans cells, wherein said polynucleotide is a conserved gene, wherein said promoter is a MET3 promoter, wherein said promoter suppressor is methionine, wherein said promoter suppressor is cysteine, and/or wherein said promoter suppressor is both cysteine and methionine.
Additionally provided herein is a method for inducing drug hypersensitivity in diploid fungal cells, said method including the steps of:
(a) inactivating a first copy of an essential polynucleotide in diploid fungal cells by recombination using a PCR-based polynucleotide disruption cassette, thereby providing heterozygous diploid fungal cells;
(b) modifying the second copy of the polynucleotide in the heterozygous diploid fungal cells by recombination using a PCR-based promoter swapping cassette including a nucleotide sequence encoding a regulatable promoter, such that expression of the second copy of the polynucleotide is regulated by said promoter;
(c) culturing said cells with a promoter suppressor and with a drug; and
(d) comparing the effects of said drug on the growth of said cells in comparison to control cells.
Additionally provided herein is a method for inducing drug hypersensitivity in diploid fungal cells, said method including the steps of:
(a) inactivating a first copy of an essential polynucleotide in diploid fungal cells by recombination using a PCR-based polynucleotide disruption cassette, thereby providing heterozygous diploid fungal cells;
(b) modifying the second copy of the polynucleotide in the heterozygous diploid fungal cells by recombination using a PCR-based promoter swapping cassette including a nucleotide sequence encoding a regulatable promoter, such that expression of the second copy of the polynucleotide is regulated by said promoter;
(c) culturing said cells with a promoter suppressor and with a drug; and
(d) comparing the effects of said drug on the growth of said cells in comparison to control cells, wherein said diploid fungal cells are Candida albicans cells, wherein said polynucleotide is a conserved gene, wherein said promoter is a MET3 promoter, wherein said promoter suppressor is methionine, wherein said promoter suppressor is cysteine, and/or wherein said promoter suppressor is both cysteine and methionine.
Furthermore, the invention provides a method for titrating the expression of a fungal cell essential polynucleotide product, said method including the steps of:
(a) inactivating a first copy of a polynucleotide in diploid fungal cells by recombination using a PCR-based polynucleotide disruption cassette, thereby providing heterozygous diploid fungal cells;
(b) modifying the second copy of the polynucleotide in the heterozygous diploid fungal cells by recombination using a PCR-based promoter swapping cassette including a nucleotide sequence encoding a MET3 promoter, such that expression of the second copy of the polynucleotide is regulated by said promoter;
(c) adding varying concentrations of a promoter suppressor which modulates said MET3 promoter; and
(d) correlating cell growth with each of said concentrations, thereby ascertaining the amount of promoter suppressor required to result in a particular reduction in cell growth.
Furthermore, the invention provides a method for titrating the expression of a fungal cell essential polynucleotide product, said method including the steps of:
(a) inactivating a first copy of a polynucleotide in diploid fungal cells by recombination using a PCR-based polynucleotide disruption cassette, thereby providing heterozygous diploid fungal cells;
(b) modifying the second copy of the polynucleotide in the heterozygous diploid fungal cells by recombination using a PCR-based promoter swapping cassette including a nucleotide sequence encoding a MET3 promoter, such that expression of the second copy of the polynucleotide is regulated by said promoter;
(c) adding varying concentrations of a promoter suppressor which modulates said MET3 promoter; and
(d) correlating cell growth with each of said concentrations, thereby ascertaining the amount of promoter suppressor required to result in a particular reduction in cell growth,
wherein the promoter suppressor is methionine, wherein the promoter suppressor is cysteine, and/or wherein the promoter suppressor is both cysteine and methionine.
Also provided herein is a nucleic acid molecule microarray having a plurality of nucleic acid molecules, said plurality including at least one nucleic acid molecule having a nucleotide sequence that is hybridizable under stringent conditions to a target nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11.
The invention also provides a fusion protein having a fragment of a first polypeptide fused to a second polypeptide, said fragment consisting of at least 5 consecutive residues of an amino acid sequence selected from one of SEQ ID NO: 12 to SEQ ID NO:22.
Additionally provided herein is a method of producing a polypeptide, said method comprising introducing into a cell, a vector comprising a promoter operably linked to a nucleotide sequence encoding a polypeptide consisting of an amino acid sequence selected from the group consisting of one of SEQ ID NO:12 to 22; and culturing the cell such that the nucleotide sequence is expressed.
Additionally, the invention provides a method for identifying a compound which modulates the activity of a polynucleotide product encoded by a nucleic acid molecule including a nucleotide sequence selected from the group consisting of one of SEQ ID NO: 1 to 11; said method including:
(a) contacting said polynucleotide product with a compound; and
(b) determining whether said compound modulates the activity of said polynucleotide product.
Also included herein is a method for identifying a compound which modulates the activity of an essential polynucleotide product said method including the steps of:
(a) inactivating a first copy of a polynucleotide in diploid fungal cells by recombination using a PCR-based polynucleotide disruption cassette, thereby providing heterozygous diploid fungal cells;
(b) modifying the second copy of the polynucleotide in the heterozygous diploid fungal cells by recombination using a PCR-based promoter swapping cassette including a nucleotide sequence encoding a MET3 promoter, such that expression of the second copy of the polynucleotide is regulated by said promoter;
(c) contacting said cells with a compound; and
(d) determining whether said compound modulates the activity of said polynucleotide product.
Additionally provided is a method of eliciting an immune response in an animal including introducing into the animal a composition including an isolated polypeptide, the amino acid sequence of which includes at least 6 consecutive residues of one of SEQ ID NO: 12 to 22 or one of SEQ ID NO: 48 to SEQ ID NO: 73.
Also included is a method of identifying a compound or binding partner that binds to a polypeptide having an amino acid sequence selected from the group consisting of one of SEQ ID NO: 12 to 22 or one of SEQ ID NO 48 to SEQ ID NO 73 or a fragment thereof, said method including:
(a) contacting the polypeptide or fragment thereof with a plurality of compounds or a preparation comprising one or more binding partners; and
(b) identifying a compound or binding partner that binds to the polypeptide or fragment thereof.
Furthermore, provided herein is a method for identifying a compound having the ability to inhibit growth of Candida albicans, said method including the steps of:
(a) reducing the level or activity of a polynucleotide product encoded by a nucleic acid selected from group consisting of SEQ ID NO: 1 to 11 in Candida albicans cells relative to a wild type cells, wherein said reduced level is not lethal to said cells;
(b) contacting said cell with a compound; and
(c) determining whether said compound inhibits the growth of said cells.
Furthermore, provided herein is a method for inhibiting growth of Candida albicans cells comprising contacting the cells with a compound that (i) reduces the level of or inhibits the activity of a nucleotide sequence selected from the group consisting of SEQ ID NO 1 to 11 or (ii) reduces the level of or inhibits the activity of a polynucleotide product encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 11, alternatively wherein said compound is an antisense molecule.
Additionally included herein is a method for treating an infection of a subject by Candida albicans comprising administering a pharmaceutical composition including a therapeutically effective amount of a compound that reduces the activity or level of a polynucleotide product encoded by a nucleic acid including a sequence selected from the group consisting of SEQ ID NO: 1 toll and a pharmaceutically acceptable carrier to said subject.
Also provided is a pharmaceutical composition including a therapeutically effective amount of an agent which reduces the activity or level of a polynucleotide product encoded by a nucleic acid selected from the group consisting of SEQ ID NO 1 to 11 in a pharmaceutically acceptable carrier.
The present invention also provides structure coordinates of the homology model of the CaYLR100w polypeptide (SEQ ID NO:12) provided in FIG. 27. The complete coordinates are listed in Table 8. The model of the present invention further provide a basis for designing stimulators and inhibitors or antagonists of one or more of the biological functions of CaYLR100w, or of mutants with altered specificity.
The present invention also provides structure coordinates of the homology model of the CaYDR341c polypeptide (SEQ ID NO:13) provided in FIG. 30. The complete coordinates are listed in Table 9. The models present in this invention further provide a basis for designing stimulators and inhibitors or antagonists of one or more of the biological functions of CaYDR341c, or of mutants with altered specificity.
The present invention also provides structure coordinates of the homology model of the CaYOL010w polypeptide (SEQ ID NO:19) provided in FIG. 33. The complete coordinates are listed in Table 10. The models present in this invention further provide a basis for designing stimulators and inhibitors or antagonists of one or more of the biological functions of CaYOL010w, or of mutants with altered specificity.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: alignment of C. albicans essential polypeptides with S. cervisiae and other sequences. FIG. 1A provides an alignment between the encoded CaYLR100w polypeptide (“A”; SEQ ID NO:12) with P1—170752 (“B”; SEQ ID NO:23) and AAB19089 (“C”; SEQ ID NO:24); FIG. 1B provides an alignment between the encoded CaYDR341c polypeptide (“A”; SEQ ID NO:13) with P1—168653 (“B”; SEQ ID NO:25) and AAB94675 (“C”; SEQ ID NO:26); FIG. 1C provides an alignment between the encoded CaYLR022c polypeptide (“A”; SEQ ID NO:14) with P1—178714 (“B”; SEQ ID NO:27) and AAB42957 (“C”; SEQ ID NO:28); FIG. 1D provides an alignment between the encoded CaYOL077c polypeptide (“A”; SEQ ID NO:15) with P1—182338 (“B”; SEQ ID NO:29) and AAB62453 (“C”; SEQ ID NO:30); FIG. 1E provides an alignment between the encoded CaYNL132w polypeptide (“A”; SEQ ID NO:16) with P1—137216 (“B”; SEQ ID NO:31) and AAB93917 (“C”; SEQ ID NO:32); FIG. 1F provides an alignment between the encoded CaYGR145w polypeptide (“A”; SEQ ID NO:17) with P1—150732 (“B”; SEQ ID NO:33) and AAB95680 (“C”; SEQ ID NO:26); FIG. 1G provides an alignment between the encoded CaYDR412w polypeptide (“A”; SEQ ID NO:18) with P1—142340 (“B”; SEQ ID NO:35) and AAW33110 (“C”; SEQ ID NO:36); FIG. 1H provides an alignment between the encoded CaYOL010w polypeptide (“A”; SEQ ID NO:19) with P1—182291 (“B”; SEQ ID NO:37) and AAW60075 (“C”; SEQ ID NO:38); FIG. 1I provides an alignment between the encoded CaYOR004w polypeptide (“A”; SEQ ID NO:20) with P1—161797 (“B”; SEQ ID NO:39) and AAG48012 (“C”; SEQ ID NO:40); FIG. 1J provides an alignment between the encoded CaYOR056c polypeptide “A”; SEQ ID NO:21) with P1—182387 (“B”; SEQ ID NO:41) and AAB09929 (“C”; SEQ ID NO:42); FIG. 1K provides an alignment between the encoded CaYRL009W polypeptide (“A”; SEQ ID NO:22) with P1—178703 (“B”; SEQ ID NO:43) and AAB43803 (“C”; SEQ ID NO:44); and FIG. 1L provides an alignment between the encoded CaYJR072c polypeptide (“A”; SEQ ID NO:45) with P1—149006 (“B”; SEQ ID NO:46) and AAG46965 (“C”; SEQ ID NO:47).
FIG. 2: alignment of C. albicans essential polypeptides with A. fumigatus sequences. FIG. 2A provides an alignment between the encoded CaYLR100w polypeptide (“A”; SEQ ID NO:12) with two versions of the analogous sequence in A. fumigatus (“B” and “C”; SEQ ID NO:48 and 49); FIG. 2B provides an alignment between the encoded CaYDR341c polypeptide (“A”; SEQ ID NO:13) with three versions of the analogous sequence in A. fumigatus (“B”, “C”, and “D”; SEQ ID NO:50, 51 and 52); FIG. 2C provides an alignment between the encoded CaYLR022c polypeptide (“A”; SEQ ID NO:14) with two versions of the analogous sequence in A. fumigatus (“B” and “C”; SEQ ID NO:53 and 54); FIG. 2D provides an alignment between the encoded CaYOL077c polypeptide (“A”; SEQ ID NO:15) with two versions of the analogous sequence in A. fumigatus (“B” and “C”; SEQ ID NO:55 and 56); FIG. 2E provides an alignment between the encoded CaYNL132w polypeptide (“A”; SEQ ID NO:16) with two versions of the analogous sequence in A. fumigatus (“B” and “C”; SEQ ID NO:57 and 58); FIG. 2F provides an alignment between the encoded CaYDR412w polypeptide (“A”; SEQ ID NO:18) with two versions of the analogous sequence in A. fumigatus (“B” and “C”; SEQ ID NO:62 and 63); FIG. 2G provides an alignment between the encoded CaYOR004w polypeptide (“A”; SEQ ID NO:20) with two versions of the analogous sequence in A. fumigatus (“B” and “C”; SEQ ID NO:64 and 65); FIG. 2H provides an alignment between the encoded CaYOR056c polypeptide (“A”; SEQ ID NO:21) with three versions of the analogous sequence in A. fumigatus (“B”, “C”, and “D”; SEQ ID NO:66, 67, and 68); FIG. 2I provides an alignment between the encoded CaYLR009w polypeptide (“A”; SEQ ID NO:22) with two versions of the analogous sequence in A. fumigatus (“B” and “C”; SEQ ID NO:69 and 70); FIG. 2J provides an alignment between the encoded CaYOL010w polypeptide (“A”; SEQ ID NO:19) with three versions of the analogous sequence in A. fumigatus (“B”, “C”, and “D”; SEQ ID NO:71, 72, and 73); FIG. 2K provides an alignment between the encoded CaYOL010w polypeptide (“A”; SEQ ID NO:19) two versions of the analogous sequence in A. fumigatus (“B” and “C”; SEQ ID NO:71 and 73); and FIG. 2L provides an alignment between the encoded CaYJR072c polypeptide (“A”; SEQ ID NO:45) with six versions of the analogous sequence in A. fumigatus (“B”, “C”, “D”, “E”, “F”, and “G”; SEQ ID NO:74, 75, 76, 77, 78, and 79).
FIG. 3: PCR-based polynucleotide disruption in C. albicans. 
FIG. 4: PCT confirmation of AAH/disruption strains that were obtained via PRC-based polynucleotide disruption in C. albicans. 
FIG. 5: Plasmid maps of pUMP and pAMP used for promoter swapping.
FIG. 6: Scheme of promoter swapping.
FIG. 7: Confirmation PCR for C. albicans MET3P-E RG1/erg1::ARG4 strains.
FIG. 8: Down regulation phenotypes by methionine and cysteine.
FIG. 9: Methionine titration of MET3P-ERG1 construct.
FIG. 10: Sensitivity of MET3P-ERG1 cells to terbinafine in the absence and presence of methionine.
FIG. 11: The polynucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:12) of the novel fungal essential gene, CaYLR100w (also referred to as FCG5), of the present invention. The CaYLR100w polypeptide (SEQ ID NO:12) is encoded by nucleotides 1 to 1038 of SEQ ID NO:1 and has a predicted molecular weight of 39.0 kDa. The conserved 3-keto sterol reductase catalytic residues, Y247 and S183, are denoted by light shading.
FIG. 12: The polynucleotide sequence (SEQ ID NO:2) and deduced amino acid sequence (SEQ ID NO:13) of the novel fungal essential gene, CaYDR341c (also referred to as FCG6), of the present invention. The CaYDR341c polypeptide (SEQ ID NO:13) is encoded by nucleotides 1 to 1866 of SEQ ID NO:2 and has a predicted molecular weight of 70.8 kDa. The conserved amino acids comprising the adenylate binding site of arginyl-tRNA synthetases, P151-H161, is denoted by light shading. The conserved amino acids comprising the Ω loop of arginyl-tRNA synthetases, S496-G502, is denoted by double underlining.
FIG. 13: The polynucleotide sequence (SEQ ID NO:3) and deduced amino acid sequence (SEQ ID NO:14) of the novel fungal essential gene, CaYLR022c (also referred to as FCG7), of the present invention. The CaYLR022c polypeptide (SEQ ID NO:14) is encoded by nucleotides 1 to 765 of SEQ ID NO:3 and has a predicted molecular weight of 29.2 kDa.
FIG. 14: The polynucleotide sequence (SEQ ID NO:4) and deduced amino acid sequence (SEQ ID NO:15) of the novel fungal essential gene, CaYOL077c (also referred to as FCG8), of the present invention. The CaYOL077c polypeptide (SEQ ID NO:15) is encoded by nucleotides 1 to 876 of SEQ ID NO:4 and has a predicted molecular weight of 34.0 kDa.
FIG. 15: The polynucleotide sequence (SEQ ID NO:5) and deduced amino acid sequence (SEQ ID NO:16) of the novel fungal essential gene, CaYNL132w (also referred to as FCG10), of the present invention. The CaYNL132w polypeptide (SEQ ID NO:16) is encoded by nucleotides 1 to 3126 of SEQ ID NO:5 and has a predicted molecular weight of 117.3 kDa.
FIG. 16: The polynucleotide sequence (SEQ ID NO:6) and deduced amino acid sequence (SEQ ID NO:17) of the novel fungal essential gene, CaYGR145w (also referred to as FCG12), of the present invention. The CaYGR145w polypeptide (SEQ ID NO:17) is encoded by nucleotides 1 to 2250 of SEQ ID NO:6 and has a predicted molecular weight of 85.0 kDa.
FIG. 17: The polynucleotide sequence (SEQ ID NO:7) and deduced amino acid sequence (SEQ ID NO:18) of the novel fungal essential gene, CaYDR412w (also referred to as FCG13), of the present invention. The CaYDR412w polypeptide (SEQ ID NO:18) is encoded by nucleotides 1 to 804 of SEQ ID NO:7 and has a predicted molecular weight of 31.3 kDa.
FIG. 18: The polynucleotide sequence (SEQ ID NO:8) and deduced amino acid sequence (SEQ ID NO:19) of the novel fungal essential gene, CaYOL010w (also referred to as FCG14), of the present invention. The CaYOL010w polypeptide (SEQ ID NO:19) is encoded by nucleotides 1 to 1113 of SEQ ID NO:8 and has a predicted molecular weight of 40.6 kDa. The conserved RNA 3′-terminal phosphate cyclase comprising the nucleotide binding site residues, R158-V168, are denoted by light shading.
FIG. 19: The polynucleotide sequence (SEQ ID NO:9) and deduced amino acid sequence (SEQ ID NO:20) of the novel fungal essential gene, CaYOR004w (also referred to as FCG15), of the present invention. The CaYOR004w polypeptide (SEQ ID NO:20) is encoded by nucleotides 1 to 771 of SEQ ID NO:9 and has a predicted molecular weight of 29.5 kDa.
FIG. 20: The polynucleotide sequence (SEQ ID NO:10) and deduced amino acid sequence (SEQ ID NO:21) of the novel fungal essential gene, CaYOR056c (also referred to as FCG16), of the present invention. The CaYOR056c polypeptide (SEQ ID NO:21) is encoded by nucleotides 1 to 1398 of SEQ ID NO:10 and has a predicted molecular weight of 52.6 kDa.
FIG. 21: The polynucleotide sequence (SEQ ID NO:11) and deduced amino acid sequence (SEQ ID NO:22) of the novel fungal essential gene, CaYLR009w (also referred to as FCG17), of the present invention. The CaYLR009w polypeptide (SEQ ID NO:22) is encoded by nucleotides 1 to 585 of SEQ ID NO:11 and has a predicted molecular weight of 23.1 kDa.
FIG. 22. TLC autoradiogram of labeled sterols from strains caerg1Δ/PMET3-CaERG1 (A) and fcg5Δ/PMET3-FCG5 (B). The position of ergosterol, lanosterol and squalene are indicated by arrows, as labelled. The squalene epoxidase (Erg1) inhibitor, terbinafine (“Ter”), was added to both a set of control tubes and a set of Met/Cys-containing tubes for a 90 minute induction period. Note that [14C]-cholesterol was used as a migration standard for ergosterol in Lane 11. The results demonstrate that CaYLR100w is a 3-keto sterol reductase. Additional experimental details may be found in Example 11, and described elsewhere herein.
FIG. 23. Growth curve of strains caerg1Δ/PMET3-CaERG1 (A) and fcg5Δ/PMET3-FCG5 (B) in the absence and presence of methionine and cysteine. The results further demonstrate that CaYLR100w is a 3-keto sterol reductase. Additional experimental details may be found in Example 11, and described elsewhere herein.
FIG. 24. Incorporation of [14C]-acetate into ergosterol and lanosterol in strains caerg1Δ/PMET3-CaERG1 (A) and fcg5Δ/PMET3-FCG5 (B) in the absence and presence of methionine and cysteine. The results further demonstrate that CaYLR100w is a 3-keto sterol reductase involved in C-4 sterol demethylation. Additional experimental details may be found in Example 11, and described elsewhere herein.
FIG. 25. Incorporation of radio-labeled arginine and leucine into proteins in cells of fcg6Δ/PMET3-FCG6 in the absence and presence of methionine and cysteine. (A.) raw counts (cpm) of [3H]-leu and [3H]-arg in the absence and presence of methionine and cysteine. (B.) percent protein synthesis by using the counts of untreated cells as 100%. The results demonstrate that CaYDR341c is involved in whole cell protein synthesis. Additional experimental details may be found in Example 12, and described elsewhere herein.
FIG. 26. Sequence alignment of the conceptual translated sequence of CaYLR100w (FCG5) polypeptide of the present invention (SEQ ID NO:12) with porcine carbonyl reductase (Protein Data Bank entry 1HU4; Genbank Accession No. gi|15826210; SEQ ID NO:251). The alignment was used as the basis for building the CaYLR100w homology model described herein. The coordinates of the CaYLR100w model are provided in Table 8. Amino acids conserved from the short chain dehydrogenase/reductase (SDR) catalytic triad are highlighted with an asterisk (*). Homologous residues in the functional active site domain that are conserved by identity are illustrated in bold.
FIG. 27 shows the three-dimensional homology model of the CaYLR100w (FCG5) polypeptide of the present invention (SEQ ID NO:12). The model is based upon an alignment to a structural homologue porcine carbonyl reductase (Protein Data Bank entry 1HU4; Genbank Accession No. gi|15826210; SEQ ID NO:251) that was used as the basis for building the CaYLR100w homology model. The coordinates of the CaYLR100w model are provided in Table 8.
FIG. 28 shows a comparison of the energy of CaYLR100w 3-keto sterol reductase homology model to the crystal structure of the porcine carbonyl reductase (Protein Data Bank entry 1HU4; Genbank Accession No. gi|5826210; SEQ ID NO:251) on which the CaYLR100w model was based. The CaYLR100w homology model is represented by the dotted (dashed) line and the porcince carbonyl reductase crystal structure is represented by the solid line.
FIG. 29: Sequence alignment of the conceptual translated sequence of CaYDR341c (FCG6) polypeptide of the present invention (SEQ ID NO:13) with Saccharomyces cerevisiae arginyl-tRNA synthetase, (chain A) (Protein Data Bank entry 1F7U; Genbank Accession No. gi|14719542; SEQ ID NO:252). The alignment was used as the basis for building the CaYDR341c homology model described herein. The coordinates of the CaYDR341c model are provided in Table 9. Amino acids defining the ADP binding site region and Q loop in both the model and the 1F7U structure are highlighted with either asterisk (“*”), or plus (“+”) sign, respectively. Homologous residues in the functional active site domain that are conserved by identity are illustrated in bold.
FIG. 30 shows the three-dimensional homology model of the CaYDR341c (FCG6) polypeptide of the present invention (SEQ ID NO:13). The model is based upon an alignment to a structural homologue Saccharomyces cerevisiae arginyl-tRNA synthetase, (chain A) (Protein Data Bank entry 1F7U; Genbank Accession No. gi|4719542; SEQ ID NO:252) that was used as the basis for building the CaYDR341c homology model. The coordinates of the CaYDR341c model are provided in Table 9.
FIG. 31 shows a comparison of the energy of the CaYDR341c arginyl-tRNA synthetase homology model to the crystal structure of the Saccharomyces cerevisiae arginyl-tRNA synthetase, (chain A) (Protein Data Bank entry 1F7U; Genbank Accession No. gi|14719542; SEQ ID NO:252) on which the CaYDR341c model was based. The CaYDR341c homology model is represented by the dotted (dashed) line and the Saccharomyces cerevisiae arginyl-tRNA synthetase crystal structure is represented by the solid line.
FIG. 32: Sequence alignment of the conceptual translated sequence of CaYOL010w (FCG14) of the present invention (SEQ ID NO:19) with Escherichia coli RNA 3′-terminal phosphate cyclase (Protein Data Bank entry 1QMH; Genbank Accession No. gi|12644279; SEQ ID NO:253). The alignment was used as the basis for building the CaYOL010w homology model described herein. The coordinates of the CaYOL010w model are provided in Table 10. Amino acids defining the nucleotide binding site region in both the model and the 1 QMH structure are highlighted with an asterisk (“*”). Homologous residues in the functional active site domain that are conserved by identity are illustrated in bold.
FIG. 33 shows the three-dimensional homology model of the CaYOL010w (FCG6) polypeptide of the present invention (SEQ ID NO:19). The model is based upon an alignment to a structural homologue Escherichia coli RNA 3′-terminal phosphate cyclase (Protein Data Bank entry 1QMH; Genbank Accession No. gi|12644279; SEQ ID NO:253) that was used as the basis for building the CaYOL010w homology model. The coordinates of the CaYOL010w model are provided in Table 10.
FIG. 34 shows a comparison of the energy of the CaYOL010w arginyl-tRNA synthetase homology model to the crystal structure of the Escherichia coli RNA 3′-terminal phosphate cyclase (Protein Data Bank entry 1QMH; Genbank Accession No. gi|12644279; SEQ ID NO:253) on which the CaYOL010w model was based. The CaYOL010w homology model is represented by the dotted (dashed) line and the Saccharomyces cerevisiae arginyl-tRNA synthetase crystal structure is represented by the solid line.



Table 1 provides a summary of the percent similarity between the Candida albicans CURFs of the present invention to the homologous sequences in S. cerevisiae. 
Table 2 provides a summary of the percent identity to sequences homologous to the fungal CURFs of the present invention in addition to the functional annotation of the same.
Table 3 identifies the Genbank Accession No. and/or patent or patent application number of homologous sequences that aligning with the CURFs of the present invention.
Table 4 lists the SEQ ID Nos. for the PCR primers used for knockout experiments.
Table 5 lists the predicted function of the novel conserved essential fungal polypeptides of the present invention.
Table 6 lists the SEQ ID NOs of the MET3 promoter swapping primers which may be used to remove the promoters associated with the essential polynucleotides encoded by SEQ ID NO: 1 through to SEQ ID NO: 11.
Table 7 shows the results of experiments designed to assess the sensitivity of the MET3P-ERG1 construct to antifungal drugs.
Table 8 provides the structural coordinates of the three dimensional structure of the CaYLR100w (FCG5) polypeptide of the present invention (SEQ ID NO:12).
Table 9 provides the structural coordinates of the three dimensional structure of the CaYDR341c (FCG6) polypeptide of the present invention (SEQ ID NO:13).
Table 10 provides the structural coordinates of the three dimensional structure of the CaYOL010w (FCG14) polypeptide of the present invention (SEQ ID NO:19).
Table 11: Illustrates the preferred hybridization conditions for the polynucleotides of the present invention. Other hybridization conditions may be known in the art or are described elsewhere herein.
Table 12: Provides a summary of various conservative substitutions encompassed by the present invention.
DETAILED DESCRIPTION OF THE INVENTION
Nucleic Acids Encoding Essential Polynucleotides
The present invention is directed to polynucleotides that encode polypeptides that have been shown to be essential for Candida albicans growth and survival. Such polypeptides are referred to herein as Fungal Conserved Genes (“FCG”), or Conserved Unknown Reading Frames (“CURFs”).
The present invention is also directed to the homologous polynucleotide and polypeptide sequences of the fungal conserved genes of the present invention in A. fumigatus. 
In one embodiment, the invention provides nucleotide sequences encoding polypeptides that are essential to Candida albicans. A polynucleotide is generally considered essential when the viability of the organism is substantially coupled to or dependent on the expression of the polynucleotide. Such nucleotide sequences include, but are not limited to, SEQ ID NO: 1 through SEQ ID NO:11, fragments and homologues thereof.
The term “essential polynucleotides” refers to a nucleotide sequence that encodes a polynucleotide product (mRNA or protein) having a function which is required for cell viability. The term “essential protein” refers to a polypeptide that is encoded by an essential polynucleotide and has a function that is required for cell viability. Accordingly, a mutation that disrupts the function of the essential polynucleotide or essential proteins results in a loss of viability of cells harboring the mutation.
In another embodiment, the invention provides an isolated nucleic acid molecule having a nucleotide sequence encoding a polypeptide essential to Candida albicans, wherein said polypeptide comprises an amino acid sequence of one of SEQ ID NO: 12 to 22.
The invention also includes the complements of SEQ ID 1 to SEQ ID 11 and fragments thereof.
A nucleic acid molecule is said to be the “complement” of another nucleic acid molecule if two single-stranded polynucleotides anneal by bases-pairing. For example, 5′-ACG-3′ pairs with its complement 3′-TGC-5′. 100% complementarity occurs when every nucleotide of one of the molecules is complementary to a corresponding nucleotide of the other.
The essential polynucleotides represent potential drug targets for Candida albicans and can be used individually or as a collection in various methods of drug screening herein described. The essential polynucleotides provided were found to be homologous with polynucleotides in S. cervisiae and/or other fungi and lacked significant similarity with human polynucleotides. Such an essential polynucleotide set can be conveniently investigated as a group in a drug screening program.
As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising a nucleotide sequence encoding a polypeptide or a biologically active ribonucleic acid (RNA). The term can further include nucleic acid molecules comprising upstream, downstream and/or intron nucleotide sequences. The term “open reading frame (ORF)” means a series of nucleotide triplets coding for amino acids without any termination codons and the triplet sequence is translatable into protein using the codon usage information appropriate for a particular organism.
As used herein, the terms “polynucleotide”, “nucleotide sequence”, “nucleic acid molecule”, “nucleic acid” or “oligonucleotide” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand. In the sequences herein, A is adenine, C is cytosine, G is guanine, T is thymine and N is G, A, C, or T(U). It is contemplated that where the polynucleotide is RNA, the T (thymine) in the sequences provide herein may be substituted with U (uracil).
By “isolated” polynucleotide(s) is intended a polynucleotide, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated polynucleotide molecules according to the present invention further include such molecules produced synthetically.
“Substantially purified” refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
The invention also provides a composition including SEQ ID NO: 1 to SEQ ID NO: 11 or fragments or variants thereof. A “composition including a given polynucleotide sequence or polypeptide sequence” refers broadly to any composition containing the given polynucleotide or polypeptide sequence. The composition may comprise a dry formulation or an aqueous solution. Compositions including polynucleotide sequences, and polynucleotide sequences encoding essential polynucleotides may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS) and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
Fragment nucleic acid molecules may encode significant portion(s) of, or most of, the polypeptides of the present invention. A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide. For example, a fragment may comprise from 5 to 3128 contiguous nucleotides. Fragments of the target polynucleotides of the invention can also refer to portions of the coding regions of such nucleic acid molecules that encode functional domains such as signal sequences, extracellular domains, transmembrane domains and cytoplasmic domains.
Fragment nucleic molecules of the present invention also include primers and probes. “Probe” refers to polynucleotides encoding essential polynucleotides of the invention, their complements or fragments thereof, which are used to detect allelic or related polynucleotides. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, chemiluminescent agents and enzymes (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories (1989), which is incorporated herein by reference in its entirety).
Probes and primers as used in the present invention typically comprise at least 10 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed polynucleotides. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification including the tables, FIGs and Sequence Listing may be used.
The primers of the invention may be used in conjunction with the polymerase chain reaction (PCR). PCR exploits certain features of DNA replication. DNA polymerase uses single-stranded DNA as a template for the synthesis of a complementary new strand. These single-stranded DNA templates can be produced by heating double-stranded DNA to temperatures near boiling. DNA polymerase also requires a small section of double-stranded DNA to initiate (“prime”) synthesis. Therefore, the starting point for DNA synthesis can be specified by supplying a PCR primer that anneals to the template at that point. Methods for designing PCR primers are well-known in the art. (See, for example, Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic, N.Y. (1990), herein incorporated by reference). Computer programs may also be used to design PCR primers. For example, Primer3.
In addition to the nucleotide sequences of Candida albicans described above, homologues of these target polynucleotide sequences in other species can be identified and isolated by molecular biological techniques well-known in the art and without undue experimentation.
To isolate homologous target polynucleotides, the C. albicans target polynucleotide sequences described above can be labeled and used to screen a cDNA library constructed from mRNA obtained from the organism of interest. Hybridization conditions should be of a lower stringency when the cDNA library is derived from an organism different from the type of organism from which the labeled sequence was derived. cDNA screening can also identify clones derived from alternatively spliced transcripts in the same or different species. Alternatively, the labeled fragment can be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions. Reduced stringency conditions will be well-known to those of skill in the art and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. (See, for example, Sambrook et al., supra)
Further, a homologous target polynucleotide sequence can be isolated by performing a polymerase chain reaction (PCR) using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the target polynucleotide of interest. The template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from the organism of interest. The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a homologous target polynucleotide sequence.
Alternatively, homologous target polynucleotides or polypeptides may be identified by searching a dataset to identify sequences having a desired level of homology to an essential polynucleotide of the invention. A variety of such databases are available to those skilled in the art including GenBank. In various embodiments, the databases are screened to identify nucleic acids with at least 97%, at least 95%, at least 90%, at least 85%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, or at least 30% identity to an essential polynucleotide of the invention.
“Homologous sequences” or “homologues” as used herein are those sequences in which a first amino acid or nucleotide sequence contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity when optimally aligned. For example, amino acid or nucleotide sequences which share common structural domains have at least about 30-40% homology, preferably 40-50% homology, more preferably 50-60%, and even more preferably 60-70%, 70-80%, or 80-90% or 95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs. Furthermore, amino acid or nucleotide sequences which share at least 30-40%, preferably 40-50%, more preferably 50-60%, 60-70%, 70-80%, or 80-90% or 95% homology and share a common functional activity are homologous.
In one embodiment, the invention provides for homologues of the essential polynucleotides of the invention encoded by SEQ ID NO: 1 through to SEQ ID NO: 11 in species including, but not limited to, Aspergillus fumigatus, Aspergillus falvus, Aspergillus niger, Coccidiodes immitis, Cryptoccoccus neoformans, Histoplasma capsulatum, Phytophthora infestans, Puccinia seconditii, Pneumocystis carinii or any species falling within the genera of any of the above species. Other yeasts in the genera of Candida, Saccharomyces, Schizosaccharomyces Sporobolomyces, Torulopsis, Trichosporon, Tricophyton, Dermatophytes, Microsproum, Wickerhamia, Ashbya, Blastomyces, Citeromyces, Crebrothecium, Cryptococcus, Debaryomyces, Endomycopsis, Geotrichum, Hansenula, Kloecker, Kluveromyces, Libomyces, Pichia, Rhodosporidium, Rhodotorula, and Yarrowia are also contemplated.
Preferably, homologues of the essential polynucleotides of the invention encoded by SEQ ID NO: 1 through to SEQ ID NO: 11 are from Absidia corymbigera, Aspergillus flavis, Aspergillus fumigatus, Aspergillus niger, Botrytis cinerea, Candida dublinensis, Candida glabrata, Candida krusei, Candia parapsilopsis, Candia tropicalis, Coccidioides immitis, Cryptococcus neoformans, Erysiphe graminis, Exophalia dermatiditis, Fusarium osysproum, Histoplasma capsulatum, Magnaporthe grisea, Mucor rouxii, Pneumocystis carinii, Puccinia graminis, Puccinia recodita, Rhizomucor pusillus, Puccinia striiformis, Rhizopus arrhizus, Septoria avenae, Septoria nodorum, Septoria triticii, Tilletia controversa, Tilletia tritici, Trichospoon beigelii and Ustilago maydis. Particularly preferred are homologues from Aspergillus fumigatus. 
The invention also provides nucleotide sequences that are hybridizable under stringent conditions to the polynucleotides of SEQ ID NO: 1 through SEQ ID NO: 11 and that are of a species other than Saccharomyces cerevisiae and Candida albicans. 
The term “stringent conditions” or “hybridizable under stringent conditions” includes reference to conditions under which a probe will selectively hybridize to its target sequence, to a detectably greater degree than to other sequences (e.g., at least two-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe. Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected. Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na+ ion, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984): Tm=81.5° C.+16.6(log M)+0.41(% GC)−0.61(% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with 90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995), herein incorporated by reference. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.
Additional examples of stringency conditions are shown in Table 11 below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.











TABLE 11








Hyridization



Stringency
Polynucleotide
Hybrid Length
Temperature and
Wash Temperature


Condition
Hybrid±
(bp)‡
Buffer†
and Buffer†







A
DNA:DNA
>or equal to 50
65° C.; 1xSSC -
65° C.;





or- 42° C.;
0.3xSSC





1xSSC, 50%





formamide


B
DNA:DNA
<50
Tb*; 1xSSC
Tb*; 1xSSC


C
DNA:RNA
>or equal to 50
67° C.; 1xSSC -
67° C.;





or- 45° C.;
0.3xSSC





1xSSC, 50%





formamide


D
DNA:RNA
<50
Td*; 1xSSC
Td*; 1xSSC


E
RNA:RNA
>or equal to 50
70° C.; 1xSSC -
70° C.;





or- 50° C.;
0.3xSSC





1xSSC, 50%





formamide


F
RNA:RNA
<50
Tf*; 1xSSC
Tf*; 1xSSC


G
DNA:DNA
>or equal to 50
65° C.; 4xSSC -
65° C.;





or- 45° C.;
1xSSC





4xSSC, 50%





formamide


H
DNA:DNA
<50
Th*; 4xSSC
Th*; 4xSSC


I
DNA:RNA
>or equal to 50
67° C.; 4xSSC -
67° C.;





or- 45° C.;
1xSSC





4xSSC, 50%





formamide


J
DNA:RNA
<50
Tj*; 4xSSC
Tj*; 4xSSC


K
RNA:RNA
>or equal to 50
70° C.; 4xSSC -
67° C.;





or- 40° C.;
1xSSC





6xSSC, 50%





formamide


L
RNA:RNA
<50
Tl*; 2xSSC
Tl*; 2xSSC


M
DNA:DNA
>or equal to 50
50° C.; 4xSSC -
50° C.;





or- 40° C. 6xSSC,
2xSSC





50% formamide


N
DNA:DNA
<50
Tn*; 6xSSC
Tn*; 6xSSC


O
DNA:RNA
>or equal to 50
55° C.; 4xSSC -
55° C.;





or- 42° C.;
2xSSC





6xSSC, 50%





formamide


P
DNA:RNA
<50
Tp*; 6xSSC
Tp*; 6xSSC


Q
RNA:RNA
>or equal to 50
60° C.; 4xSSC -
60° C.; 2Xssc





or- 45° C.;





6xSSC, 50%





formamide


R
RNA:RNA
<50
Tr*; 4xSSC
Tr*; 4xSSC





‡The “hybrid length” is the anticipated length for the hybridized region(s) of the hybridizing polynucleotides. When hybridizing a polynucletotide of unknown sequence, the hybrid is assumed to be that of the hybridizing polynucleotide of the present invention. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.Methods of aligning two or more polynucleotide sequences and/or determining the percent identity between two polynucleotide sequences are well known in the art (e.g., MegAlign program of the DNA*Star suite of programs, etc).


†SSPE (1xSSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is 0.15M NaCl anmd 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. The hydridizations and washes may additionally include 5X Denhardt's reagent, .5-1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide.


*Tb-Tr: The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature Tm of the hybrids there Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(° C.) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C.) = 81.5 +16.6(log10[Na+]) + 0.41 (% G + C) − (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([NA+] for 1xSSC = .165 M).


±The present invention encompasses the substitution of any one, or more DNA or RNA hybrid partners with either a PNA, or a modified polynucleotide. Such modified polynucleotides are known in the art and are more particularly described elsewhere herein.






Additional examples of stringency conditions for polynucleotide hybridization are provided, for example, in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel et al., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, which are hereby incorporated by reference herein.
In another embodiment, the present invention encompasses isolated nucleic acids comprising a nucleotide sequence that has at least 40%, 45%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more nucleotide sequence identity to the nucleotide sequences set forth in SEQ ID NO 1 to SEQ ID NO 11. The nucleotide sequences of the invention also include nucleotide sequences that encode polypeptides having at least 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or higher amino acid sequence identity or similarity to the amino acid sequences set forth in SEQ ID NO 12 to SEQ ID NO 22.
The following terms are used to describe the sequence relationships between a polynucleotide or polypeptide of the present invention with a reference polynucleotide or a polypeptide to determine sequence identity: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, and (d) “percentage of sequence identity”.
As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison with a polynucleotide or a polypeptide of the present invention. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or polynucleotide sequence, or the complete cDNA or polynucleotide sequence.
As used herein. “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the nucleotides or residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. As used herein, “comparison window” includes reference to a contiguous and specified segment of a polynucleotide or polypeptide sequence, wherein the polynucleotide or polypeptide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides/amino acids residues in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide or polypeptide sequence, a gap penalty is typically introduced and is subtracted from the number of matches.
When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988), e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
Tolerated conservative amino acid substitutions of the present invention involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
In addition, the present invention also encompasses the conservative substitutions provided in Table 12 below.









TABLE 12





For Amino




Acid
Code
Replace with any of:







Alanine
A
D-Ala, Gly, beta-Ala, L-Cys, D-Cys


Arginine
R
D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-




Met, D-Ile, Orn, D-Orn


Asparagine
N
D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln


Aspartic
D
D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln


Acid


Cysteine
C
D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr


Glutamine
Q
D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp


Glutamic
E
D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln


Acid


Glycine
G
Ala, D-Ala, Pro, D-Pro, β-Ala, Acp


Isoleucine
I
D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met


Leucine
L
D-Leu, Val, D-Val, Met, D-Met


Lysine
K
D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met,




Ile, D-Ile, Orn, D-Orn


Methionine
M
D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val


Phenylalanine
F
D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-




3,4, or 5-phenylproline, cis-3,4, or 5-phenylproline


Proline
P
D-Pro, L-1-thioazolidine-4-carboxylic acid, D- or L-1-




oxazolidine-4-carboxylic acid


Serine
S
D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O),




L-Cys, D-Cys


Threonine
T
D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O),




Val, D-Val


Tyrosine
Y
D-Tyr, Phe, D-Phe, L-Dopa, His, D-His


Valine
V
D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met









Aside from the uses described above, such amino acid substitutions may also increase protein or peptide stability. The invention encompasses amino acid substitutions that contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the protein or peptide sequence. Also included are substitutions that include amino acid residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids.
Both identity and similarity can be readily calculated by reference to the following publications: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Informatics Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.
In addition, the present invention also encompasses substitution of amino acids based upon the probability of an amino acid substitution resulting in conservation of function. Such probabilities are determined by aligning multiple polynucleotides with related function and assessing the relative penalty of each substitution to proper polynucleotide function. Such probabilities are often described in a matrix and are used by some algorithms (e.g., BLAST, CLUSTALW, GAP, etc.) in calculating percent similarity wherein similarity refers to the degree by which one amino acid may substitute for another amino acid without lose of function. An example of such a matrix is the PAM250 or BLOSUM62 matrix.
Aside from the canonical chemically conservative substitutions referenced above, the invention also encompasses substitutions which are typically not classified as conservative, but that may be chemically conservative under certain circumstances. Analysis of enzymatic catalysis for proteases, for example, has shown that certain amino acids within the active site of some enzymes, may have highly perturbed pKa's due to the unique microenvironment of the active site. Such perturbed pKa's could enable some amino acids to substitute for other amino acids while conserving enzymatic structure and function. Examples of amino acids that are known to have amino acids with perturbed pKa's are the Glu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, the His-159 residue of Papain, etc. The conservation of function relates to either anomalous protonation or anomalous deprotonation of such amino acids, relative to their canonical, non-perturbed pKa. The pKa perturbation may enable these amino acids to actively participate in general acid-base catalysis due to the unique ionization environment within the enzyme active site. Thus, substituting an amino acid capable of serving as either a general acid or general base within the microenvironment of an enzyme active site or cavity, as may be the case, in the same or similar capacity as the wild-type amino acid, would effectively serve as a conservative amino substitution.
As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J Mol. Biol. 48: 443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Nad. Acad. Sci. 85: 2444 (1988); by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA; the CLUSTAL program is well described by Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5. 151.153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al., Computer Applications in the Biosciences 8: 155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24: 307-331 (1994). These references are herein incorporated.
Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the Smith-Waterman algorithm (supra) on a DeCypher system using default parameters (Matrix=Blosum62, Gap Opening penalty: 12, Gap Extension Penalty: 2).
Additionally, whether any particular nucleic acid molecule or polypeptide is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. However, the CLUSTALW algorithm automatically converts U's to T's when comparing RNA sequences to DNA sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA sequences to calculate percent identity via pairwise alignments are: Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of the subject nucleotide sequence, whichever is shorter. For multiple alignments, the following CLUSTALW parameters are preferred: Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation Penalty Range=8; End Gap Separation Penalty=Off; % Identity for Alignment Delay=40%; Residue Specific Gaps: Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. The pairwise and multiple alignment parameters provided for CLUSTALW above represent the default parameters as provided with the AlignX software program (Vector NTI suite of programs, version 6.0).
The present invention encompasses the application of a manual correction to the percent identity results, in the instance where the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions. If only the local pairwise percent identity is required, no manual correction is needed. However, a manual correction may be applied to determine the global percent identity from a global polynucleotide alignment. Percent identity calculations based upon global polynucleotide alignments are often preferred since they reflect the percent identity between the polynucleotide molecules as a whole (i.e., including any polynucleotide overhangs, not just overlapping regions), as opposed to, only local matching polynucleotides. Manual corrections for global percent identity determinations are required since the CLUSTALW program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This corrected score may be used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALW alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the CLUSTALW alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the CLUSTALW program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by CLUSTALW is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are required for the purposes of the present invention.
By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for instance, an amino acid sequence referenced in herein (SEQ ID NO:12-22) or to the amino acid sequence encoded by cDNA contained in a deposited clone, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment the query and subject sequences are both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA sequences to calculate percent identity via pairwise alignments are: Matrix=BLOSUM, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of the subject nucleotide sequence, whichever is shorter. For multiple alignments, the following CLUSTALW parameters are preferred: Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation Penalty Range=8; End Gap Separation Penalty=Off, % Identity for Alignment Delay-40%; Residue Specific Gaps: Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. The pairwise and multiple alignment parameters provided for CLUSTALW above represent the default parameters as provided with the AlignX software program (Vector NTI suite of programs, version 6.0).
The present invention encompasses the application of a manual correction to the percent identity results, in the instance where the subject sequence is shorter than the query sequence because of N- or C-terminal deletions, not because of internal deletions. If only the local pairwise percent identity is required, no manual correction is needed. However, a manual correction may be applied to determine the global percent identity from a global polypeptide alignment. Percent identity calculations based upon global polypeptide alignments are often preferred since they reflect the percent identity between the polypeptide molecules as a whole (i.e., including any polypeptide overhangs, not just overlapping regions), as opposed to, only local matching polypeptides. Manual corrections for global percent identity determinations are required since the CLUSTALW program does not account for N- and C-terminal truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what may be used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.
For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the CLUSTALW alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the CLUSTALW program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence, which are not matched/aligned with the query. In this case the percent identity calculated by CLUSTALW is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the CLUSTALW alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are required for the purposes of the present invention.
In addition to the above method of aligning two or more polynucleotide or polypeptide sequences to arrive at a percent identity value for the aligned sequences, it may be desirable in some circumstances to use a modified version of the CLUSTALW algorithm which takes into account known structural features of the sequences to be aligned, such as for example, the SWISS-PROT designations for each sequence. The result of such a modified CLUSTALW algorithm may provide a more accurate value of the percent identity for two polynucleotide or polypeptide sequences. Support for such a modified version of CLUSTALW is provided within the CLUSTALW algorithm and would be readily appreciated to one of skill in the art of bioinformatics.
Although the nucleotide sequences and amino acid sequences from S. cervisiae which are homologues of the essential polynucleotide encoded by SEQ ID NO: 1 to SEQ ID NO: 11 is mostly published, uses of such homologues in S. cerevisae in drug screening are not known and are thus specifically provided by the invention. To use such nucleotide and/or amino acid sequences of S. cervisiae, public databases, such as Stanford Genomic Resources or Proteome may be used to identify and retrieve the sequences.
The nucleic acid molecules of the invention also include peptide nucleic acids (PNAs), or derivative molecules such as phosphorothioate, phosphotriester, phosphoramidate, and methylphosphonate, that specifically bind to single-stranded DNA or RNA in a base pair-dependent manner (Zamecnik, P. C. et al., Proc. Natl. Acad. Sci. 75:280 284 (1978); Goodchild, P. C., et al., Proc. Natl. Acad. Sci. 83:4143-4146).
PNA molecules comprise a nucleic acid oligomer to which an amino acid residue, such as lysine, and an amino group have been added. These small molecules, also designated anti-polynucleotide agents, stop transcript elongation by binding to their complementary (template) strand of nucleic acid (Nielsen, P. E. et al., Anticancer Drug Des 8:53-63 (1993)). For example, reviews of methods for synthesis of DNA, RNA and their analogues can be found in: Oligonucleotides and Analogues, eds. F. Eckstein, IRL Press, New York (1991); Oligonucleotide Synthesis, ed. M. J. Gait, IRL Press, Oxford, England (1984). Additionally, methods for antisense RNA technology are described in U.S. Pat. Nos. 5,194,428 and 5,110, 802. A skilled artisan can readily obtain these classes of nucleic acid molecules using the herein described polynucleotide sequences; see, for example, Innovative and Perspectives in Solid Phase Synthesis, Egholm, et al. pp 325-328 (1992) or U.S. Pat. No. 5,539,082.
As will be appreciated by the skilled practitioner, should the amino acid fragment comprise an antigenic epitope, for example, biological function per se need not be maintained. The terms fungal essential polypeptide and fungal essential protein are used interchangeably herein to refer to the encoded product of the fungal essential nucleic acid sequence according to the present invention.
It is another aspect of the present invention to provide modulators of the fungal essential polypeptides and fungal essential peptide targets which can affect the function or activity of fungal essential polynucleotides in a cell in which fungal essential polynucleotide function or activity is to be modulated or affected. In addition, modulators of fungal essential polypeptides can affect downstream systems and molecules that are regulated by, or which interact with, fungal essential polypeptides in the cell. Modulators of fungal essential polypeptides include compounds, materials, agents, drugs, and the like, that antagonize, inhibit, reduce, block, suppress, diminish, decrease, or eliminate fungal essential polypeptides function and/or activity. Such compounds, materials, agents, drugs and the like can be collectively termed “antagonists”. Alternatively, modulators of fungal essential polypeptides include compounds, materials, agents, drugs, and the like, that agonize, enhance, increase, augment, or amplify fungal essential polypeptides function in a cell. Such compounds, materials, agents, drugs and the like can be collectively termed “agonists”.
As used herein the terms “modulate” or “modulates” refer to an increase or decrease in the amount, quality or effect of a particular activity, DNA, RNA, or protein. The definition of “modulate” or “modulates” as used herein is meant to encompass agonists and/or antagonists of a particular activity, DNA, RNA, or protein.
The present invention is also directed to polynucleotides encoding the fungal essential polynucleotides of the present invention lacking a start methionine. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. It is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
In specific embodiments, the present invention is directed to the following polynucleotides which encode fungal essential polypeptides of the present invention lacking a start methionine: nucleotides 4 to 1038 of SEQ ID NO:1 (CaYLR100w); nucleotides 4 to 1866 of SEQ ID NO:2 (CaYDR341c); nucleotides 4 to 765 of SEQ ID NO:3 (CaYLR022c); nucleotides 4 to 876 of SEQ ID NO:4 (CaYOL077c); nucleotides 4 to 3126 of SEQ ID NO:5 (CaYNL132w); nucleotides 4 to 2250 of SEQ ID NO:6 (CaYGR145w); nucleotides 4 to 804 of SEQ ID NO:7 (CaYDR412w); nucleotides 4 to 1113 of SEQ ID NO:8 (CaYOL010w); nucleotides 4 to 771 of SEQ ID NO:9 (CaYOR004w); nucleotides 4 to 1398 of SEQ ID NO:10 (CaYOR056c); and/or nucleotides 4 to 585 of SEQ ID NO:11 (CaYLR009w).
In another embodiment, the present invention is directed to representative clones containing all or most of the sequence for SEQ ID NO:1 to SEQ ID NO:11 (encoding the polypeptides provided as SEQ ID NO:12 to SEQ ID NO:22) that were deposited with the American Type Culture Collection (“ATCC”). The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure. The deposited clone is inserted in the pSport1 plasmid (Life Technologies) using the NotI and SalI restriction endonuclease cleavage sites.
Polypeptides
The polypeptides of the invention used and encompassed in the methods and compositions of the present invention include those polypeptides that are encoded by the essential polynucleotide sequences as described above, such as the essential polynucleotide sequences set forth in SEQ ID NO: 1 through to SEQ ID NO: 11. The amino acid sequences of SEQ ID NO: 12 to SEQ ID NO: 22 are deduced using the codon usage of C. albicans from the respective nucleotide sequences of SEQ ID NO: 12 to SEQ ID NO: 22. However, when expressed in an organism other than C. albicans, protein products for the target polynucleotides having the amino acid sequences of SEQ ID NO: 12 to 22 may be encoded by nucleotide sequences that are translated using the universal genetic code. One of skill in the art would know the modifications that are necessary to accommodate for such a difference in codon usage.
As used herein, the term “polypeptide” refers to any peptide or protein including two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. “Polypeptide” refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. “Polypeptides” may contain amino acids other than the 20 gene-encoded amino acids. “Polypeptides” include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well-known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in research literature.
Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. A given polypeptide may contain many types of modifications. Polypeptides may be branched, or cyclic, with or without branching. Cyclic, branched and branched-cyclic polypeptides may result from posttranslational natural processes or may be made by synthetic methods. Modifications include but are not limited to acetylation, acylations, amidation, covalent attachment of flavin, disulfide bond formation, formation of covalent cross-links, and glycosylation. See, for instance, Proteins—structure and molecular properties, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York (1993); F. Wold, Posttranslational protein modifications: perspectives and prospects, pgs 1-12 in Posttranslational covalent modification of proteins, B. C. Johnson, Ed., Academic Press, New York (1983); S. Seifter and S. Englard, Analysis for protein modifications and nonprotein cofactors, 182 Methods of Enzymology 626 (1990); and S. I. Rattan et al., Protein synthesis, posttranslational modifications, and aging, 663 Ann NY Acad Sci 48 (1992).
In addition, however, the methods and compositions of the invention also use and encompass proteins and polypeptides that represent functionally equivalent polynucleotide products. Such functionally equivalent polynucleotide products include, but are not limited to, natural variants of the polypeptides having an amino acid sequence set forth in SEQ ID NO; 12 to SEQ ID NO: 22.
The term “variant” (or analog) as used herein is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequence of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions and/or deletions in any combination.
Variants of the defined sequence and fragments thereof also form part of the present invention. Preferred variants are those that vary from the reference sequence by conservative amino acid substitutions, i.e., those that substitute a residue with another of like characteristics. Typical conservative substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the acid residues Asp and Glu; among Asn and Gln; among the basic residues Lys and Arg; and among the aromatic residues Phe and Tyr.
The term “functionally equivalent”, as utilized herein, refers to a polypeptide capable of exhibiting a substantially similar in vivo activity as the Candida albicans essential polypeptide encoded by one or more of the essential polynucleotide sequences described herein. Alternatively, when utilized as part of assays described herein below, the term “functionally equivalent” can refer to peptides or polypeptides that are capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the corresponding portion of the target polynucleotide product would interact with such other molecules. Preferably, the functionally equivalent essential polynucleotide polypeptide of the invention are also the same size or about the same size as a essential polynucleotide polypeptide encoded by one or more of the essential polynucleotide sequences described herein.
Fragments of the essential polynucleotide polypeptides are also included in the invention. A fragment is a polypeptide having an amino acid sequence that is entirely the same as part, but not all, of the amino acid sequence of the aforementioned essential polynucleotide polypeptides. As with essential polynucleotide polypeptides, fragments may be “free-standing” or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region.
Preferred fragments of the invention are biologically active fragments. The term “active” refers to those forms of the polypeptide that retain the biologic and/or immunologic activities of any naturally occurring polypeptide. According to the invention, the terms “biologically active” or “biological activity” refer to a protein or peptides having structural, regulatory or biochemical functions of a naturally occurring molecule. Likewise “biologically active” or “biological activity” refers to the capability of the natural, recombinant or synthetic essential polynucleotide peptide, or any peptide thereof, to include a specific biological response in appropriate animals or cells and to bind with specific antibodies.
Additionally, preferred polypeptides are those containing fragments including at least about a contiguous 5 amino acid region, more preferably including at least a contiguous 10, 40, 50, 75 or 125 amino acid region of a protein or fragment thereof of the present invention. In another preferred embodiment, the proteins of the present invention include between about 10 and about 25 contiguous amino acid region, more preferably between about 20 and about 50 contiguous amino acid region and even more preferably between about 40 and about 80 contiguous amino acid region.
Such fragments are conventionally employed by themselves or with unrelated proteins as part of fusion proteins. As used herein, a fusion protein comprises all or part (preferably a biologically active part) of a polypeptide of the invention operably linked to a heterologous or unrelated polypeptide. The unrelated polypeptide may be a detectable label for enabling detection of the polypeptide of the invention or a matrix-binding domain for immobilizing the fusion protein. The fusion proteins can be produced by standard recombinant DNA techniques.
Possible fusion protein expression vectors include but are not limited to vectors incorporating sequences that encode beta-galactosidease and trpE fusions, maltose-binding protein fusions (pMal series; New England Biolabs), glutathionie-S-transferase fusions (pGEX series; Pharmacia) polyhistidine fusions (PET series; Novagen Inc., Madison, Wis.), and thioredoxin fusion s(pTrxFus; Invitrogen, Carlsbad, Calif.).
Expression vectors may be constructed that will express a fusion protein including any protein or polypeptide of the present invention including fragments or variants thereof. Such fusion proteins can be used, e.g., to raise antisera against the protein, to study the biochemical properties of the protein, to engineer a protein exhibiting different immunological or functional properties, to aid in the identification or purification of the protein, to improve the stability of a recombinantly-expressed protein or as therapeutic agents. Methods are well-known in the art for constructing expression vectors encoding these and other fusion proteins.
The essential polynucleotide polypeptides of the invention can be prepared in any suitable manner. The polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides and polypeptides produced by a combination of these methods. These methods are well understood in the art.
The present invention also provides for homologous proteins. A homologue protein may be derived from, but not limited to, Aspergillus fumigatus, Aspergillus falvus, Aspergillus niger, Coccidiodes immitis, Cryptoccoccus neoformans, Histoplasma capsulatum, Phytophthora infestans, Puccinia seconditii, Pneumocystis carinii or any species falling within the genera of any of the above species. Other yeasts in the genera of Candida, Saccharomyces, Schizosaccharomyces Sporobolomyces, Torulopsis, Trichosporon, Tricophyton, Dermatophytes, Microsproum, Wickerhamia, Ashbya, Blastomyces, Citeromyces, Crebrothecium, Cryptococcus, Debaryomyces, Endomycopsis, Geotrichum, Hansenula, Kloecker, Kluveromyces, Libomyces, Pichia are also contemplated.
Preferably, homologues of the polypeptides of the invention encoded by SEQ ID NO: 1 through to SEQ ID NO: 11 are from Absidia corymbigera, Aspergillus flavis, Aspergillus fumigatus, Aspergillus niger, Botrytis cinerea, Candida dublinensis, Candida glabrata, Candida krusei, Candia parapsilopsis, Candia tropicalis, Coccidioides immitis, Cryptococcus neoformans, Erysiphe graminis, Exophalia dermatiditis, Fusarium osysproum, Histoplasma capsulatum, Magnaporthe grisea, Mucor rouxii, Pneumocystis carinii, Puccinia graminis, Puccinia recodita, Rhizomucor pusillus, Puccinia striiformis, Rhizopus arrhizus, Septoria avenae, Septoria nodorum, Septoria triticii, Tilletia controversa, Tilletia tritici, Trichospoon beigelii and Ustilago maydis fumigatus. 
Particularly preferred homologues of the present invention are from Aspergillus fumigatus. Particularly preferred homologous have an amino acid sequence comprising SEQ ID NO: 48 to SEQ ID NO: 73.
Desirably, a homologue can be derived by using one or more of the disclosed sequences to define a pair of primers to isolate the homologue-encoding nucleic acid molecules from any desired species. Such molecules can be expressed to yield protein homologues by recombinant means.
A homologue of an essential polynucleotide polypeptide is a polypeptide having an amino acid sequence that is homologous to a natural essential polynucleotide polypeptide amino acid sequence that a nucleic acid sequence encoding the homologue is capable of hybridizing under reduced and/or high stringent conditions to a nucleic acid sequence encoding the natural essential polynucleotide polypeptide amino acid sequence disclosed herein. Preferably the homologue retains one or more biological activities of essential polynucleotide.
Essential polynucleotide protein homologues of the invention include allelic variations of the natural polynucleotide encoding the essential polynucleotide protein. A “natural” polynucleotide is one that is found in nature. Essential polynucleotide protein homologues can be produced using techniques known in the art, including but not limited to direct modifications to a polynucleotide encoding a protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutapolynucleotidesis.
The present invention encompasses the essential polynucleotide proteins that have undergone posttranslational modification. Such modification can include, for example, glycosylation (e.g., including the addition of N-linked and/or O-lined oligosaccharides) or post translation conformation changes or post translation deletions.
The present invention is also directed to polypeptides lacking a start methionine. It is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
In specific embodiments, the present invention is directed to the following polypeptides which correspond to fungal essential polypeptides of the present invention lacking a start methionine: amino acids 2 to 346 of SEQ ID NO:12 (CaYLR100w); amino acids 2 to 622 of SEQ ID NO:13 (CaYDR341c); amino acids 2 to 255 of SEQ ID NO:14 (CaYLR022c); amino acids 2 to 292 of SEQ ID NO:15 (CaYOL077c); amino acids 2 to 1042 of SEQ ID NO:16 (CaYNL132w); amino acids 2 to 750 of SEQ ID NO:17 (CaYGR145w); amino acids 2 to 268 of SEQ ID NO:18 (CaYDR412w); amino acids 2 to 371 of SEQ ID NO:19 (CaYOL010w); amino acids 2 to 257 of SEQ ID NO:20 (CaYOR004w); amino acids 2 to 466 of SEQ ID NO:21 (CaYOR056c); and/or amino acids 2 to 195 of SEQ ID NO:22 (CaYLR009w).
Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the protein without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)).
Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].”. In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.
Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the protein will likely be retained when less than the majority of the residues of the protein are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.
Alternatively, such N-terminus or C-terminus deletions of a polypeptide of the present invention may, in fact, result in a significant increase in one or more of the biological activities of the polypeptide(s). For example, biological activity of many polypeptides are governed by the presence of regulatory domains at either one or both termini. Such regulatory domains effectively inhibit the biological activity of such polypeptides in lieu of an activation event (e.g., binding to a cognate ligand or receptor, phosphorylation, proteolytic processing, etc.). Thus, by eliminating the regulatory domain of a polypeptide, the polypeptide may effectively be rendered biologically active in the absence of an activation event.
Features of the Polypeptide Encoded by Polynucleotide No:1
The polynucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO:12) of the novel fungal essential gene, CaYLR100w (also referred to as FCG5), of the present invention. The CaYLR100w polypeptide (SEQ ID NO:12) is encoded by nucleotides 1 to 1038 of SEQ ID NO:1 and has a predicted molecular weight of 39.0 kDa.
In preferred embodiments, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of CaYLR100w. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 4 thru 1038 of SEQ ID NO:1, and the polypeptide corresponding to amino acids 2 thru 346 of SEQ ID NO:13. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.
As illustrated in FIGS. 1 and 2 and described elsewhere herein, the C. albicans CaYLR100w (FCG5) polynucleotide of the present invention, has been found to share 60% identity at the protein level with the S. cerevisiae ERG27 (“ScERG27”) polynucleotide which encodes 3-keto sterol reductase involved in ergosterol biosynthesis. The CaYLR100w has been demonstrated biochemically to represent a 3-keto sterol reductase involved in C-4 sterol demethylation.
Briefly, CaYLR100w was determined to represent a homolog of ScERG27 by using a genetically modified strain where the target CaYLR100w polynucleotide was placed under the control of the CaMET3 promoter. Using this system, reduced synthetic activity of the ergosterol pathway was observed upon reduced CaYLR100w expression via down-regulation of the CaMET3 promoter by methionine and cysteine (see FIG. 22B). A similar effect was also observed with a known polynucleotide CaERG1 (encoding squalene epoxidase) used as a control (see FIG. 22A).
As shown in FIG. 22A, where a control strain caerg1Δ/PMET3-CaERG1 was used (see FIG. 22A), downregulation of the CaERG1 promoter in the presence of methionine and cysteine decreased incorporation of [14C]-acetate into the ergosterol biosynthetic pathway. The observed decrease, ranging from 62 to 88% compared with untreated cells (FIG. 24A), in incorporation of [14C]-acetate into ergosterol was observed beginning within 1 hour of methionine and cysteine addition and continued to decrease over the 4.5 hour experimental period (FIG. 22A). Downregulation of CaYLR100w also had a significant effect on cell growth as demonstrated by decreased absorbance at OD600 (FIG. 23A). However, the decrease in ergosterol synthesis by methionine and cysteine was not the result of having fewer cells present compared with untreated cells over the 4.5 hour time frame since all samples were adjusted to the same density prior to addition of radolabeled actetate.
Terbinofine is a specific inhibitor of CaErg1 (squalene epoxidase) and therefore is capable of blocking ergosterol synthesis at squalene in the biosynthetic pathway. The caerg1Δ/PMET3-CaERG1 strain treated with terbinofine demonstrated a block in ergosterol biosynthesis at squalene as can be visualized in lanes 1 and 2 in FIG. 22A. Downregulation of the ergosterol biosynthetic pathway in the caerg1Δ/PMET3-CaERG1 strain with methionine and cysteine resulted in the decreased accumulation of [14C]-acetate into squalene as compared with the untreated cells (FIG. 22A, lanes 5 and 6).
Results obtained with the test strain fcg5Δ/PMET3-FCG5 were similar to those obtained with the caerg1Δ/PMET3-CaERG1 control strain as seen in FIG. 22B and FIG. 23B. [14C]-acetate counts incorporated into the ergosterol and lanosterol intermediates decreased between 62 to 88% compared with untreated cells following the 45 minute to 4.5 hour downregulation period (FIGS. 22B and 24B). Cell growth of this strain was also retarded in the presence of methionine and cysteine, as expected (FIG. 23B). As seen with the caerg1Δ/PMET3-CaERG1 control strain, terbinofine treatment blocked the incorporation of radiolabelled acetate into ergosterol, with a majority of the counts trapped in squalene. Methionine and cysteine treated fcg5Δ/PMET3-FCG5 cells that were subsequently treated with terbinofine prior to the incorporation of [14C]-acetate demonstrated decreased counts associated with squalene as compared with non-downregulated, terbinofine treated cells (FIG. 22B, lanes 5 and 6). These results demonstrate that downregulation of either the CaERG1 or the CaFCG5 genes will result in decreased activity of the ergosterol biosynthetic pathway in general.
Given its high homology with the S. cerevisiae counterpart, CaYLR100w or FCG5, in conjunction with the biochemical data provided herein, is therefore an otholog of ScERG27 that encodes 3-keto sterol reductase in C. albicans. 
The CaYLR100w (FCG5) has been formally renamed “CaERG27”. As CaERG27 is an essential polynucleotide in C. albicans, downregulation with methionine and cysteine would presumably render cells more susceptible to inhibition by antifungal agents and thus would be extremely useful in drug discovery for fungal therapeutics.
CaYLR100w polynucleotides and polypeptides, including fragments and modualtors thereof, are useful for the treatment, amelioration, and/or detection of fungal diseases and/or disorders, and are also useful in drug discovery for identifying additional fungal therapeutics.
The invention also encompasses N- and/or C-terminal deletions of the CaYLR100w polypeptide of the present invention.
In preferred embodiments, the following N-terminal CaYLR100w deletion polypeptides are encompassed by the present invention: M1-P346, S2-P346, L3-P346, L4-P346, K5-P346, D6-P346, S7-P346, T8-P346, V9-P346, A10-P346, V11-P346, I12-P346, T13-P346, G14-P346, T15-P346, S16-P346, S17-P346, N18-P346, L19-P346, G20-P346, F21-P346, N22-P346, I23-P346, A24-P346, V25-P346, R26-P346, L27-P346, L28-P346, E29-P346, G30-P346, L31-P346, P32-P346, D33-P346, N34-P346, K35-P346, E36-P346, I37-P346, T38-P346, L39-P346, V40-P346, V41-P346, T42-P346, S43-P346, R44-P346, T45-P346, L46-P346, P47-P346, K48-P346, V49-P346, K50-P346, E51-P346, V52-P346, I53-P346, S54-P346, D55-P346, I56-P346, K57-P346, K58-P346, Y59-P346, I60-P346, V61-P346, A62-P346, K63-P346, I64-P346, P65-P346, T66-P346, K67-P346, V68-P346, N69-P346, K70-P346, V71-P346, E72-P346, F73-P346, D74-P346, Y75-P346, L76-P346, L77-P346, V78-P346, D79-P346, F80-P346, T81-P346, D82-P346, M83-P346, V84-P346, S85-P346, I86-P346, L87-P346, S88-P346, A89-P346, Y90-P346, Y91-P346, E92-P346, L93-P346, N94-P346, K95-P346, R96-P346, Y97-P346, K98-P346, H99-P346, I100-P346, D101-P346, Y102-P346, L103-P346, F104-P346, I105-P346, N106-P346, A107-P346, A108-P346, Q109-P346, G110-P346, V111-P346, Y112-P346, G113-P346, G114-P346, I115-P346, D116-P346, W117-P346, T118-P346, G119-P346, A120-P346, V121-P346, L122-P346, E123-P346, V124-P346, L125-P346, Q126-P346, S127-P346, P128-P346, I129-P346, E130-P346, A131-P346, V132-P346, T133-P346, N134-P346, P135-P346, T136-P346, Y137-P346, K138-P346, L139-P346, Q140-P346, K141-P346, V142-P346, G143-P346, V144-P346, E145-P346, S146-P346, G147-P346, D148-P346, K149-P346, L150-P346, G151-P346, L152-P346, V153-P346, F154-P346, Q155-P346, A156-P346, N157-P346, V158-P346, F159-P346, G160-P346, P161-P346, Y162-P346, Y163-P346, F164-P346, I165-P346, H166-P346, R167-P346, I168-P346, K169-P346, H170-P346, L171-P346, L172-P346, E173-P346, N174-P346, G175-P346, G176-P346, K177-P346, I178-P346, V179-P346, W180-P346, V181-P346, S182-P346, S183-P346, L184-P346, M185-P346, S186-P346, S187-P346, P188-P346, K189-P346, Y190-P346, L191-P346, S192-P346, F193-P346, N194-P346, D195-P346, L196-P346, Q197-P346, L198-P346, L199-P346, R200-P346, S201-P346, P202-P346, A203-P346, S204-P346, Y205-P346, E206-P346, G207-P346, S208-P346, K209-P346, R210-P346, L211-P346, V212-P346, D213-P346, L214-P346, M215-P346, H216-P346, F217-P346, G218-P346, T219-P346, Y220-P346, N221-P346, K222-P346, L223-P346, E224-P346, R225-P346, E226-P346, H227-P346, G228-P346, I229-P346, K230-P346, Q231-P346, Y232-P346, L233-P346, V234-P346, H235-P346, P236-P346, G237-P346, I238-P346, F239-P346, T240-P346, S241-P346, F242-P346, S243-P346, F244-P346, F245-P346, Q246-P346, Y247-P346, L248-P346, N249-P346, V250-P346, F251-P346, T252-P346, Y253-P346, Y254-P346, G255-P346, M256-P346, L257-P346, F258-P346, L259-P346, F260-P346, Y261-P346, L262-P346, A263-P346, R264-P346, F265-P346, L266-P346, G267-P346, S268-P346, P269-P346, Y270-P346, H271-P346, N272-P346, I273-P346, S274-P346, G275-P346, Y276-P346, I277-P346, A278-P346, A279-P346, N280-P346, A281-P346, P282-P346, V283-P346, A284-P346, A285-P346, A286-P346, L287-P346, G288-P346, Q289-P346, T290-P346, K291-P346, Q292-P346, N293-P346, C294-P346, K295-P346, T296-P346, A297-P346, S298-P346, A299-P346, C300-P346, T301-P346, R302-P346, S303-P346, G304-P346, K305-P346, E306-P346, Y307-P346, L308-P346, L309-P346, E310-P346, E311-P346, E312-P346, I313-P346, D314-P346, S315-P346, T316-P346, G317-P346, L318-P346, D319-P346, D320-P346, V321-P346, V322-P346, L323-P346, Y324-P346, L325-P346, D326-P346, T327-P346, L328-P346, T329-P346, K330-P346, E331-P346, W332-P346, D333-P346, E334-P346, K335-P346, L336-P346, K337-P346, D338-P346, Q339-P346, and/or I340-P346 of SEQ ID NO:12. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CaYLR100w deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CaYLR100w deletion polypeptides are encompassed by the present invention: M1-P346, M1-Q345, M1-R344, M1-T343, M1-N342, M1-V341, M1-I340, M1-Q339, M1-D338, M1-K337, M1-L336, M1-K335, M1-E334, M1-D333, M1-W332, M1-E331, M1-K330, M1-T329, M1-L328, M1-T327, M1-D326, M1-L325, M1-Y324, M1-L323, M1-V322, M1-V321, M1-D320, M1-D319, M1-L318, M1-G317, M1-T316, M1-S315, M1-D314, M1-I313, M1-E312, M1-E311, M1-E310, M1-L309, M1-L308, M1-Y307, M1-E306, M1-K305, M1-G304, M1-S303, M1-R302, M1-T301, M1-C300, M1-A299, M1-S298, M1-A297, M1-T296, M1-K295, M1-C294, M1-N293, M1-Q292, M1-K291, M1-T290, M1-Q289, M1-G288, M1-L287, M1-A286, M1-A285, M1-A284, M1-V283, M1-P282, M1-A281, M1-N280, M1-A279, M1-A278, M1-I277, M1-Y276, M1-G275, M1-S274, M1-I273, M1-N272, M1-H271, M1-Y270, M1-P269, M1-S268, M1-G267, M1-L266, M1-F265, M1-R264, M1-A263, M1-L262, M1-Y261, M1-F260, M1-L259, M1-F258, M1-L257, M1-M256, M1-G255, M1-Y254, M1-Y253, M1-T252, M1-F251, M1-V250, M1-N249, M1-L248, M1-Y247, M1-Q246, M1-F245, M1-F244, M1-S243, M1-F242, M1-S241, M1-T240, M1-F239, M1-I238, M1-G237, M1-P236, M1-H235, M1-V234, M1-L233, M1-Y232, M1-Q231, M1-K230, M1-I229, M1-G228, M1-H227, M1-E226, M1-R225, M1-E224, M1-L223, M1-K222, M1-N221, M1-Y220, M1-T219, M1-G218, M1-F217, M1-H216, M1-M215, M1-L214, M1-D213, M1-V212, M1-L211, M1-R210, M1-K209, M1-S208, M1-G207, M1-E206, M1-Y205, M1-S204, M1-A203, M1-P202, M1-S201, M1-R200, M1-L199, M1-L198, M1-Q197, M1-L196, M1-D195, M1-N194, M1-F193, M1-S192, M1-L191, M1-Y190, M1-K189, M1-P188, M1-S187, M1-S186, M1-M185, M1-L184, M1-S183, M1-S182, M1-V181, M1-W180, M1-V179, M1-I178, M1-K177, M1-G176, M1-G175, M1-N174, M1-E173, M1-L172, M1-L171, M1-H170, M1-K169, M1-I168, M1-R167, M1-H166, M1-I165, M1-F164, M1-Y163, M1-Y162, M1-P161, M1-G160, M1-F159, M1-V158, M1-N157, M1-A156, M1-Q155, M1-F154, M1-V153, M1-L152, M1-G151, M1-L150, M1-K149, M1-D148, M1-G147, M1-S146, M1-E145, M1-V144, M1-G143, M1-V142, M1-K141, M1-Q140, M1-L139, M1-K138, M1-Y137, M1-T136, M1-P135, M1-N134, M1-T133, M1-V132, M1-A131, M1-E130, M1-I129, M1-P128, M1-S127, M1-Q126, M1-L125, M1-V124, M1-E123, M1-L122, M1-V121, M1-A120, M1-G119, M1-T118, M1-W117, M1-D116, M1-I115, M1-G114, M1-G113, M1-Y112, M1-V111, M1-G110, M1-Q109, M1-A108, M1-A107, M1-N106, M1-I105, M1-F104, M1-L103, M1-Y102, M1-D101, M1-I100, M1-H99, M1-K98, M1-Y97, M1-R96, M1-K95, M1-N94, M1-L93, M1-E92, M1-Y91, M1-Y90, M1-A89, M1-S88, M1-L87, M1-I86, M1-S85, M1-V84, M1-M83, M1-D82, M1-T81, M1-F80, M1-D79, M1-V78, M1-L77, M1-L76, M1-Y75, M1-D74, M1-F73, M1-E72, M1-V71, M1-K70, M1-N69, M1-V68, M1-K67, M1-T66, M1-P65, M1-I64, M1-K63, M1-A62, M1-V61, M1-I60, M1-Y59, M1-K58, M1-K57, M1-I56, M1-D55, M1-S54, M1-I53, M1-V52, M1-E51, M1-K50, M1-V49, M1-K48, M1-P47, M1-L46, M1-T45, M1-R44, M1-S43, M1-T42, M1-V41, M1-V40, M1-L39, M1-T38, M1-I37, M1-E36, M1-K35, M1-N34, M1-D33, M1-P32, M1-L31, M1-G30, M1-E29, M1-L28, M1-L27, M1-R26, M1-V25, M1-A24, M1-I23, M1-N22, M1-F21, M1-G20, M1-L19, M1-N18, M1-S17, M1-S16, M1-T15, M1-G14, M1-T13, M1-I12, M1-V11, M1-A10, M1-V9, M1-T8, and/or M1-S7 of SEQ ID NO:12. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CaYLR100w deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses polynucleotides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polynucleotide sequence provided in SEQ ID NO:1, and in particular to the coding region of the polynucleotide sequence provided in SEQ ID NO:1. Preferably such polynucleotides encode polypeptides that have biological activity, particularly 3-keto sterol reductase activity.
The present invention also encompasses polypeptides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polypeptide sequence provided in SEQ ID NO:12.
Most preferred are polypeptides that share at least about 99.5% identity with the polypeptide sequence provided in SEQ ID NO:12.
The present invention is also directed to a homology model detailing the three-dimensional structure of the CaYLR100w polypeptide (SEQ ID NO:12) of the present invention.
Protein threading and molecular modeling of CaYLR100w suggest that CaYLR100w has a three dimensional fold similar to that of the porcine carbonyl reductase EC number 1.1.1.- (Ghosh et al., 2001), Protein Data Bank (PDB, Bernstein et. al., 1977 & Berman et. al., 2000) entry 1HU4. Based on sequence, structure, motifs and known short chain dehydrogenase/reductase family signature sequences, CaYLR100w contains a novel known short chain dehydrogenase/reductase domain found also in 3-keto sterol reductases.
The polypeptide CaYLR100w contains a distinct structural domain know as the short chain dehydrogenase/reductase (SDR) superfamily catalytic domain which contains the active site. The three dimensional crystallographic structure for several short chain dehydrogenase/reductases have been reported and are deposited into the Protein Data Bank (Ghosh et al., 2001, 2000, Bernstein et. al., 1977, Berman et. al., 2000). The structure (Protein Data Bank, PDB entry 1HU4) of the carbonyl reductase from pig (porcine) is similar to the other short chain dehydrogenase/reductases (EC 1.1.1.-) and is the closest structural homolog of CaYLR100w.
The short chain dehydrogenase/reductase (SDR) family is a very large family of enzymes that are known to be NAD- or NADP-dependent oxidoreductases. Most members of this family are 250 to 300 amino acids in length. This family of proteins uses a Tyr-Lys-Ser triad as catalytic residues. The SDRs catalyze the activation and inactivation of steroids, vitamins, protstaglandins and other bioactive molecules by oxidation and reduction of hydroxyl and carbonyl groups, respectively. CaYLR100w is thought to have the 3-keto sterol reductase activity and the 3-keto sterol reductases are members of the SDR superfamily.
The basic SDR fold includes a seven stranded parallel beta sheet flanked by three parallel helices on each side. The core of this domain contains the classic “Rossman fold” that has been associated with coenzyme NADPH binding. Mutagenesis and modeling experiments have suggested that a Tyr and Lys (part of a catalytic triad) comprising the YXXXK (SEQ ID NO:254) motif, demonstrate that the Tyr proton as a donor in electrophilic attack on the substrate carbonyl in a reduction reaction. These SDRs are also referred to as short chain dehydrogenase/reductases and seem to share the same core domain tertiary structure based on a Rossmann fold.
This structure-based information and sequence information from novel genes can be used to identify other protein family members that share this same fold.
The present invention provides a three dimensional model of the CaYLR100w polypeptide. The three dimensional model provides for a specific description of the catalytic core and functional sites in the 3-keto sterol CaYLR100w polypeptide.
The catalytic core and functional sites are defined by atomic coordinates (Table 8). Based on these data, the inventors have ascribed the CaYLR100w polypeptide as having dehydrogenase/reductase activity(s), specifically the 3-keto sterol reductase activity and cellular and systemic regulatory function(s). Specifically the reductase activity relates to the activation and/or inactivation of steroids, vitamins, protstaglandins and other bioactive molecules by reduction of hydroxyl groups. For CaYLR100w it is the reductase activity at the 3-keto position of steroids during the biosynthesis of ergosterol.
Homology models are useful when there is no experimental information available on the protein of interest. A three dimensional model can be constructed on the basis of the known structure of a homologous protein (Greer et. al., 1991, Lesk, et. al., 1992, Levitt, 1992, Cardozo, et. al., 1995, Sali, et. al., 1995).
Those of skill in the art will understand that a homology model is constructed on the basis of first identifying a template, or, protein of known structure which is similar to the protein without known structure. This can be accomplished by pairwise alignment of sequences using such programs as FASTA (Pearson, et. al. 1990) and BLAST (Altschul, et. al., 1990). In cases where sequence similarity is high (greater than 30%) these pairwise comparison methods may be adequate. Likewise, multiple sequence alignments or profile-based methods can be used to align a query sequence to an alignment of multiple (structurally and biochemically) related proteins. When the sequence similarity is low, more advanced techniques are used such as fold recognition (protein threading; Hendlich, et. al., 1990, Koppensteiner et. al. 2000, Sippl & Weitckus 1992, Sippl 1993), where the compatibility of a particular sequence with the three dimensional fold of a potential template protein is gauged on the basis of a knowledge-based potential. Following the initial sequence alignment, the query template can be optimally aligned by manual manipulation or by incorporation of other features (motifs, secondary structure predictions, and allowed sequence conservation, etc.). Next, structurally conserved regions can be identified and are used to construct the core secondary structure (Levitt, 1992, Sali, et. al., 1995) elements in the three dimensional model. Variable regions, called “unconserved regions” and loops can be added using knowledge-based techniques. The complete model with variable regions and loops can be refined performing forcefield calculations (Sali, et. al., 1995, Cardozo, et. al., 1995).
For CaYLR100w, a multiple sequence alignment generated manually by combining results from protein threading pairwise alignments and these pairwise alignments were used to align the sequence of CaYLR100w with the sequence of porcine carbonyl reductase, alcohol dehydrogenases, 17β hydroxysteroid dehydrogenases and other SDRs for which three dimensional structures exist. The alignment produced a sequence identity of 20% between the porcine carbonyl reductase (Gosh et. al. 2001, Protein Data Bank entry 1HU4; Genbank Accession No. gi|15826210; SEQ ID NO:251). The alignment of CaYLR100w with PDB entry 1 HU4 chain A is set forth in FIG. 26.
For the present invention, the homology model of CaYLR100w was derived from the sequence alignment set forth in FIG. 26. An overall atomic model including plausible sidechain orientations was generated using the program LOOK (Levitt, 1992). The three dimensional model for CaYLR100w is defined by the set of structure coordinates as set forth in Table 8 and is shown in FIG. 27 rendered by backbone secondary structures.
In order to recognize errors in three-dimensional structures, knowledge based mean fields can be used to judge the quality of protein folds (Sippl 1993). The methods can be used to recognize misfolded structures as well as faulty parts of structural models. The technique generates an energy graph where the energy distribution for a given protein fold is displayed on the y-axis and residue position in the protein fold is displayed on the x-axis. The knowledge based mean fields compose a force field derived from a set of globular protein structures taken as a subset from the Protein Data Bank (Bernstein et. al. 1977). To analyze the quality of a model, the energy distribution is plotted and compared to the energy distribution of the template from which the model was generated. FIG. 28 shows the energy graph for the CaYLR100w model (dotted line) and the template (porcine carbonyl reductase) from which the model was generated. The model has virtually an identical energy plot when compared to the short chain dehydrogenase/reductase template demonstrating that CaYLR100w has similar structural characteristics except for one region corresponding to residues 100-150 of CaYLR 100w. However the energy plot suggests the overall model three-dimensional fold for CaYLR 100w is “native-like”. This graph supports the motif and sequence alignments described herein in confirming that the three dimensional structure coordinates of CaYLR100w are an accurate and useful representation for the polypeptide.
The term “structure coordinates” refers to Cartesian coordinates generated from the building of a homology model.
Those of skill in the art will understand that a set of structure coordinates for a protein is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates, as emanate from generation of similar homology models using different alignment templates (i.e., other than the structure coordinates of 1HU4), and/or using different methods in generating the homology model, will have minor effects on the overall shape. Variations in coordinates may also be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in Table 8 could be manipulated by fractionalization of the structure coordinates; integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
Various computational analyses are therefore necessary to determine whether a molecule or a portion thereof is sufficiently similar to all or parts of CaYLR100w described above as to be considered the same. Such analyses may be carried out in current software applications, such as INSIGHTII (Accelrys Inc., San Diego, Calif.) version 2000 as described in the User's Guide, online or software applications available in the SYBYL software suite (Tripos Inc., St. Louis, Mo.).
Using the superimposition tool in the program INSIGHTII comparisons can be made between different structures and different conformations of the same structure. The procedure used in INSIGHTII to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); the second structure (i.e., moving structure) is identified as the source structure. Since atom equivalency within INSIGHTII is defined by user input, for the purpose of this invention equivalent atoms are defined as protein backbone atoms. (N, Cα, C and O) for all conserved residues between the two structures being compared. Also, only rigid fitting operations were considered. When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by INSIGHTII.
For the purpose of this invention, any homology model of a CaYLR100w that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) of less than about 2.0 A when superimposed on the relevant backbone atoms described by structure coordinates listed in Table 8 are considered identical. More preferably, the root mean square deviation is less than about 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 Angstroms.
The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the “root mean square deviation” defines the variation in the backbone of a protein from the relevant portion of the backbone of CaYLR100w as defined by the structure coordinates described herein.
This invention as embodied by the three-dimensional model enables the structure-based design of modulators of the biological function of CaYLR100w, as well as mutants with altered biological function and/or specificity.
The sequence alignment (FIG. 26) used as a template for creating the three-dimensional model of CaYLR100w short chain dehydrogenase/reductase domain shows 20% sequence identity between catalytic domain of CaYLR100w and porcine carbonyl reductase, PDB code 1HU4.
In the active site of short chain dehydrogenase/reductases (“SDRS”), there is a catalytic triad (YKS) that catalyzes the activation and inactivation of steroids, vitamins, protstaglandins and other bioactive molecules by oxidation and reduction of hydroxyl and carbonyl groups, respectively. The tyrosine side chain is thought to act as the proton donor in electrophilic attack on the substrate carbonyl in a reduction reaction. In porcine carbonyl reductase, the catalytic triad consists of Y193, K197 and S139. In the model and alignment, FIG. 26 and FIG. 27 of CaYLR100w shows that two of the three catalytic residues are conserved and are displayed in the active site. The tyrosine and serine are conserved in CaYLR100w (Y247 and S183) but the lysine position is a phenylalanine (F251) in CaYLR100w. The fact that two of the three catalytic residues are conserved supports the assignment of function for CaYLR100w as a 3-keto sterol reductase member of the SDR superfamily.
The conservation of the catalytic amino acids as part of the active site and the overall 20% sequence identity emphasizes the significance of the three-dimensional model of the CaYLR100w polypeptide. The conserved residues are located in the functional sites that are essential for coenzyme and substrate binding. These active site residues play critical roles in the mechanism of catalysis, substrate specificity, and coenzyme binding.
The structure coordinates of the CaYLR100w homology model, portion thereofs, are preferably stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery and target prioritization and validation.
Accordingly, in one embodiment of this invention is provided a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Table 8.
For the first time, the present invention permits the use, through homology modeling based upon the sequence of CaYLR100w (FIGS. 26 and 27) of structure-based or rational drug design techniques to design, select, and synthesizes chemical entities that are capable of modulating the biological function of CaYLR100w. Comparison of the CaYLR100w homology model with the structures of other SDRs enable the use of rational or structure based drug design methods to design, select or synthesize specific chemical modulators of CaYLR100w.
Accordingly, the present invention is also directed to the entire sequence in FIG. 11, or any portion thereof for the purpose of generating a homology model for the purpose of three dimensional structure-based drug designs.
The present invention also encompasses mutants or homologues of the sequence in FIG. 11, or any portion thereof. In a preferred embodiment, the mutants or homologues have at least 25% identity, more preferably 50% identity, more preferably 75% identity, and most preferably 90% identity to the amino acid residues in FIG. 11 (SEQ ID NO:12).
The three-dimensional model structure of the CaYLR100w will also provide methods for identifying modulators of biological function. Various methods or combination thereof can be used to identify these compounds.
Structure coordinates of the active site region defined above can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential CaYLR100w modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic interactions, and dipole interaction. Alternatively, or in conjunction, the three-dimensional structural model can be employed to design or select compounds as potential CaYLR100w modulators. Compounds identified as potential CaYLR100w modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the CaYLR100w, or in characterizing CaYLR100w deactivation in the presence of a small molecule. Examples of assays useful in screening of potential CaYLR100w modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids from CaYLR100w according to Table 8.
However, as will be understood by those of skill in the art upon this disclosure, other structure based design methods can be used. Various computational structure based design methods have been disclosed in the art.
For example, a number of computer modeling systems are available in which the sequence of the CaYLR100w and the CaYLR100w structure (i.e., atomic coordinates of CaYLR100w and/or the atomic coordinates of the active site region as provided in Table 8) can be input. The computer system then generates the structural details of one or more these regions in which a potential CaYLR100w modulator binds so that complementary structural details of the potential modulators can be determined. Design in these modeling systems is generally based upon the compound being capable of physically and structurally associating with CaYLR100w. In addition, the compound must be able to assume a conformation that allows it to associate with CaYLR100w. Some modeling systems estimate the potential inhibitory or binding effect of a potential CaYLR100w modulator prior to actual synthesis and testing.
Methods for screening chemical entities or fragments for their ability to associate with a given protein target are well known. Often these methods begin by visual inspection of the binding site on the computer screen. Selected fragments or chemical entities are then positioned in one or more positions and orientations within the active site region in CaYLR100w. Molecular docking is accomplished using software such as INSIGHTII, ICM (Molsoft LLC, La Jolla, Calif.), and SYBYL, following by energy minimization and molecular dynamics with standard molecular mechanic forcefields such as CHARMM and MMFF. Examples of computer programs which assist in the selection of chemical fragment or chemical entities useful in the present invention include, but are not limited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntz et. al. 1982).
Alternatively, compounds may be designed de novo using either an empty active site or optionally including some portion of a known inhibitor. Methods of this type of design include, but are not limited to LUDI (Bohm 1992), LeapFrog (Tripos Associates, St. Louis Mo.) and DOCK (Kuntz et. al., 1982). Programs such as DOCK (Kuntz et. al. 1982) can be used with the atomic coordinates from the homology model to identify potential ligands from databases or virtual databases which potentially bind the in the active site region, and which may therefore be suitable candidates for synthesis and testing. The computer programs may utilize a combination of the following steps:
1) Selection of fragments or chemical entities from a database and then positioning the chemical entity in one or more orientations within the CaYLR100w catalytic domain defined by Table 8
2) Characterization of the structural and chemical features of the chemical entity and active site including van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interactions
3) Search databases for molecular fragments which can be joined to or replace the docked chemical entity and spatially fit into regions defined by the said CaYLR100w catalytic domain or catalytic domain active/functional sites
4) Evaluate the docked chemical entity and fragments using a combination of scoring schemes which account for van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic interactions
Databases that may be used include ACD (Molecular Designs Limited), Aldrich (Aldrich Chemical Company), NCI (National Cancer Institute), Maybridge (Maybridge Chemical Company Ltd), CCDC (Cambridge Crystallographic Data Center), CAST (Chemical Abstract Service), Derwent (Derwent Information Limited).
Upon selection of preferred chemical entities or fragments, their relationship to each other and CaYLR100w, can be visualized and then assembled into a single potential modulator. Programs useful in assembling the individual chemical entities include, but are not limited to SYBYL and LeapFrog (Tripos Associates, St. Louis Mo.), LUDI (Bohm 1992) as well as 3D Database systems (Martin 1992).
Additionally, the three-dimensional homology model of CaYLR100w will aid in the design of mutants with altered biological activity. Site directed mutagenesis can be used to generate proteins with similar or varying degrees of biological activity compared to native CaYLR100w. This invention also relates to the generation of mutants or homologs of CaYLR100w. It is clear that molecular modeling using the three dimensional structure coordinates set forth in Table 8 and visualization of the CaYLR100w model, FIG. 27 and alignment in FIG. 26 can be utilized to design homologs or mutant polypeptides of CaYLR100w that have similar or altered biological activities, function or reactivities.
The invention also relates to in silico screening methods including in silico docking and methods of structure based drug design which utilize the three dimensional coordinates of CaYLR100w (Table 8). Also provided are methods of identifying modulators of CaYLR100w that include modulator building or searching utilizing computer programs and algorithms. In an embodiment of the invention a method is provided for designing potential modulators of CaYLR100w comprising any combination of steps which utilize said three dimensional structure to design or select potential modulators.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:1 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1024 of SEQ ID NO:1, b is an integer between 15 to 1038, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:1, and where b is greater than or equal to a+14
Features of the Polypeptide Encoded by Polynucleotide No:2
The polynucleotide sequence (SEQ ID NO:2) and deduced amino acid sequence (SEQ ID NO:13) of the novel fungal essential gene, CaYDR341c (also referred to as FCG6), of the present invention. The CaYDR341c polypeptide (SEQ ID NO:13) is encoded by nucleotides 1 to 1866 of SEQ ID NO:2 and has a predicted molecular weight of 70.8 kDa.
In preferred embodiments, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of CaYDR341c. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 4 thru 1866 of SEQ ID NO:2, and the polypeptide corresponding to amino acids 2 thru 622 of SEQ ID NO:14. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.
As illustrated in FIGS. 1 and 2 and described elsewhere herein, the CaYDR341c polypeptide of the present invention shares 65% identity with S. cerevisiae CaYDR341c. Based upon homology to known proteins, CaYDR341c has been predicted to encode an arginine tRNA synthetase involved in protein synthesis. Experiments described herein demonstrate that downregulation of a genetically manipulated strain having CaYDR341c (FCG6) placed under the control of the CaMET3 promoter with methionine and cysteine affects protein synthesis. As shown in FIG. 25, incorporation of radiolabelled leucine and arginine into charged tRNAs and protein was greatly reduced in the presence of methionine and cysteine after a 3.5 hour induction period as compared with strains in the absence of methionine and cysteine.
Based upon homology to known arginine tRNA synthetases, in conjunction with the biochemical data shown herein, it is clear that CaYDR341c is involved in protein synthesis.
Briefly, the results illustrated in FIG. 25 clearly show a dramatic effect of the 3.5 hour methionine/cysteine downregulation on general protein synthesis. This is demonstrated by the fact that arginine incorporation into polypeptide is almost completely inhibited (98.6% inhibition as seen in FIGS. 25A and 25B) in the presence of methionine and cysteine as compared with that in the absence of methionine and cysteine. Leucine incorporation is also greatly impaired by as much as 96.6% with methionine and cysteine treated cells compared to untreated cells (FIGS. 25A and 25B). This result is expected since a block obtained by insufficient argininyl-tRNA would be expected to halt the further elongation of polypeptide synthesis, including the further incorporation of [3H]-leucine. Therefore, the results provide direct biochemical evidence that CaYDR341c is indeed involved in protein synthesis, and encodes an argininyl-tRNA synthetase.
CaYDR341c polynucleotides and polypeptides, including fragments and modualtors thereof, are useful for the treatment, amelioration, and/or detection of fungal diseases and/or disorders, and are also useful in drug discovery for identifying additional fungal therapeutics.
The invention also encompasses N- and/or C-terminal deletions of the CaYDR341c polypeptide of the present invention.
In preferred embodiments, the following N-terminal CaYDR341c deletion polypeptides are encompassed by the present invention: M1-M622, S2-M622, V3-M622, E4-M622, T5-M622, I6-M622, S7-M622, D8-M622, S9-M622, L10-M622, K11′-M622, Q12-M622, L13-M622, G14-M622, L15-M622, S16-M622, Q17-M622, P18-M622, A19-M622, A20-M622, I21-M622, E22-M622, G23-M622, T24-M622, H25-M622, P26-M622, Q27-M622, Y28-M622, N29-M622, V30-M622, V31-M622, D32-M622, V33-M622, F34-M622, R35-M622, N36-M622, Y37-M622, I38-M622, A39-M622, E40-M622, E41-M622, L42-M622, H43-M622, R44-M622, I45-M622, S46-M622, S47-M622, V48-M622, D49-M622, K50-M622, S51-M622, I52-M622, I53-M622, I54-M622, Q55-M622, A56-M622, L57-M622, D58-M622, T59-M622, P60-M622, K61-M622, V62-M622, L63-M622, D64-M622, Q65-M622, G66-M622, D67-M622, I68-M622, I69-M622, V70-M622, P71-M622, I72-M622, P73-M622, K74-M622, L75-M622, R76-M622, L77-M622, K78-M622, G79-M622, I80-M622, N81-M622, P82-M622, N83-M622, E84-M622, K85-M622, S86-M622, K87-M622, E88-M622, W89-M622, A90-M622, E91-M622, N92-M622, F93-M622, N94-M622, K95-M622, G96-M622, K97-M622, F98-M622, I99-M622, S100-M622, E101-M622, I102-M622, K103-M622, P104-M622, Q105-M622, G106-M622, V107-M622, F108-M622, L109-M622, Q 110-M622, F111-M622, Y112-M622, F113-M622, A114-M622, K115-M622, T116-M622, L117-M622, L118-M622, Y119-M622, N120-M622, L121-M622, V122-M622, I123-M622, E124-M622, D125-M622, V126-M622, L127-M622, K128-M622, R129-M622, K130-M622, S131-M622, D132-M622, Y133-M622, G134-M622, Y135-M622, L136-M622, P137-M622, L138-M622, G139-M622, V140-M622, G141-M622, K142-M622, K143-M622, A144-M622, I145-M622, V146-M622, E147-M622, F148-M622, S149-M622, S150-M622, P151-M622, N152-M622, I153-M622, A154-M622, K155-M622, P156-M622, F157-M622, H158-M622, A159-M622, G160-M622, H161-M622, L162-M622, R163-M622, S164-M622, T165-M622, I166-M622, I167-M622, G168-M622, G169-M622, F170-M622, I171-M622, S172-M622, N173-M622, L174-M622, Y175-M622, E176-M622, K177-M622, V178-M622, G179-M622, W180-M622, D181-M622, V182-M622, T183-M622, R184-M622, I185-M622, N186-M622, Y187-M622, L188-M622, G189-M622, D190-M622, W191-M622, G192-M622, K193-M622, Q194-M622, F195-M622, G196-M622, L197-M622, L198-M622, A199-M622, V200-M622, G201-M622, F202-M622, E203-M622, R204-M622, Y205-M622, G206-M622, D207-M622, E208-M622, S209-M622, K210-M622, L211-M622, A212-M622, S213-M622, D214-M622, P215-M622, I216-M622, N217-M622, H218-M622, L219-M622, F220-M622, E221-M622, V222-M622, Y223-M622, V224-M622, K225-M622, I226-M622, N227-M622, Q228-M622, D229-M622, V230-M622, T231-M622, K232-M622, E233-M622, T234-M622, S235-M622, E236-M622, A237-M622, T238-M622, G239-M622, E240-M622, T241-M622, P242-M622, A243-M622, E244-M622, T245-M622, I246-M622, D247-M622, A248-M622, S249-M622, E250-M622, Q251-M622, D252-M622, E253-M622, K254-M622, K255-M622, I256-M622, Q257-M622, S258-M622, S259-M622, T260-M622, N261-M622, E262-M622, E263-M622, A264-M622, R265-M622, R266-M622, F267-M622, F268-M622, R269-M622, R270-M622, M271-M622, E272-M622, D273-M622, G274-M622, D275-M622, E276-M622, S277-M622, A278-M622, L279-M622, K280-M622, I281-M622, W282-M622, A283-M622, R284-M622, F285-M622, R286-M622, D287-M622, L288-M622, S289-M622, I290-M622, E291-M622, K292-M622, Y293-M622, V294-M622, D295-M622, T296-M622, Y297-M622, G298-M622, R299-M622, L300-M622, N301-M622, I302-M622, K303-M622, Y304-M622, D305-M622, V306-M622, Y307-M622, S308-M622, G309-M622, E310-M622, S311-M622, Q312-M622, V313-M622, P314-M622, Q315-M622, E316-M622, K317-M622, M318-M622, K319-M622, E320-M622, A321-M622, T322-M622, K323-M622, L324-M622, F325-M622, E326-M622, D327-M622, K328-M622, G329-M622, L330-M622, I331-M622, D332-M622, I333-M622, D334-M622, R335-M622, G336-M622, A337-M622, K338-M622, L339-M622, I340-M622, D341-M622, L342-M622, T343-M622, K344-M622, F345-M622, N346-M622, K347-M622, K348-M622, L349-M622, G350-M622, K351-M622, A352-M622, L353-M622, V354-M622, E355-M622, K356-M622, S357-M622, D358-M622, G359-M622, T360-M622, S361-M622, L362-M622, Y363-M622, L364-M622, T365-M622, R366-M622, D367-M622, V368-M622, G369-M622, E370-M622, A371-M622, I372-M622, K373-M622, R374-M622, Y375-M622, E376-M622, T377-M622, Y378-M622, K379-M622, F380-M622, D381-M622, K382-M622, M383-M622, I384-M622, Y385-M622, V386-M622, I387-M622, A388-M622, A389-M622, Q390-M622, Q391-M622, D392-M622, L393-M622, H394-M622, C395-M622, A396-M622, Q397-M622, F398-M622, F399-M622, E400-M622, I401-M622, L402-M622, K403-M622, Q404-M622, M405-M622, G406-M622, F407-M622, E408-M622, W409-M622, A410-M622, H411-M622, N412-M622, L413-M622, E414-M622, H415-M622, V416-M622, N417-M622, F418-M622, G419-M622, M420-M622, V421-M622, Q422-M622, G423-M622, M424-M622, S425-M622, T426-M622, R427-M622, K428-M622, G429-M622, T430-M622, V431-M622, V432-M622, F433-M622, L434-M622, D435-M622, N436-M622, I437-M622, L438-M622, Q439-M622, E440-M622, T441-M622, K442-M622, E443-M622, K444-M622, M445-M622, H446-M622, E447-M622, V448-M622, M449-M622, Q450-M622, K451-M622, N452-M622, E453-M622, E454-M622, K455-M622, Y456-M622, A457-M622, Q458-M622, I459-M622, E460-M622, D461-M622, P462-M622, D463-M622, K464-M622, I465-M622, A466-M622, D467-M622, L468-M622, I469-M622, G470-M622, I471-M622, S472-M622, A473-M622, V474-M622, M475-M622, I476-M622, Q477-M622, D478-M622, M479-M622, Q480-M622, S481-M622, K482-M622, R483-M622, I484-M622, H485-M622, N486-M622, Y487-M622, E488-M622, F489-M622, K490-M622, W491-M622, D492-M622, R493-M622, M494-M622, T495-M622, S496-M622, F497-M622, E498-M622, G499-M622, D500-M622, T501-M622, G502-M622, P503-M622, Y504-M622, L505-M622, Q506-M622, Y507-M622, A508-M622, H509-M622, S510-M622, R511-M622, L512-M622, C513-M622, S514-M622, M515-M622, Q516-M622, R517-M622, K518-M622, S519-M622, G520-M622, I521-M622, S522-M622, I523-M622, E524-M622, E525-M622, L526-M622, E527-M622, H528-M622, A529-M622, N530-M622, F531-M622, D532-M622, L533-M622, L534-M622, V535-M622, E536-M622, P537-M622, C538-M622, A539-M622, S540-M622, A541-M622, L542-M622, A543-M622, R544-M622, T545-M622, L546-M622, A547-M622, Q548-M622, Y549-M622, P550-M622, D551-M622, V552-M622, I553-M622, K554-M622, K555-M622, A556-M622, V557-M622, K558-M622, G559-M622, L560-M622, E561-M622, P562-M622, S563-M622, T564-M622, I565-M622, V566-M622, T567-M622, Y568-M622, L569-M622, F570-M622, S571-M622, V572-M622, T573-M622, H574-M622, I575-M622, V576-M622, S577-M622, Q578-M622, C579-M622, Y580-M622, D581-M622, I582-M622, L583-M622, W584-M622, V585-M622, S586-M622, G587-M622, Q588-M622, E589-M622, K590-M622, D591-M622, V592-M622, A593-M622, I594-M622, A595-M622, R596-M622, L597-M622, A598-M622, L599-M622, Y600-M622, E601-M622, A602-M622, A603-M622, R604-M622, Q605-M622, V606-M622, I607-M622, N608-M622, N609-M622, G610-M622, M611-M622, T612-M622, L613-M622, L614-M622, G615-M622, and/or L616-M622 of SEQ ID NO:13. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CaYDR341c deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CaYDR341c deletion polypeptides are encompassed by the present invention: M1-M622, M1-R621, M1-N620, M1-V619, M1-P618, M1-T617, M1-L616, M1-G615, M1-L614, M1-L613, M1-T612, M1-M611, M1-G610, M1-N609, M1-N608, M1-I607, M1-V606, M1-Q605, M1-R604, M1-A603, M1-A602, M1-E601, M1-Y600, M1-L599, M1-A598, M1-L597, M1-R596, M1-A595, M1-I594, M1-A593, M1-V592, M1-D591, M1-K590, M1-E589, M1-Q588, M1-G587, M1-S586, M1-V585, M1-W584, M1-L583, M1-I582, M1-D581, M1-Y580, M1-C579, M1-Q578, M1-S577, M1-V576, M1-I575, M1-H574, M1-T573, M1-V572, M1-S571, M1-F570, M1-L569, M1-Y568, M1-T567, M1-V566, M1-I565, M1-T564, M1-S563, M1-P562, M1-E561, M1-L560, M1-G559, M1-K558, M1-V557, M1-A556, M1-K555, M1-K554, M1-I553, M1-V552, M1-D551, M1-P550, M1-Y549, M1-Q548, M1-A547, M1-L546, M1-T545, M1-R544, M1-A543, M1-L542, M1-A541, M1-S540, M1-A539, M1-C538, M1-P537, M1-E536, M1-V535, M1-L534, M1-L533, M1-D532, M1-F531, M1-N530, M1-A529, M1-H528, M1-E527, M1-L526, M1-E525, M1-E524, M1-I523, M1-S522, M1-I521, M1-G520, M1-S519, M1-K518, M1-R517, M1-Q516, M1-M515, M1-S514, M1-C513, M1-L512, M1-R511, M1-S510, M1-H509, M1-A508, M1-Y507, M1-Q506, M1-L505, M1-Y504, M1-P503, M1-G502, M1-T501, M1-D500, M1-G499, M1-E498, M1-F497, M1-S496, M1-T495, M1-M494, M1-R493, M1-D492, M1-W491, M1-K490, M1-F489, M1-E488, M1-Y487, M1-N486, M1-H485, M1-I484, M1-R483, M1-K482, M1-S481, M1-Q480, M1-M479, M1-D478, M1-Q477, M1-I476, M1-M475, M1-V474, M1-A473, M1-S472, M1-I471, M1-G470, M1-I469, M1-L468, M1-D467, M1-A466, M1-I465, M1-K464, M1-D463, M1-P462, M1-D461, M1-E460, M1-I459, M1-Q458, M1-A457, M1-Y456, M1-K455, M1-E454, M1-E453, M1-N452, M1-K451, M1-Q450, M1-M449, M1-V448, M1-E447, M1-H446, M1-M445, M1-K444, M1-E443, M1-K442, M1-T441, M1-E440, M1-Q439, M1-L438, M1-I437, M1-N436, M1-D435, M1-L434, M1-F433, M1-V432, M1-V431, M1-T430, M1-G429, M1-K428, M1-R427, M1-T426, M1-S425, M1-M424, M1-G423, M1-Q422, M1-V421, M1-M420, M1-G419, M1-F418, M1-N417, M1-V416, M1-H415, M1-E414, M1-L413, M1-N412, M1-H411, M1-A410, M1-W409, M1-E408, M1-F407, M1-G406, M1-M405, M1-Q404, M1-K403, M1-L402, M1-I401, M1-E400, M1-F399, M1-F398, M1-Q397, M1-A396, M1-C395, M1-H394, M1-L393, M1-D392, M1-Q391, M1-Q390, M1-A389, M1-A388, M1-I387, M1-V386, M1-Y385, M1-I384, M1-M383, M1-K382, M1-D381, M1-F380, M1-K379, M1-Y378, M1-T377, M1-E376, M1-Y375, M1-R374, M1-K373, M1-I372, M1-A371, M1-E370, M1-G369, M1-V368, M1-D367, M1-R366, M1-T365, M1-L364, M1-Y363, M1-L362, M1-S361, M1-T360, M1-G359, M1-D358, M1-S357, M1-K356, M1-E355, M1-V354, M1-L353, M1-A352, M1-K351, M1-G350, M1-L349, M1-K348, M1-K347, M1-N346, M1-F345, M1-K344, M1-T343, M1-L342, M1-D341, M1-I340, M1-L339, M1-K338, M1-A337, M1-G336, M1-R335, M1-D334, M1-I333, M1-D332, M1-I331, M1-L330, M1-G329, M1-K328, M1-D327, M1-E326, M1-F325, M1-L324, M1-K323, M1-T322, M1-A321, M1-E320, M1-K319, M1-M318, M1-K317, M1-E316, M1-Q315, M1-P314, M1-V313, M1-Q312, M1-S311, M1-E310, M1-G309, M1-S308, M1-Y307, M1-V306, M1-D305, M1-Y304, M1-K303, M1-I302, M1-N301, M1-L300, M1-R299, M1-G298, M1-Y297, M1-T296, M1-D295, M1-V294, M1-Y293, M1-K292, M1-E291, M1-I290, M1-S289, M1-L288, M1-D287, M1-R286, M1-F285, M1-R284, M1-A283, M1-W282, M1-I281, M1-K280, M1-L279, M1-A278, M1-S277, M1-E276, M1-D275, M1-G274, M1-D273, M1-E272, M1-M271, M1-R270, M1-R269, M1-F268, M1-F267, M1-R266, M1-R265, M1-A264, M1-E263, M1-E262, M1-N261, M1-T260, M1-S259, M1-S258, M1-Q257, M1-I256, M1-K255, M1-K254, M1-E253, M1-D252, M1-Q251, M1-E250, M1-S249, M1-A248, M1-D247, M1-I246, M1-T245, M1-E244, M1-A243, M1-P242, M1-T241, M1-E240, M1-G239, M1-T238, M1-A237, M1-E236, M1-S235, M1-T234, M1-E233, M1-K232, M1-T231, M1-V230, M1-D229, M1-Q228, M1-N227, M1-I226, M1-K225, M1-V224, M1-Y223, M1-V222, M1-E221, M1-F220, M1-L219, M1-H218, M1-N217, M1-I216, M1-P215, M1-D214, M1-S213, M1-A212, M1-L211, M1-K210, M1-S209, M1-E208, M1-D207, M1-G206, M1-Y205, M1-R204, M1-E203, M1-F202, M1-G201, M1-V200, M1-A199, M1-L198, M1-L197, M1-G196, M1-F195, M1-Q194, M1-K193, M1-G192, M1-W191, M1-D190, M1-G189, M1-L188, M1-Y187, M1-N186, M1-I185, M1-R184, M1-T183, M1-V182, M1-D181, M1-W180, M1-G179, M1-V178, M1-K177, M1-E176, M1-Y175, M1-L174, M1-N173, M1-S172, M1-I171, M1-F170, M1-G169, M1-G168, M1-I167, M1-I166, M1-T165, M1-S164, M1-R163, M1-L162, M1-H161, M1-G160, M1-A159, M1-H158, M1-F157, M1-P156, M1-K155, M1-A154, M1-I153, M1-N152, M1-P151, M1-S150, M1-S149, M1-F148, M1-E147, M1-V146, M1-I145, M1-A144, M1-K143, M1-K142, M1-G141, M1-V140, M1-G139, M1-L138, M1-P137, M1-L136, M1-Y135, M1-G134, M1-Y133, M1-D132, M1-S131, M1-K130, M1-R129, M1-K128, M1-L127, M1-V126, M1-D125, M1-E124, M1-I123, M1-V122, M1-L121, M1-N120, M1-Y119, M1-L118, M1-L117, M1-T116, M1-K115, M1-A114, M1-F113, M1-Y112, M1-F111, M1-Q110, M1-L109, M1-F108, M1-V107, M1-G106, M1-Q105, M1-P104, M1-K103, M1-I102, M1-E101, M1-S100, M1-I99, M1-F98, M1-K97, M1-G96, M1-K95, M1-N94, M1-F93, M1-N92, M1-E91, M1-A90, M1-W89, M1-E88, M1-K87, M1-S86, M1-K85, M1-E84, M1-N83, M1-P82, M1-N81, M1-I80, M1-G79, M1-K78, M1-L77, M1-R76, M1-L75, M1-K74, M1-P73, M1-I72, M1-P71, M1-V70, M1-I69, M1-I68, M1-D67, M1-G66, M1-Q65, M1-D64, M1-L63, M1-V62, M1-K61, M1-P60, M1-T59, M1-D58, M1-L57, M1-A56, M1-Q55, M1-I54, M1-I53, M1-I52, M1-S51, M1-K50, M1-D49, M1-V48, M1-S47, M1-S46, M1-I45, M1-R44, M1-H43, M1-L42, M1-E41, M1-E40, M1-A39, M1-I38, M1-Y37, M1-N36, M1-R35, M1-F34, M1-V33, M1-D32, M1-V31, M1-V30, M1-N29, M1-Y28, M1-Q27, M1-P26, M1-H25, M1-T24, M1-G23, M1-E22, M1-I21, M1-A20, M1-A19, M1-P18, M1-Q17, M1-S16, M1-L15, M1-G14, M1-L13, M1-Q12, M1-K11, M1-L10, M1-S9, M1-D8, and/or M1-S7 of SEQ ID NO:13. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CaYDR341c deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses polynucleotides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polynucleotide sequence provided in SEQ ID NO:2, and in particular to the coding region of the polynucleotide sequence provided in SEQ ID NO:2. Preferably such polynucleotides encode polypeptides that have biological activity, particularly arginyl-tRNA synthetase activity.
Most preferred are polynucleotides that share at least about 96.8% identity with the polynucleotide sequence provided in SEQ ID NO:2.
The present invention also encompasses polypeptides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polypeptide sequence provided in SEQ ID NO:12.
Most preferred are polypeptides that share at least about 96.8% identity with the polypeptide sequence provided in SEQ ID NO:12.
The present invention is also directed to a homology model detailing the three-dimensional structure of the CaYDR341c polypeptide (SEQ ID NO:13) of the present invention.
Protein threading and molecular modeling of CaYDR341c suggest that CaYDR341c has a three dimensional fold similar to that of the arginyl-tRNA synthetase (EC number 6.1.1.-) from yeast (Delagoutte et. al., 2000), Protein Data Bank (PDB, Bernstein et. al., 1977 & Berman et. al., 2000) (Protein Data Bank entry 1F7U; Genbank Accession No. gi|14719542; SEQ ID NO:252). Based on sequence, structure, motifs and known RNA binding signature sequences, CaYDR341c contains a novel RNA synthetase class 1 domain.
The polypeptide CaYDR341c contains several distinct structural domains including the arginyl-tRNA synthetase catalytic domain which contains the active site.
The three dimensional crystallographic structure for several arginyl-tRNA synthetases have been reported and are deposited into the Protein Data Bank (Delagoutte et. al., 2000, Bernstein et. al., 1977, Berman et. al., 2000). The structure (Protein Data Bank, PDB entry 1F7u) of the arginyl-tRNA synthetase from yeast (Saccharomyces cerevisiae) is similar to the other aminoacyl-tRNA synthetases (EC 6.1.1.-). They constitute a family of RNA-binding proteins that are responsible for the correct translation of the genetic code by linking the 3′ end of the correct tRNA. In most organisms there are 20 aminoacyl-tRNA synthetases, each one responsible for its cognate tRNA(s). The structure of the yeast (Saccharomyces cerevisiae) arginyl-tRNA synthetase has the catalytic domain at the core to which four structurally defined domains are appended. Domains attached to the N-terminus and C-terminus are defined as Add1 and Add2, respectively. The other two domains (Ins1 and Ins2) are inserted into regions of the catalytic core.
The arginyl-tRNA synthetase enzyme is a ternery complex with tRNAarg and forms an extensive interface with a large burried surface area described in detail by Delagoutte et. al., 2000. The interactions between the enzyme and RNA include (I) exposed aromatic and alaphatic interactions that are involved in van der Waals and hydrophobic intereactions; (II) positively charged residues that interact with the sugar phosphate backbone; (III) polar side chains for hydrogen bonds directly with nucleic acids or water-mediated hydrogen bonds. The recognition surface is marked by a series of amino acids that are conserved in all known arginyl-tRNA synthetases (e.g. Y491, A495, R501, Y565, and M607). In addition a structural motif known as the Ω loop, which contains residues 480-485, forms a critical protruding loop that has a dual functional role by forming part of a binding pocket and stabilizing the tRNA conformation by correct positioning of the RNA anticodon. A second structural feature that several aminoacyl-tRNA synthetases share is located in the N-terminal region and is a tetrapeptide sequence ‘HIGH’ (SEQ ID NO:255). The ‘HIGH’ (SEQ ID NO:255) region has been shown (Brick et al. 1988) to be part of the adenylate binding site. The ‘HIGH’ (SEQ ID NO:255) signature has been found in the aminoacyl-tRNA synthetases specific for arginine, cysteine, glutamic acid, glutamine, isoleucine, leucine, methionine, tyrosine, tryptophan, and valine. These aminoacyl-tRNA synthetases are referred to as class-I synthetases (Delarue and Moras, 1993; Schimmel, 1991) and seem to share the same core domain tertiary structure based on a Rossmann fold.
This structure-based information and sequence information from novel genes can be used to identify other protein family members that share this same fold.
The three dimensional model of the CaYDR341c polypeptide provides for a specific description of the catalytic core and functional sites in the arginyl-tRNA synthetase, CaYDR341c polypeptide.
The catalytic core and functional sites are defined by atomic coordinates (Table 9). Based on this data, the inventors have ascribed the CaYDR341c polypeptide as having arginyl-tRNA synthetase activity(s) and cellular and systemic regulatory function(s).
The invention also relates to in silico screening methods including in silico docking and methods of structure based drug design which utilize the three dimensional coordinates of CaYDR341c (Table 9). Also provided are methods of identifying modulators of CaYDR341c that include modulator building or searching utilizing computer programs and algorithms. In an embodiment of the invention a method is provided for designing potential modulators of CaYDR341c comprising any combination of steps which utilize said three dimensional structure to design or select potential modulators.
Homology models are useful when there is no experimental information available on the protein of interest. A three dimensional model can be constructed on the basis of the known structure of a homologous protein (Greer et. al., 1991, Lesk, et. al., 1992, Levitt, 1992, Cardozo, et. al., 1995, Sali, et. al., 1995).
Those of skill in the art will understand that a homology model is constructed on the basis of first identifying a template, or, protein of known structure which is similar to the protein without known structure. This can be accomplished by pairwise alignment of sequences using such programs as FASTA (Pearson, et. al. 1990) and BLAST (Altschul, et. al., 1990). In cases where sequence similarity is high (greater than 30%), these pairwise comparison methods may be adequate. Likewise, multiple sequence alignments or profile-based methods can be used to align a query sequence to an alignment of multiple (structurally and biochemically) related proteins. When the sequence similarity is low, more advanced techniques are used such as fold recognition (protein threading; Hendlich, et. al., 1990, Koppensteiner et. al. 2000, Sippl & Weitckus 1992, Sippl 1993), where the compatibility of a particular sequence with the three dimensional fold of a potential template protein is gauged on the basis of a knowledge-based potential. Following the initial sequence alignment, the query template can be optimally aligned by manual manipulation or by incorporation of other features (motifs, secondary structure predictions, and allowed sequence conservation). Next, structurally conserved regions can be identified and are used to construct the core secondary structure (Levitt, 1992, Sali, et. al., 1995) elements in the three dimensional model. Variable regions, called “unconserved regions” and loops can be added using knowledge-based techniques. The complete model with variable regions and loops can be refined performing forcefield calculations (Sali, et. al., 1995, Cardozo, et. al., 1995).
For CaYDR341c a pairwise alignment generated by FASTA was used to align the sequence of CaYDR341c with the sequence the arginyl-tRNA synthetase, EC number 6.1.1.- from yeast, Saccharomyces cerevisiae (Delagoutte et. al., 2000), (Protein Data Bank code 1F7U). The alignment of CaYDR341c with PDB entry 1F7U chain A is set forth in FIG. 29. In this invention, the homology model of CaYDR341c was derived from the sequence alignment set forth in FIG. 29. An overall atomic model including plausible sidechain orientations was generated using the program LOOK (Levitt, 1992). The three dimensional model for CaYDR341c is defined by the set of structure coordinates as set forth in Table 9 and is shown in FIG. 31 rendered by backbone secondary structures.
In order to recognize errors in three-dimensional structures, knowledge based mean fields can be used to judge the quality of protein folds (Sippl 1993). The methods can be used to recognize misfolded structures as well as faulty parts of structural models. The technique generates an energy graph where the energy distribution for a given protein fold is displayed on the y-axis and residue position in the protein fold is displayed on the x-axis. The knowledge based mean fields compose a force field derived from a set of globular protein structures taken as a subset from the Protein Data Bank (Bernstein et. al. 1977). To analyze the quality of a model, the energy distribution is plotted and compared to the energy distribution of the template from which the model was generated. FIG. 31 shows the energy graph for the CaYDR341c model (dotted line) and the template (arginyl-tRNA synthetase) from which the model was generated. The model has virtually an identical energy plot when compared to arginyl-tRNA synthetase template demonstrating that CaYDR341c has similar structural characteristics and suggest the overall three-dimensional fold is “native-like”. This graph supports the motif and sequence alignments in confirming that the three dimensional structure coordinates of CaYDR341c are an accurate and useful representation for the polypeptide.
The term “structure coordinates” refers to Cartesian coordinates generated from the building of a homology model.
Those of skill in the art will understand that a set of structure coordinates for a protein is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates, as emanate from generation of similar homology models using different alignment templates (i.e., other than the structure coordinates of 1F7u), and/or using different methods in generating the homology model, will have minor effects on the overall shape. Variations in coordinates may also be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in Table 9 could be manipulated by fractionalization of the structure coordinates; integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
Various computational analyses are therefore necessary to determine whether a molecule, or a portion thereof, is sufficiently similar to all or parts of CaYDR341c described above as to be considered the same. Such analyses may be carried out in current software applications, such as INSIGHTII (Accelrys Inc., San Diego, Calif.) version 2000 as described in the User's Guide, online or software applications available in the SYBYL software suite (Tripos Inc., St. Louis, Mo.).
Using the superimposition tool in the program INSIGHTII comparisons can be made between different structures and different conformations of the same structure. The procedure used in INSIGHTII to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); the second structure (i.e., moving structure) is identified as the source structure. Since atom equivalency within INSIGHTII is defined by user input, for the purpose of this invention, equivalent atoms are defined as protein backbone atoms (N, Cα, C and O) for all conserved residues between the two structures being compared. Also, only rigid fitting operations are considered. When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by INSIGHTII.
For the purpose of this invention, any homology model of a CaYDR341c that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) of less than about 2.0 A when superimposed on the relevant backbone atoms described by structure coordinates listed in Table 9 are considered identical. More preferably, the root mean square deviation is less than about 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 Angstroms.
The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the “root mean square deviation” defines the variation in the backbone of a protein from the relevant portion of the backbone of CaYDR341c as defined by the structure coordinates described herein.
This invention as embodied by the three-dimensional model enables the structure-based design of modulators of the biological function of CaYDR341c, as well as mutants with altered biological function and/or specificity.
The sequence alignment (FIG. 29) used as a template for creating the three-dimensional model of CaYDR341c arginyl-tRNA synthetase domain shows 67% sequence identity between catalytic domain of CaYDR341c and yeast arginyl-tRNA synthetase, PDB code 1F7U. For the arginyl-tRNA synthetases there are at least two functional regions that are critical. In the N-terminal region of the enzyme the adenylate binding site has been shown to be highly conserved. The motifs and structure of this region are similar for class 1 synthetases aminoacyl-tRNA synthetases specific for arginine, cysteine, glutamic acid, glutamine, isoleucine, leucine, methionine, tyrosine, tryptophan, and valine (Brick et al. 1988). FIG. 29 shows this region highlighted by (*) and corresponds to H158-H161 in the three dimensional model for CaYDR341c (Table 9). The adenylate binding site, including the canonical “HIGH” (SEQ ID NO:255) motif, and surrounding sequence is completely conserved at the sequence and structure level. The Ω loop in aminoacyl-tRNA synthetases forms a critical protruding structure that functions by forming part of a binding pocket for the tRNA molecule and stabilizing the tRNA conformation by correct positioning of the tRNA anticodon. FIG. 29 shows that the Ω loop in CaYDR341c is completely conserved (denoted by “+”) both in sequence and structurally.
The conservation of the amino acids in both of these functional sites, and the overall 67% sequence identity, emphasizes the significance of the CaYDR341c three-dimensional model. The conserved residues are located in the functional sites at the tRNA interface presenting a well structured catalytic domain. These functional site residues play critical roles in the mechanism of catalysis, substrate specificity and tRNA binding.
The structure coordinates of a CaYDR341c homology model, and portions thereof, are stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery and target prioritization and validation.
Accordingly, in one embodiment of this invention is provided a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Table 9.
For the first time, the present invention permits the use, through homology modeling based upon the sequence of CaYDR341c (FIG. 12) of structure-based or rational drug design techniques to design, select, and synthesizes chemical entities that are capable of modulating the biological function of CaYDR341c. Comparison of the CaYDR341c homology model with the structures of other the arginyl-tRNA synthetases enable the use of rational or structure based drug design methods to design, select or synthesize specific chemical modulators of CaYDR341c.
Accordingly, the present invention is also directed to the entire sequence in FIG. 12, or any portion thereof, for the purpose of generating a homology model for the purpose of three dimensional structure-based drug designs.
The present invention also encompasses mutants or homologues of the sequence in FIG. 12, or any portion thereof. In a preferred embodiment, the mutants or homologues have at least 25% identity, more preferably 50% identity, more preferably 75% identity, and most preferably 90% identity to the amino acid residues in FIG. 12.
The three-dimensional model structure of the CaYDR341c will also provide methods for identifying modulators of biological function. Various methods or combination thereof can be used to identify these compounds.
Structure coordinates of the active site region defined above can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential CaYDR341c modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic interactions, and dipole interaction. Alternatively, or in conjunction, the three-dimensional structural model can be employed to design or select compounds as potential CaYDR341c modulators. Compounds identified as potential CaYDR341c modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the CaYDR341c, or in characterizing CaYDR341c deactivation in the presence of a small molecule. Examples of assays useful in screening of potential CaYDR341c modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids from CaYDR341c according to Table 9.
However, as will be understood by those of skill in the art upon this disclosure, other structure based design methods can be used. Various computational structure based design methods have been disclosed in the art.
For example, a number of computer modeling systems are available in which the sequence of the CaYDR341c and the CaYDR341c structure (i.e., atomic coordinates of CaYDR341c and/or the atomic coordinates of the active site region as provided in Table 9) can be input. The computer system then generates the structural details of one or more these regions in which a potential CaYDR341c modulator binds so that complementary structural details of the potential modulators can be determined. Design in these modeling systems is generally based upon the compound being capable of physically and structurally associating with CaYDR341c. In addition, the compound must be able to assume a conformation that allows it to associate with CaYDR341c. Some modeling systems estimate the potential inhibitory or binding effect of a potential CaYDR341c modulator prior to actual synthesis and testing.
Methods for screening chemical entities or fragments for their ability to associate with a given protein target are well known. Often these methods begin by visual inspection of the binding site on the computer screen. Selected fragments or chemical entities are then positioned in one or more positions and orientations within the active site region in CaYDR341c. Molecular docking is accomplished using software such as INSIGHTII, ICM (Molsoft LLC, La Jolla, Calif.), and SYBYL, following by energy minimization and molecular dynamics with standard molecular mechanic forcefields such as CHARMM and MMFF. Examples of computer programs which assist in the selection of chemical fragment or chemical entities useful in the present invention include, but are not limited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntz et. al. 1982).
Alternatively, compounds may be designed de novo using either an empty active site or optionally including some portion of a known inhibitor. Methods of this type of design include, but are not limited to LUDI (Bohm 1992), LeapFrog (Tripos Associates, St. Louis Mo.) and DOCK (Kuntz et. al., 1982). Programs such as DOCK (Kuntz et. al. 1982) can be used with the atomic coordinates from the homology model to identify potential ligands from databases or virtual databases which potentially bind the in the active site region, and which may therefore be suitable candidates for synthesis and testing. The computer programs may utilize a combination of the following steps:
1) Selection of fragments or chemical entities from a database and then positioning the chemical entity in one or more orientations within the CaYDR341c catalytic domain defined by Table 9
2) Characterization of the structural and chemical features of the chemical entity and active site including van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interactions
3) Search databases for molecular fragments which can be joined to or replace the docked chemical entity and spatially fit into regions defined by the said CaYDR341c catalytic domain or catalytic domain functional sites
4) Evaluate the docked chemical entity and fragments using a combination of scoring schemes which account for van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic interactions
Databases that may be used include ACD (Molecular Designs Limited), Aldrich (Aldrich Chemical Company), NCI (National Cancer Institute), Maybridge (Maybridge Chemical Company Ltd), CCDC (Cambridge Crystallographic Data Center), CAST (Chemical Abstract Service), Derwent (Derwent Information Limited).
Upon selection of preferred chemical entities or fragments, their relationship to each other and CaYDR341c can be visualized and then assembled into a single potential modulator. Programs useful in assembling the individual chemical entities include, but are not limited to SYBYL and LeapFrog (Tripos Associates, St. Louis Mo.), LUDI (Bohm 1992) as well as 3D Database systems (Martin 1992).
Additionally, the three-dimensional homology model of CaYDR341c will aid in the design of mutants with altered biological activity. Site directed mutagenesis can be used to generate proteins with similar or varying degrees of biological activity compared to native CaYDR341c. This invention also relates to the generation of mutants or homologs of CaYDR341c. It is clear that molecular modeling using the three dimensional structure coordinates set forth in Table 9 and visualization of the CaYDR341c model, FIG. 31 can be utilized to design homologs or mutant polypeptides of CaYDR341c that have similar or altered biological activities, function or reactivities.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:2 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1852 of SEQ ID NO:2, b is an integer between 15 to 1866, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:2, and where b is greater than or equal to a+14.
Features of the Polypeptide Encoded by Polynucleotide No:3
The polynucleotide sequence (SEQ ID NO:3) and deduced amino acid sequence (SEQ ID NO:14) of the novel fungal essential gene, CaYLR022c (also referred to as FCG7), of the present invention. The CaYLR022c polypeptide (SEQ ID NO:14) is encoded by nucleotides 1 to 765 of SEQ ID NO:3 and has a predicted molecular weight of 29.2 kDa.
In preferred embodiments, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of CaYLR022c. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 4 thru 765 of SEQ ID NO:3, and the polypeptide corresponding to amino acids 2 thru 255 of SEQ ID NO:15. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.
The invention also encompasses N- and/or C-terminal deletions of the CaYLR022c polypeptide of the present invention.
In preferred embodiments, the following N-terminal CaYLR022c deletion polypeptides are encompassed by the present invention: M1-E255, A2-E255, V3-E255, I4-E255, N5-E255, Q6-E255, P7-E255, N8-E255, S9-E255, Q10-E255, I11-E255, R12-E255, L13-E255, T14-E255, N15-E255, V16-E255, S17-E255, L18-E255, V19-E255, R20-E255, M21-E255, K22-E255, K23-E255, G24-E255, K25-E255, K26-E255, R27-E255, F28-E255, E29-E255, I30-E255, A31-E255, C32-E255, Y33-E255, Q34-E255, N35-E255, K36-E255, V37-E255, Q38-E255, D39-E255, W40-E255, R41-E255, L42-E255, K43-E255, V44-E255, E45-E255, K46-E255, D47-E255, I48-E255, D49-E255, E50-E255, V51-E255, L52-E255, Q53-E255, I54-E255, P55-E255, Q56-E255, V57-E255, F58-E255, I59-E255, N60-E255, V61-E255, S62-E255, K63-E255, G64-E255, Q65-E255, V66-E255, A67-E255, N68-E255, N69-E255, D70-E255, D71-E255, L72-E255, Q73-E255, K74-E255, C75-E255, F76-E255, G77-E255, T78-E255, T79-E255, N80-E255, Q81-E255, D82-E255, E83-E255, I84-E255, I85-E255, A86-E255, E87-E255, I88-E255, L89-E255, N90-E255, K91-E255, G92-E255, E93-E255, I94-E255, Q95-E255, L96-E255, N97-E255, E98-E255, K99-E255, E100-E255, R101-E255, N102-E255, A103-E255, N104-E255, L105-E255, Q106-E255, Q107-E255, K108-E255, Q109-E255, N110-E255, E111-E255, F112-E255, L113-E255, N114-E255, I115-E255, I116-E255, S117-E255, T118-E255, K119-E255, C120-E255, I121-E255, N122-E255, P123-E255, R124-E255, S125-E255, K126-E255, K127-E255, R128-E255, Y129-E255, P130-E255, P131-E255, S132-E255, M133-E255, I134-E255, E135-E255, K136-E255, V137-E255, L138-E255, N139-E255, E140-E255, V141-E255, K142-E255, F143-E255, H144-E255, L145-E255, N146-E255, P147-E255, T148-E255, K149-E255, P150-E255, T151-E255, K152-E255, I153-E255, Q154-E255, A155-E255, L156-E255, D157-E255, A158-E255, I159-E255, K160-E255, L161-E255, L162-E255, V163-E255, E164-E255, K165-E255, Q166-E255, I167-E255, I168-E255, P169-E255, I170-E255, A171-E255, R172-E255, A173-E255, Q174-E255, M175-E255, K176-E255, V177-E255, R178-E255, I179-E255, T180-E255, L181-E255, S182-E255, K183-E255, K184-E255, A185-E255, Y186-E255, L187-E255, K188-E255, T189-E255, F190-E255, Q191-E255, D192-E255, E193-E255, I194-E255, K195-E255, P196-E255, V197-E255, I198-E255, D199-E255, Q200-E255, I201-E255, V202-E255, E203-E255, E204-E255, D205-E255, N206-E255, N207-E255, G208-E255, K209-E255, Q210-E255, Y211-E255, E212-E255, I213-E255, V214-E255, G215-E255, I216-E255, I217-E255, D218-E255, P219-E255, I220-E255, N221-E255, Y222-E255, R223-E255, V224-E255, L225-E255, V226-E255, T227-E255, L228-E255, I229-E255, E230-E255, N231-E255, T232-E255, D233-E255, G234-E255, S235-E255, N236-E255, K237-E255, V238-E255, A239-E255, K240-E255, G241-E255, E242-E255, G243-E255, S244-E255, I245-E255, E246-E255, V247-E255, L248-E255, and/or D249-E255 of SEQ ID NO:14. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CaYLR022c deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CaYLR022c deletion polypeptides are encompassed by the present invention: M1-E255, M1-K254, M1-I253, M1-A252, M1-S251, M1-M250, M1-D249, M1-L248, M1-V247, M1-E246, M1-I245, M1-S244, M1-G243, M1-E242, M1-G241, M1-K240, M1-A239, M1-V238, M1-K237, M1-N236, M1-S235, M1-G234, M1-D233, M1-T232, M1-N231, M1-E230, M1-I229, M1-L228, M1-T227, M1-V226, M1-L225, M1-V224, M1-R223, M1-Y222, M1-N221, M1-I220, M1-P219, M1-D218, M1-I217, M1-I216, M1-G215, M1-V214, M1-I213, M1-E212, M1-Y211, M1-Q210, M1-K209, M1-G208, M1-N207, M1-N206, M1-D205, M1-E204, M1-E203, M1-V202, M1-I201, M1-Q200, M1-D199, M1-I198, M1-V197, M1-P196, M1-K195, M1-I194, M1-E193, M1-D192, M1-Q191, M1-F190, M1-T189, M1-K188, M1-L187, M1-Y186, M1-A185, M1-K184, M1-K183, M1-S182, M1-L181, M1-T180, M1-I179, M1-R178, M1-V177, M1-K176, M1-M175, M1-Q174, M1-A173, M1-R172, M1-A171, M1-I170, M1-P169, M1-I168, M1-I167, M1-Q166, M1-K165, M1-E164, M1-V163, M1-L162, M1-L161, M1-K160, M1-I159, M1-A158, M1-D157, M1-L156, M1-A155, M1-Q154, M1-I153, M1-K152, M1-T151, M1-P150, M1-K149, M1-T148, M1-P147, M1-N146, M1-L145, M1-H144, M1-F143, M1-K142, M1-V141, M1-E140, M1-N139, M1-L138, M1-V137, M1-K136, M1-E135, M1-I134, M1-M133, M1-S132, M1-P131, M1-P130, M1-Y129, M1-R128, M1-K127, M1-K126, M1-S125, M1-R124, M1-P123, M1-N122, M1-I121, M1-C120, M1-K119, M1-T118, M1-S117, M1-I116, M1-I115, M1-N114, M1-L113, M1-F112, M1-E111, M1-N110, M1-Q109, M1-K108, M1-Q107, M1-Q106, M1-L105, M1-N104, M1-A103, M1-N102, M1-R101, M1-E100, M1-K99, M1-E98, M1-N97, M1-L96, M1-Q95, M1-I94, M1-E93, M1-G92, M1-K91, M1-N90, M1-L89, M1-I88, M1-E87, M1-A86, M1-I85, M1-I84, M1-E83, M1-D82, M1-Q81, M1-N80, M1-T79, M1-T78, M1-G77, M1-F76, M1-C75, M1-K74, M1-Q73, M1-L72, M1-D71, M1-D70, M1-N69, M1-N68, M1-A67, M1-V66, M1-Q65, M1-G64, M1-K63, M1-S62, M1-V61, M1-N60, M1-I59, M1-F58, M1-V57, M1-Q56, M1-P55, M1-I54, M1-Q53, M1-L52, M1-V51, M1-E50, M1-D49, M1-I48, M1-D47, M1-K46, M1-E45, M1-V44, M1-K43, M1-L42, M1-R41, M1-W40, M1-D39, M1-Q38, M1-V37, M1-K36, M1-N35, M1-Q34, M1-Y33, M1-C32, M1-A31, M1-I30, M1-E29, M1-F28, M1-R27, M1-K26, M1-K25, M1-G24, M1-K23, M1-K22, M1-M21, M1-R20, M1-V19, M1-L18, M1-S17, M1-V16, M1-N15, M1-T14, M1-L13, M1-R12, M1-I11, M1-Q10, M1-S9, M1-N8, and/or M1-P7 of SEQ ID NO:14. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CaYLR022c deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses polynucleotides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polynucleotide sequence provided in SEQ ID NO:3, and in particular to the coding region of the polynucleotide sequence provided in SEQ ID NO:3. Preferably such polynucleotides encode polypeptides that have biological activity.
The present invention also encompasses polypeptides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polypeptide sequence provided in SEQ ID NO:13.
Most preferred are polypeptides that share at least about 99.7% identity with the polypeptide sequence provided in SEQ ID NO:13.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:3 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 751 of SEQ ID NO:3, b is an integer between 15 to 765, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:3, and where b is greater than or equal to a+14.
Features of the Polypeptide Encoded by Polynucleotide No:4
The polynucleotide sequence (SEQ ID NO:4) and deduced amino acid sequence (SEQ ID NO:15) of the novel fungal essential gene, CaYOL077c (also referred to as FCG8), of the present invention. The CaYOL077c polypeptide (SEQ ID NO:15) is encoded by nucleotides 1 to 876 of SEQ ID NO:4 and has a predicted molecular weight of 34.0 kDa.
In preferred embodiments, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of CaYOL077c. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 4 thru 876 of SEQ ID NO:4, and the polypeptide corresponding to amino acids 2 thru 292 of SEQ ID NO:16. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.
The invention also encompasses N- and/or C-terminal deletions of the CaYOL077c polypeptide of the present invention.
In preferred embodiments, the following N-terminal CaYOL077c deletion polypeptides are encompassed by the present invention: M1-K292, S2-K292, A3-K292, I4-K292, Y5-K292, K6-K292, A7-K292, L8-K292, Q9-K292, S10-K292, K 11-K292, S12-K292, S13-K292, K14-K292, E15-K292, T16-K292, S17-K292, E18-K292, K19-K292, T20-K292, K21-K292, H22-K292, I23-K292, N24-K292, R25-K292, Q26-K292, R27-K292, L28-K292, L29-K292, V30-K292, I31-K292, S32-K292, S33-K292, R34-K292, G35-K292, I36-K292, T37-K292, Y38-K292, R39-K292, H40-K292, R41-K292, H42-K292, L43-K292, I44-K292, Q45-K292, D46-K292, L47-K292, L48-K292, A49-K292, L50-K292, L51-K292, P52-K292, H53-K292, A54-K292, R55-K292, K56-K292, E57-K292, P58-K292, K59-K292, F60-K292, D61-K292, S62-K292, K63-K292, K64-K292, N65-K292, L66-K292, H67-K292, Q68-K292, L69-K292, N70-K292, E71-K292, V72-K292, A73-K292, E74-K292, L75-K292, Y76-K292, N77-K292, C78-K292, N79-K292, N80-K292, I81-K292, F82-K292, F83-K292, F84-K292, E85-K292, C86-K292, R87-K292, K88-K292, H89-K292, Q90-K292, D91-K292, L92-K292, Y93-K292, L94-K292, W95-K292, I96-K292, S97-K292, K98-K292, P99-K292, P100-K292, N101-K292, G102-K292, P103-K292, T104-K292, L105-K292, K106-K292, F107-K292, H108-K292, I109-K292, Q110-K292, N111-K292, L112-K292, H113-K292, T114-K292, L115-K292, D116-K292, E117-K292, L118-K292, N119-K292, F120-K292, T121-K292, G122-K292, N123-K292, C124-K292, L125-K292, K126-K292, G127-K292, S128-K292, R129-K292, P130-K292, I131-K292, L132-K292, S133-K292, F134-K292, D135-K292, K136-K292, S137-K292, F138-K292, L139-K292, E140-K292, N141-K292, D142-K292, H143-K292, Y144-K292, K145-K292, L146-K292, L147-K292, K148-K292, E149-K292, M150-K292, F151-K292, L152-K292, Q153-K292, T154-K292, F155-K292, G156-K292, V157-K292, P158-K292, P159-K292, N160-K292, A161-K292, R162-K292, K163-K292, S164-K292, K165-K292, P166-K292, F167-K292, I168-K292, D169-K292, H170-K292, V171-K292, M172-K292, T173-K292, F174-K292, S175-K292, I176-K292, V177-K292, D178-K292, G179-K292, K180-K292, I181-K292, W182-K292, I183-K292, R184-K292, N185-K292, Y186-K292, Q187-K292, I188-K292, N189-K292, E190-K292, T191-K292, L192-K292, D193-K292, V194-K292, K195-K292, E196-K292, N197-K292, D198-K292, K199-K292, I200-K292, E201-K292, D202-K292, D203-K292, E204-K292, D205-K292, Y206-K292, D207-K292, V208-K292, D209-K292, Q210-K292, L211-K292, N212-K292, L213-K292, V214-K292, E215-K292, I216-K292, G217-K292, P218-K292, R219-K292, L220-K292, V221-K292, L222-K292, T223-K292, L224-K292, I225-K292, T226-K292, V227-K292, L228-K292, E229-K292, G230-K292, S231-K292, F232-K292, S233-K292, G234-K292, P235-K292, K236-K292, I237-K292, Y238-K292, E239-K292, N240-K292, K241-K292, Q242-K292, Y243-K292, V244-K292, S245-K292, P246-K292, N247-K292, F248-K292, V249-K292, R250-K292, A251-K292, Q252-K292, L253-K292, K254-K292, Q255-K292, Q256-K292, A257-K292, A258-K292, D259-K292, Q260-K292, A261-K292, K262-K292, S263-K292, R264-K292, S265-K292, Q266-K292, A267-K292, A268-K292, L269-K292, E270-K292, R271-K292, K272-K292, I273-K292, K274-K292, K275-K292, R276-K292, N277-K292, Q278-K292, V279-K292, L280-K292, K281-K292, A282-K292, D283-K292, P284-K292, L285-K292, and/or S286-K292 of SEQ ID NO:15. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CaYOL077c deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CaYOL077c deletion polypeptides are encompassed by the present invention: M1-K292, M1-F291, M1-L290, M1-A289, M1-D288, M1-N287, M1-S286, M1-L285, M1-P284, M1-D283, M1-A282, M1-K281, M1-L280, M1-V279, M1-Q278, M1-N277, M1-R276, M1-K275, M1-K274, M1-I273, M1-K272, M1-R271, M1-E270, M1-L269, M1-A268, M1-A267, M1-Q266, M1-S265, M1-R264, M1-S263, M1-K262, M1-A261, M1-Q260, M1-D259, M1-A258, M1-A257, M1-Q256, M1-Q255, M1-K254, M1-L253, M1-Q252, M1-A251, M1-R250, M1-V249, M1-F248, M1-N247, M1-P246, M1-S245, M1-V244, M1-Y243, M1-Q242, M1-K241, M1-N240, M1-E239, M1-Y238, M1-I237, M1-K236, M1-P235, M1-G234, M1-S233, M1-F232, M1-S231, M1-G230, M1-E229, M1-L228, M1-V227, M1-T226, M1-I225, M1-L224, M1-T223, M1-L222, M1-V221, M1-L220, M1-R219, M1-P218, M1-G217, M1-I216, M1-E215, M1-V214, M1-L213, M1-N212, M1-L211, M1-Q210, M1-D209, M1-V208, M1-D207, M1-Y206, M1-D205, M1-E204, M1-D203, M1-D202, M1-E201, M1-I200, M1-K199, M1-D198, M1-N197, M1-E196, M1-K195, M1-V194, M1-D193, M1-L192, M1-T191, M1-E190, M1-N189, M1-I188, M1-Q187, M1-Y186, M1-N185, M1-R184, M1-I183, M1-W182, M1-I181, M1-K180, M1-G179, M1-D178, M1-V177, M1-I176, M1-S175, M1-F174, M1-T173, M1-M172, M1-V171, M1-H170, M1-D169, M1-I168, M1-F167, M1-P166, M1-K165, M1-S164, M1-K163, M1-R162, M1-A161, M1-N160, M1-P159, M1-P158, M1-V157, M1-G156, M1-F155, M1-T154, M1-Q153, M1-L152, M1-F151, M1-M150, M1-E149, M1-K148, M1-L147, M1-L146, M1-K145, M1-Y144, M1-H143, M1-D142, M1-N141, M1-E140, M1-L139, M1-F138, M1-S137, M1-K136, M1-D135, M1-F134, M1-S133, M1-L132, M1-I131, M1-P130, M1-R129, M1-S128, M1-G127, M1-K126, M1-L125, M1-C124, M1-N123, M1-G122, M1-T121, M1-F120, M1-N119, M1-L118, M1-E117, M1-D116, M1-L115, M1-T114, M1-H113, M1-L112, M1-N111, M1-Q110, M1-I109, M1-H108, M1-F107, M1-K106, M1-L105, M1-T104, M1-P103, M1-G102, M1-N101, M1-P100, M1-P99, M1-K98, M1-S97, M1-I96, M1-W95, M1-L94, M1-Y93, M1-L92, M1-D91, M1-Q90, M1-H89, M1-K88, M1-R87, M1-C86, M1-E85, M1-F84, M1-F83, M1-F82, M1-I81, M1-N80, M1-N79, M1-C78, M1-N77, M1-Y76, M1-L75, M1-E74, M1-A73, M1-V72, M1-E71, M1-N70, M1-L69, M1-Q68, M1-H67, M1-L66, M1-N65, M1-K64, M1-K63, M1-S62, M1-D61, M1-F60, M1-K59, M1-P58, M1-E57, M1-K56, M1-R55, M1-A54, M1-H53, M1-P52, M1-L51, M1-L50, M1-A49, M1-L48, M1-L47, M1-D46, M1-Q45, M1-I44, M1-L43, M1-H42, M1-R41, M1-H40, M1-R39, M1-Y38, M1-T37, M1-I36, M1-G35, M1-R34, M1-S33, M1-S32, M1-I31, M1-V30, M1-L29, M1-L28, M1-R27, M1-Q26, M1-R25, M1-N24, M1-I23, M1-H22, M1-K21, M1-T20, M1-K19, M1-E18, M1-S17, M1-T16, M1-E15, M1-K14, M1-S13, M1-S12, M1-K11, M1-S10, M1-Q9, M1-L8, and/or M1-A7 of SEQ ID NO: 15. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CaYOL077c deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:4 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 862 of SEQ ID NO:4, b is an integer between 15 to 876, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:4, and where b is greater than or equal to a+14.
Features of the Polypeptide Encoded by Polynucleotide No:5
The polynucleotide sequence (SEQ ID NO:5) and deduced amino acid sequence (SEQ ID NO:16) of the novel fungal essential gene, CaYNL132w (also referred to as FCG10), of the present invention. The CaYNL132w polypeptide (SEQ ID NO:16) is encoded by nucleotides 1 to 3126 of SEQ ID NO:5 and has a predicted molecular weight of 117.3 kDa.
In preferred embodiments, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of CaYNL132w. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 4 thru 3126 of SEQ ID NO:5, and the polypeptide corresponding to amino acids 2 thru 1042 of SEQ ID NO:17. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.
The invention also encompasses N- and/or C-terminal deletions of the CaYNL132w polypeptide of the present invention.
In preferred embodiments, the following N-terminal CaYNL132w deletion polypeptides are encompassed by the present invention: M1-K1042, G2-K1042, K3-K1042, K4-K1042, A5-K1042, I6-K1042, D7-K1042, A8-K1042, R9-K1042, I10-K1042, P11-K1042, A12-K1042, L13-K1042, I14-K1042, R15-K1042, N16-K1042, G17-K1042, V18-K1042, Q19-K1042, E20-K1042, K21-K1042, Q22-K1042, R23-K1042, S24-K1042, F25-K1042, F26-K1042, I27-K1042, I28-K1042, V29-K1042, G30-K1042, D31-K1042, K32-K1042, A33-K1042, R34-K1042, N35-K1042, Q36-K1042, L37-K1042, P38-K1042, N39-K1042, L40-K1042, H41-K1042, Y42-K1042, L43-K1042, M44-K1042, M45-K1042, S46-K1042, A47-K1042, D48-K1042, L49-K1042, K50-K1042, M51-K1042, N52-K1042, K53-K1042, S54-K1042, V55-K1042, L56-K1042, W57-K1042, A58-K1042, Y59-K1042, K60-K1042, K61-K1042, K62-K1042, L63-K1042, L64-K1042, G65-K1042, F66-K1042, T67-K1042, S68-K1042, H69-K1042, R70-K1042, Q71-K1042, K72-K1042, R73-K1042, E74-K1042, A75-K1042, K76-K1042, I77-K1042, K78-K1042, K79-K1042, D80-K1042, I81-K1042, K82-K1042, R83-K1042, G84-K1042, I85-K1042, R86-K1042, E87-K1042, V88-K1042, N89-K1042, E90-K1042, Q91-K1042, D92-K1042, P93-K1042, F94-K1042, E95-K1042, A96-K1042, F97-K1042, I98-K1042, S99-K1042, N100-K1042, Q101-K1042, H102-K1042, I103-K1042, R104-K1042, Y105-K1042, V106-K1042, Y107-K1042, Y108-K1042, K109-K1042, E110-K1042, T111-K1042, E112-K1042, K113-K1042, I114-K1042, L115-K1042, G116-K1042, N117-K1042, T118-K1042, Y119-K1042, G120-K1042, M121-K1042, C122-K1042, I123-K1042, L124-K1042, Q125-K1042, D126-K1042, F127-K1042, E128-K1042, A129-K1042, I130-K1042, T131-K1042, P132-K1042, N133-K1042, L134-K1042, L135-K1042, A136-K1042, R137-K1042, T138-K1042, I139-K1042, E140-K1042, T141-K1042, V142-K1042, E143-K1042, G144-K1042, G145-K1042, G146-K1042, L147-K1042, V148-K1042, V149-K1042, I150-K1042, L151-K1042, L152-K1042, K153-K1042, N154-K1042, M155-K1042, T156-K1042, S157-K1042, L158-K1042, K159-K1042, Q160-K1042, L161-K1042, Y162-K1042, T163-K1042, M164-K1042, S165-K1042, M166-K1042, D167-K1042, I168-K1042, H169-K1042, S170-K1042, R171-K1042, Y172-K1042, R173-K1042, T174-K1042, E175-K1042, A176-K1042, H177-K1042, D178-K1042, D179-K1042, V180-K1042, V181-K1042, A182-K1042, R183-K1042, F184-K1042, N185-K1042, E186-K1042, R187-K1042, F188-K1042, L189-K1042, L190-K1042, S191-K1042, L192-K1042, G193-K1042, S194-K1042, C195-K1042, E196-K1042, N197-K1042, C198-K1042, L199-K1042, V200-K1042, V201-K1042, D202-K1042, D203-K1042, E204-K1042, L205-K1042, N206-K1042, V207-K1042, L208-K1042, P209-K1042, I210-K1042, S211-K1042, G212-K1042, G213-K1042, K214-K1042, H215-K1042, V216-K1042, K217-K1042, P218-K1042, L219-K1042, P220-K1042, P221-K1042, K222-K1042, D223-K1042, D224-K1042, D225-K1042, E226-K1042, L227-K1042, T228-K1042, P229-K1042, N230-K1042, A231-K1042, K232-K1042, E233-K1042, L234-K1042, K235-K1042, E236-K1042, L237-K1042, K238-K1042, E239-K1042, S240-K1042, L241-K1042, A242-K1042, D243-K1042, V244-K1042, Q245-K1042, P246-K1042, A247-K1042, G248-K1042, S249-K1042, L250-K1042, V251-K1042, A252-K1042, L253-K1042, S254-K1042, K255-K1042, T256-K1042, I257-K1042, N258-K1042, Q259-K1042, A260-K1042, Q261-K1042, A262-K1042, I263-K1042, L264-K1042, T265-K1042, F266-K1042, I267-K1042, D268-K1042, V269-K1042, I270-K1042, S271-K1042, E272-K1042, K273-K1042, T274-K1042, L275-K1042, R276-K1042, N277-K1042, T278-K1042, V279-K1042, T280-K1042, L281-K1042, T282-K1042, A283-K1042, G284-K1042, R285-K1042, G286-K1042, R287-K1042, G288-K1042, K289-K1042, S290-K1042, A291-K1042, A292-K1042, L293-K1042, G294-K1042, I295-K1042, A296-K1042, I297-K1042, A298-K1042, A299-K1042, A300-K1042, I301-K1042, S302-K1042, H303-K1042, G304-K1042, Y305-K1042, S306-K1042, N307-K1042, I308-K1042, F309-K1042, V310-K1042, T311-K1042, S312-K1042, P313-K1042, S314-K1042, P315-K1042, E316-K1042, N317-K1042, L318-K1042, K319-K1042, T320-K1042, L321-K1042, F322-K1042, E323-K1042, F324-K1042, I325-K1042, F326-K1042, K327-K1042, G328-K1042, F329-K1042, D330-K1042, A331-K1042, L332-K1042, G333-K1042, Y334-K1042, T335-K1042, E336-K1042, H337-K1042, M338-K1042, D339-K1042, Y340-K1042, D341-K1042, I342-K1042, I343-K1042, Q344-K1042, S345-K1042, T346-K1042, N347-K1042, P348-K1042, S349-K1042, F350-K1042, N351-K1042, K352-K1042, A353-K1042, I354-K1042, V355-K1042, R356-K1042, V357-K1042, D358-K1042, V359-K1042, K360-K1042, R361-K1042, E362-K1042, H363-K1042, R364-K1042, Q365-K1042, T366-K1042, I367-K1042, Q368-K1042, Y369-K1042, I370-K1042, S371-K1042, P372-K1042, N373-K1042, D374-K1042, S375-K1042, H376-K1042, V377-K1042, L378-K1042, G379-K1042, Q380-K1042, A381-K1042, E382-K1042, L383-K1042, L384-K1042, I385-K1042, I386-K1042, D387-K1042, E388-K1042, A389-K1042, A390-K1042, A391-K1042, I392-K1042, P393-K1042, L394-K1042, P395-K1042, I396-K1042, V397-K1042, K398-K1042, K399-K1042, L400-K1042, M401-K1042, G402-K1042, P403-K1042, Y404-K1042, L405-K1042, I406-K1042, F407-K1042, M408-K1042, A409-K1042, S410-K1042, T411-K1042, I412-K1042, N413-K1042, G414-K1042, Y415-K1042, E416-K1042, G417-K1042, T418-K1042, G419-K1042, R420-K1042, S421-K1042, L422-K1042, S423-K1042, L424-K1042, K425-K1042, L426-K1042, I427-K1042, Q428-K1042, Q429-K1042, L430-K1042, R431-K1042, T432-K1042, Q433-K1042, S434-K1042, N435-K1042, N436-K1042, A437-K1042, T438-K1042, P439-K1042, S440-K1042, E441-K1042, T442-K1042, T443-K1042, V444-K1042, V445-K1042, S446-K1042, R447-K1042, D448-K1042, K449-K1042, K450-K1042, S451-K1042, N452-K1042, E453-K1042, I454-K1042, T455-K1042, G456-K1042, A457-K1042, L458-K1042, T459-K1042, R460-K1042, T461-K1042, L462-K1042, K463-K1042, E464-K1042, V465-K1042, V466-K1042, L467-K1042, D468-K1042, E469-K1042, P470-K1042, I471-K1042, R472-K1042, Y473-K1042, A474-K1042, P475-K1042, G476-K1042, D477-K1042, P478-K1042, I479-K1042, E480-K1042, K481-K1042, W482-K1042, L483-K1042, N484-K1042, K485-K1042, L486-K1042, L487-K1042, C488-K1042, L489-K1042, D490-K1042, V491-K1042, S492-K1042, L493-K1042, S494-K1042, K495-K1042, N496-K1042, A497-K1042, K498-K1042, F499-K1042, A500-K1042, T501-K1042, K502-K1042, G503-K1042, T504-K1042, P505-K1042, H506-K1042, P507-K1042, S508-K1042, Q509-K1042, C510-K1042, Q511-K1042, L512-K1042, F513-K1042, Y514-K1042, V515-K1042, N516-K1042, R517-K1042, D518-K1042, T519-K1042, L520-K1042, F521-K1042, S522-K1042, Y523-K1042, H524-K1042, P525-K1042, V526-K1042, S527-K1042, E528-K1042, A529-K1042, F530-K1042, L531-K1042, Q532-K1042, K533-K1042, M534-K1042, M535-K1042, A536-K1042, L537-K1042, Y538-K1042, V539-K1042, A540-K1042, S541-K1042, H542-K1042, Y543-K1042, K544-K1042, N545-K1042, S546-K1042, P547-K1042, N548-K1042, D549-K1042, L550-K1042, Q551-K1042, L552-K1042, M553-K1042, S554-K1042, D555-K1042, A556-K1042, P557-K1042, A558-K1042, H559-K1042, Q560-K1042, L561-K1042, F562-K1042, V563-K1042, L564-K1042, L565-K1042, P566-K1042, P567-K1042, 1568-K1042, E569-K1042, A570-K1042, G571-K1042, D572-K1042, N573-K1042, R574-K1042, V575-K1042, P576-K1042, D577-K1042, P578-K1042, L579-K1042, C580-K1042, V581-K1042, I582-K1042, Q583-K1042, L584-K1042, A585-K1042, L586-K1042, E587-K1042, G588-K1042, E589-K1042, I590-K1042, S591-K1042, K592-K1042, E593-K1042, S594-K1042, V595-K1042, R596-K1042, K597-K1042, S598-K1042, L599-K1042, S600-K1042, R601-K1042, G602-K1042, Q603-K1042, R604-K1042, A605-K1042, G606-K1042, G607-K1042, D608-K1042, L609-K1042, I610-K1042, P611-K1042, W612-K1042, L613-K1042, I614-K1042, S615-K1042, Q616-K1042, Q617-K1042, F618-K1042, Q619-K1042, D620-K1042, E621-K1042, E622-K1042, F623-K1042, A624-K1042, S625-K1042, L626-K1042, S627-K1042, G628-K1042, A629-K1042, R630-K1042, V631-K1042, V632-K1042, R633-K1042, I634-K1042, A635-K1042, T636-K1042, N637-K1042, P638-K1042, E639-K1042, Y640-K1042, S641-K1042, G642-K1042, M643-K1042, G644-K1042, Y645-K1042, G646-K1042, S647-K1042, R648-K1042, A649-K1042, M650-K1042, E651-K1042, L652-K1042, L653-K1042, R654-K1042, D655-K1042, Y656-K1042, Y657-K1042, S658-K1042, G659-K1042, K660-K1042, F661-K1042, T662-K1042, D663-K1042, I664-K1042, S665-K1042, E666-K1042, S667-K1042, T668-K1042, E669-K1042, L670-K1042, N671-K1042, D672-K1042, H673-K1042, T674-K1042, I675-K1042, T676-K1042, R677-K1042, V678-K1042, T679-K1042, D680-K1042, S681-K1042, E682-K1042, L683-K1042, A684-K1042, N685-K1042, A686-K1042, S687-K1042, L688-K1042, K689-K1042, D690-K1042, E691-K1042, I692-K1042, K693-K1042, L694-K1042, R695-K1042, D696-K1042, V697-K1042, K698-K1042, T699-K1042, L700-K1042, P701-K1042, P702-K1042, L703-K1042, L704-K1042, L705-K1042, K706-K1042, L707-K1042, S708-K1042, E709-K1042, K710-K1042, A711-K1042, P712-K1042, Y713-K1042, Y714-K1042, L715-K1042, H716-K1042, Y717-K1042, L718-K1042, G719-K1042, V720-K1042, S721-K1042, Y722-K1042, G723-K1042, F724-K1042, T725-K1042, S726-K1042, Q727-K1042, L728-K1042, H729-K1042, K730-K1042, F731-K1042, W732-K1042, K733-K1042, K734-K1042, A735-K1042, G736-K1042, F737-K1042, T738-K1042, P739-K1042, V740-K1042, Y741-K1042, L742-K1042, R743-K1042, Q744-K1042, T745-K1042, P746-K1042, N747-K1042, E748-K1042, L749-K1042, T750-K1042, G751-K1042, E752-K1042, H753-K1042, T754-K1042, S755-K1042, V756-K1042, V757-K1042, I758-K1042, S759-K1042, V760-K1042, L761-K1042, P762-K1042, G763-K1042, R764-K1042, E765-K1042, D766-K1042, K767-K1042, W768-K1042, L769-K1042, H770-K1042, E771-K1042, F772-K1042, S773-K1042, K774-K1042, D775-K1042, F776-K1042, H777-K1042, K778-K1042, R779-K1042, F780-K1042, L781-K1042, S782-K1042, L783-K1042, L784-K1042, S785-K1042, Y786-K1042, E787-K1042, F788-K1042, K789-K1042, K790-K1042, F791-K1042, Q792-K1042, A793-K1042, S794-K1042, Q795-K1042, A796-K1042, L797-K1042, S798-K1042, I799-K1042, I800-K1042, E801-K1042, A802-K1042, A803-K1042, E804-K1042, Q805-K1042, G806-K1042, E807-K1042, G808-K1042, D809-K1042, E810-K1042, T811-K1042, T812-K1042, S813-K1042, Q814-K1042, K815-K1042, L816-K1042, T817-K1042, K818-K1042, E819-K1042, Q820-K1042, L821-K1042, D822-K1042, L823-K1042, L824-K1042, L825-K1042, S826-K1042, P827-K1042, F828-K1042, D829-K1042, L830-K1042, K831-K1042, R832-K1042, L833-K1042, D834-K1042, S835-K1042, Y836-K1042, A837-K1042, N838-K1042, N839-K1042, L840-K1042, L841-K1042, D842-K1042, Y843-K1042, H844-K1042, V845-K1042, I846-K1042, V847-K1042, D848-K1042, M849-K1042, L850-K1042, P851-K1042, L852-K1042, I853-K1042, S854-K1042, Q855-K1042, L856-K1042, F857-K1042, F858-K1042, S859-K1042, K860-K1042, K861-K1042, T862-K1042, G863-K1042, Q864-K1042, D865-K1042, I866-K1042, S867-K1042, L868-K1042, S869-K1042, S870-K1042, V871-K1042, Q872-K1042, S873-K1042, A874-K1042, I875-K1042, L876-K1042, L877-K1042, A878-K1042, I879-K1042, G880-K1042, L881-K1042, Q882-K1042, H883-K1042, K884-K1042, D885-K1042, M886-K1042, D887-K1042, Q888-K1042, I889-K1042, A890-K1042, K891-K1042, E892-K1042, L893-K1042, N894-K1042, L895-K1042, P896-K1042, T897-K1042, N898-K1042, Q899-K1042, A900-K1042, M901-K1042, A902-K1042, M903-K1042, F904-K1042, A905-K1042, K906-K1042, I907-K1042, I908-K1042, R909-K1042, K910-K1042, F911-K1042, S912-K1042, T913-K1042, Y914-K1042, F915-K1042, R916-K1042, K917-K1042, V918-K1042, L919-K1042, S920-K1042, K921-K1042, A922-K1042, I923-K1042, E924-K1042, E925-K1042, S926-K1042, M927-K1042, P928-K1042, D929-K1042, L930-K1042, E931-K1042, D932-K1042, E933-K1042, N934-K1042, V935-K1042, D936-K1042, A937-K1042, M938-K1042, N939-K1042, G940-K1042, K941-K1042, E942-K1042, T943-K1042, E944-K1042, Q945-K1042, I946-K1042, D947-K1042, Y948-K1042, K949-K1042, A950-K1042, I951-K1042, E952-K1042, Q953-K1042, K954-K1042, L955-K1042, Q956-K1042, D957-K1042, D958-K1042, L959-K1042, E960-K1042, E961-K1042, A962-K1042, G963-K1042, D964-K1042, E965-K1042, A966-K1042, I967-K1042, K968-K1042, E969-K1042, M970-K1042, R971-K1042, E972-K1042, K973-K1042, Q974-K1042, R975-K1042, E976-K1042, L977-K1042, I978-K1042, N979-K1042, A980-K1042, L981-K1042, N982-K1042, L983-K1042, D984-K1042, K985-K1042, Y986-K1042, A987-K1042, I988-K1042, A989-K1042, E990-K1042, D991-K1042, A992-K1042, E993-K1042, W994-K1042, D995-K1042, E996-K1042, K997-K1042, S998-K1042, M999-K1042, D1000-K1042, K1001-K1042, A1002-K1042, T1003-K1042, K1004-K1042, G1005-K1042, K1006-K1042, G1007-K1042, N1008-K1042, V1009-K1042, V1010-K1042, S1011-K1042, I1012-K1042, K1013-K1042, S1014-K1042, G1015-K1042, K1016-K1042, R1017-K1042, K1018-K1042, S1019-K1042, K1020-K1042, E1021-K1042, N1022-K1042, A1023-K1042, N1024-K1042, D1025-K1042, I1026-K1042, Y1027-K1042, E1028-K1042, K1029-K1042, E1030-K1042, M1031-K1042, K1032-K1042, A1033-K1042, V1034-K1042, K1035-K1042, and/or K1036-K1042 of SEQ ID NO:16. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CaYNL132w deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CaYNL132w deletion polypeptides are encompassed by the present invention: M1-K1042, M1-K1041, M1-S1040, M1-K1039, M1-K1038, M1-S1037, M1-K1036, M1-K1035, M1-V1034, M1-A1033, M1-K1032, M1-M1031, M1-E1030, M1-K1029, M1-E1028, M1-Y1027, M1-I1026, M1-D1025, M1-N1024, M1-A1023, M1-N1022, M1-E1021, M1-K1020, M1-S1019, M1-K1018, M1-R1017, M1-K1016, M1-G1015, M1-S1014, M1-K1013, M1-I1012, M1-S1011, M1-V1010, M1-V1009, M1-N1008, M1-G1007, M1-K1006, M1-G1005, M1-K1004, M1-T1003, M1-A1002, M1-K1001, M1-D1000, M1-M999, M1-S998, M1-K997, M1-E996, M1-D995, M1-W994, M1-E993, M1-A992, M1-D991, M1-E990, M1-A989, M1-I988, M1-A987, M1-Y986, M1-K985, M1-D984, M1-L983, M1-N982, M1-L981, M1-A980, M1-N979, M1-I978, M1-L977, M1-E976, M1-R975, M1-Q974, M1-K973, M1-E972, M1-R971, M1-M970, M1-E969, M1-K968, M1-I967, M1-A966, M1-E965, M1-D964, M1-G963, M1-A962, M1-E961, M1-E960, M1-L959, M1-D958, M1-D957, M1-Q956, M1-L955, M1-K954, M1-Q953, M1-E952, M1-I951, M1-A950, M1-K949, M1-Y948, M1-D947, M1-I946, M1-Q945, M1-E944, M1-T943, M1-E942, M1-K941, M1-G940, M1-N939, M1-M938, M1-A937, M1-D936, M1-V935, M1-N934, M1-E933, M1-D932, M1-E931, M1-L930, M1-D929, M1-P928, M1-M927, M1-S926, M1-E925, M1-E924, M1-I923, M1-A922, M1-K921, M1-S920, M1-L919, M1-V918, M1-K917, M1-R916, M1-F915, M1-Y914, M1-T913, M1-S912, M1-F911, M1-K910, M1-R909, M1-I908, M1-I907, M1-K906, M1-A905, M1-F904, M1-M903, M1-A902, M1-M901, M1-A900, M1-Q899, M1-N898, M1-T897, M1-P896, M1-L895, M1-N894, M1-L893, M1-E892, M1-K891, M1-A890, M1-I889, M1-Q888, M1-D887, M1-M886, M1-D885, M1-K884, M1-H883, M1-Q882, M1-L881, M1-G880, M1-I879, M1-A878, M1-L877, M1-L876, M1-I875, M1-A874, M1-S873, M1-Q872, M1-V871, M1-S870, M1-S869, M1-L868, M1-S867, M1-I866, M1-D865, M1-Q864, M1-G863, M1-T862, M1-K861, M1-K860, M1-S859, M1-F858, M1-F857, M1-L856, M1-Q855, M1-S854, M1-I853, M1-L852, M1-P851, M1-L850, M1-M849, M1-D848, M1-V847, M1-I846, M1-V845, M1-H844, M1-Y843, M1-D842, M1-L841, M1-L840, M1-N839, M1-N838, M1-A837, M1-Y836, M1-S835, M1-D834, M1-L833, M1-R832, M1-K831, M1-L830, M1-D829, M1-F828, M1-P827, M1-S826, M1-L825, M1-L824, M1-L823, M1-D822, M1-L821, M1-Q820, M1-E819, M1-K818, M1-T817, M1-L816, M1-K815, M1-Q814, M1-S813, M1-T812, M1-T811, M1-E810, M1-D809, M1-G808, M1-E807, M1-G806, M1-Q805, M1-E804, M1-A803, M1-A802, M1-E801, M1-I800, M1-I799, M1-S798, M1-L797, M1-A796, M1-Q795, M1-S794, M1-A793, M1-Q792, M1-F791, M1-K790, M1-K789, M1-F788, M1-E787, M1-Y786, M1-S785, M1-L784, M1-L783, M1-S782, M1-L781, M1-F780, M1-R779, M1-K778, M1-H777, M1-F776, M1-D775, M1-K774, M1-S773, M1-F772, M1-E771, M1-H770, M1-L769, M1-W768, M1-K767, M1-D766, M1-E765, M1-R764, M1-G763, M1-P762, M1-L761, M1-V760, M1-S759, M1-I758, M1-V757, M1-V756, M1-S755, M1-T754, M1-H753, M1-E752, M1-G751, M1-T750, M1-L749, M1-E748, M1-N747, M1-P746, M1-T745, M1-Q744, M1-R743, M1-L742, M1-Y741, M1-V740, M1-P739, M1-T738, M1-F737, M1-G736, M1-A735, M1-K734, M1-K733, M1-W732, M1-F731, M1-K730, M1-H729, M1-L728, M1-Q727, M1-S726, M1-T725, M1-F724, M1-G723, M1-Y722, M1-S721, M1-V720, M1-G719, M1-L718, M1-Y717, M1-H716, M1-L715, M1-Y714, M1-Y713, M1-P712, M1-A711, M1-K710, M1-E709, M1-S708, M1-L707, M1-K706, M1-L705, M1-L704, M1-L703, M1-P702, M1-P701, M1-L700, M1-T699, M1-K698, M1-V697, M1-D696, M1-R695, M1-L694, M1-K693, M1-I692, M1-E691, M1-D690, M1-K689, M1-L688, M1-S687, M1-A686, M1-N685, M1-A684, M1-L683, M1-E682, M1-S681, M1-D680, M1-T679, M1-V678, M1-R677, M1-T676, M1-I675, M1-T674, M1-H673, M1-D672, M1-N671, M1-L670, M1-E669, M1-T668, M1-S667, M1-E666, M1-S665, M1-I664, M1-D663, M1-T662, M1-F661, M1-K660, M1-G659, M1-S658, M1-Y657, M1-Y656, M1-D655, M1-R654, M1-L653, M1-L652, M1-E651, M1-M650, M1-A649, M1-R648, M1-S647, M1-G646, M1-Y645, M1-G644, M1-M643, M1-G642, M1-S641, M1-Y640, M1-E639, M1-P638, M1-N637, M1-T636, M1-A635, M1-I634, M1-R633, M1-V632, M1-V631, M1-R630, M1-A629, M1-G628, M1-S627, M1-L626, M1-S625, M1-A624, M1-F623, M1-E622, M1-E621, M1-D620, M1-Q619, M1-F618, M1-Q617, M1-Q616, M1-S615, M1-I614, M1-L613, M1-W612, M1-P611, M1-I610, M1-L609, M1-D608, M1-G607, M1-G606, M1-A605, M1-R604, M1-Q603, M1-G602, M1-R601, M1-S600, M1-L599, M1-S598, M1-K597, M1-R596, M1-V595, M1-S594, M1-E593, M1-K592, M1-S591, M1-I590, M1-E589, M1-G588, M1-E587, M1-L586, M1-A585, M1-L584, M1-Q583, M1-I582, M1-V581, M1-C580, M1-L579, M1-P578, M1-D577, M1-P576, M1-V575, M1-R574, M1-N573, M1-D572, M1-G571, M1-A570, M1-E569, M1-I568, M1-P567, M1-P566, M1-L565, M1-L564, M1-V563, M1-F562, M1-L561, M1-Q560, M1-H559, M1-A558, M1-P557, M1-A556, M1-D555, M1-S554, M1-M553, M1-L552, M1-Q551, M1-L550, M1-D549, M1-N548, M1-P547, M1-S546, M1-N545, M1-K544, M1-Y543, M1-H542, M1-S541, M1-A540, M1-V539, M1-Y538, M1-L537, M1-A536, M1-M535, M1-M534, M1-K533, M1-Q532, M1-L531, M1-F530, M1-A529, M1-E528, M1-S527, M1-V526, M1-P525, M1-H524, M1-Y523, M1-S522, M1-F521, M1-L520, M1-T519, M1-D518, M1-R517, M1-N516, M1-V515, M1-Y514, M1-F513, M1-L512, M1-Q511, M1-C510, M1-Q509, M1-S508, M1-P507, M1-H506, M1-P505, M1-T504, M1-G503, M1-K502, M1-T501, M1-A500, M1-F499, M1-K498, M1-A497, M1-N496, M1-K495, M1-S494, M1-L493, M1-S492, M1-V491, M1-D490, M1-L489, M1-C488, M1-L487, M1-L486, M1-K485, M1-N484, M1-L483, M1-W482, M1-K481, M1-E480, M1-I479, M1-P478, M1-D477, M1-G476, M1-P475, M1-A474, M1-Y473, M1-R472, M1-I471, M1-P470, M1-E469, M1-D468, M1-L467, M1-V466, M1-V465, M1-E464, M1-K463, M1-L462, M1-T461, M1-R460, M1-T459, M1-L458, M1-A457, M1-G456, M1-T455, M1-I454, M1-E453, M1-N452, M1-S451, M1-K450, M1-K449, M1-D448, M1-R447, M1-S446, M1-V445, M1-V444, M1-T443, M1-T442, M1-E441, M1-S440, M1-P439, M1-T438, M1-A437, M1-N436, M1-N435, M1-S434, M1-Q433, M1-T432, M1-R431, M1-L430, M1-Q429, M1-Q428, M1-I427, M1-L426, M1-K425, M1-L424, M1-S423, M1-L422, M1-S421, M1-R420, M1-G419, M1-T418, M1-G417, M1-E416, M1-Y415, M1-G414, M1-N413, M1-I412, M1-T411, M1-S410, M1-A409, M1-M408, M1-F407, M1-I406, M1-L405, M1-Y404, M1-P403, M1-G402, M1-M401, M1-L400, M1-K399, M1-K398, M1-V397, M1-I396, M1-P395, M1-L394, M1-P393, M1-I392, M1-A391, M1-A390, M1-A389, M1-E388, M1-D387, M1-I386, M1-I385, M1-L384, M1-L383, M1-E382, M1-A381, M1-Q380, M1-G379, M1-L378, M1-V377, M1-H376, M1-S375, M1-D374, M1-N373, M1-P372, M1-S371, M1-I370, M1-Y369, M1-Q368, M1-I367, M1-T366, M1-Q365, M1-R364, M1-H363, M1-E362, M1-R361, M1-K360, M1-V359, M1-D358, M1-V357, M1-R356, M1-V355, M1-I354, M1-A353, M1-K352, M1-N351, M1-F350, M1-S349, M1-P348, M1-N347, M1-T346, M1-S345, M1-Q344, M1-I343, M1-I342, M1-D341, M1-Y340, M1-D339, M1-M338, M1-H337, M1-E336, M1-T335, M1-Y334, M1-G333, M1-L332, M1-A331, M1-D330, M1-F329, M1-G328, M1-K327, M1-F326, M1-I325, M1-F324, M1-E323, M1-F322, M1-L321, M1-T320, M1-K319, M1-L318, M1-N317, M1-E316, M1-P315, M1-S314, M1-P313, M1-S312, M1-T311, M1-V310, M1-F309, M1-I308, M1-N307, M1-S306, M1-Y305, M1-G304, M1-H303, M1-S302, M1-I301, M1-A300, M1-A299, M1-A298, M1-I297, M1-A296, M1-I295, M1-G294, M1-L293, M1-A292, M1-A291, M1-S290, M1-K289, M1-G288, M1-R287, M1-G286, M1-R285, M1-G284, M1-A283, M1-T282, M1-L281, M1-T280, M1-V279, M1-T278, M1-N277, M1-R276, M1-L275, M1-T274, M1-K273, M1-E272, M1-S271, M1-I270, M1-V269, M1-D268, M1-I267, M1-F266, M1-T265, M1-L264, M1-I263, M1-A262, M1-Q261, M1-A260, M1-Q259, M1-N258, M1-I257, M1-T256, M1-K255, M1-S254, M1-L253, M1-A252, M1-V251, M1-L250, M1-S249, M1-G248, M1-A247, M1-P246, M1-Q245, M1-V244, M1-D243, M1-A242, M1-L241, M1-S240, M1-E239, M1-K238, M1-L237, M1-E236, M1-K235, M1-L234, M1-E233, M1-K232, M1-A231, M1-N230, M1-P229, M1-T228, M1-L227, M1-E226, M1-D225, M1-D224, M1-D223, M1-K222, M1-P221, M1-P220, M1-L219, M1-P218, M1-K217, M1-V216, M1-H215, M1-K214, M1-G213, M1-G212, M1-S211, M1-I210, M1-P209, M1-L208, M1-V207, M1-N206, M1-L205, M1-E204, M1-D203, M1-D202, M1-V201, M1-V200, M1-L199, M1-C198, M1-N197, M1-E196, M1-C195, M1-S194, M1-G193, M1-L192, M1-S191, M1-L190, M1-L189, M1-F188, M1-R187, M1-E186, M1-N185, M1-F184, M1-R183, M1-A182, M1-V181, M1-V180, M1-D179, M1-D178, M1-H177, M1-A176, M1-E175, M1-T174, M1-R173, M1-Y172, M1-R171, M1-S170, M1-H169, M1-I168, M1-D167, M1-M166, M1-S165, M1-M164, M1-T163, M1-Y162, M1-L161, M1-Q160, M1-K159, M1-L158, M1-S157, M1-T156, M1-M155, M1-N154, M1-K153, M1-L152, M1-L151, M1-I150, M1-V149, M1-V148, M1-L147, M1-G146, M1-G145, M1-G144, M1-E143, M1-V142, M1-T141, M1-E140, M1-I139, M1-T138, M1-R137, M1-A136, M1-L135, M1-L134, M1-N133, M1-P132, M1-T131, M1-I130, M1-A129, M1-E128, M1-F127, M1-D126, M1-Q125, M1-L124, M1-I123, M1-C122, M1-M121, M1-G120, M1-Y119, M1-T118, M1-N117, M1-G116, M1-L115, M1-I114, M1-K113, M1-E112, M1-T111, M1-E110, M1-K109, M1-Y108, M1-Y107, M1-V106, M1-Y105, M1-R104, M1-I103, M1-H102, M1-Q101, M1-N100, M1-S99, M1-I98, M1-F97, M1-A96, M1-E95, M1-F94, M1-P93, M1-D92, M1-Q91, M1-E90, M1-N89, M1-V88, M1-E87, M1-R86, M1-I85, M1-G84, M1-R83, M1-K82, M1-I81, M1-D80, M1-K79, M1-K78, M1-I77, M1-K76, M1-A75, M1-E74, M1-R73, M1-K72, M1-Q71, M1-R70, M1-H69, M1-S68, M1-T67, M1-F66, M1-G65, M1-L64, M1-L63, M1-K62, M1-K61, M1-K60, M1-Y59, M1-A58, M1-W57, M1-L56, M1-V55, M1-S54, M1-K53, M1-N52, M1-M51, M1-K50, M1-L49, M1-D48, M1-A47, M1-S46, M1-M45, M1-M44, M1-L43, M1-Y42, M1-H41, M1-L40, M1-N39, M1-P38, M1-L37, M1-Q36, M1-N35, M1-R34, M1-A33, M1-K32, M1-D31, M1-G30, M1-V29, M1-I28, M1-I27, M1-F26, M1-F25, M1-S24, M1-R23, M1-Q22, M1-K21, M1-E20, M1-Q19, M1-V18, M1-G17, M1-N16, M1-R15, M1-I14, M1-L13, M1-A12, M1-P11, M1-I10, M1-R9, M1-A8, and/or M1-D7 of SEQ ID NO:16. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CaYNL132w deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:5 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 3112 of SEQ ID NO:5, b is an integer between 15 to 3126, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:5, and where b is greater than or equal to a+14.
Features of the Polypeptide Encoded by Polynucleotide No:6
The polynucleotide sequence (SEQ ID NO:6) and deduced amino acid sequence (SEQ ID NO:17) of the novel fungal essential gene, CaYGR145w (also referred to as FCG12), of the present invention. The CaYGR145w polypeptide (SEQ ID NO:17) is encoded by nucleotides 1 to 2250 of SEQ ID NO:6 and has a predicted molecular weight of 85.0 kDa.
In preferred embodiments, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of CaYGR145w. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 4 thru 2250 of SEQ ID NO:6, and the polypeptide corresponding to amino acids 2 thru 1042 of SEQ ID NO:18. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.
The invention also encompasses N- and/or C-terminal deletions of the CaYGR145w polypeptide of the present invention.
In preferred embodiments, the following N-terminal CaYGR145w deletion polypeptides are encompassed by the present invention: M1-M750, V2-M750, L3-M750, K4-M750, S5-M750, T6-M750, T7-M750, A8-M750, G9-M750, N10-M750, V11-M750, S12-M750, V13-M750, Y14-M750, Q15-M750, V16-M750, S17-M750, G18-M750, T19-M750, N20-M750, V21-M750, S22-M750, R23-M750, S24-M750, L25-M750, P26-M750, D27-M750, W28-M750, I29-M750, D30-M750, K31-M750, K32-M750, R33-M750, K34-M750, R35-M750, A36-M750, L37-M750, K38-M750, H39-M750, D40-M750, L41-M750, E42-M750, Y43-M750, Q44-M750, N45-M750, R46-M750, I47-M750, E48-M750, L49-M750, I50-M750, Q51-M750, D52-M750, F53-M750, E54-M750, F55-M750, S56-M750, E57-M750, A58-M750, S59-M750, N60-M750, K61-M750, I62-M750, K63-M750, V64-M750, T65-M750, N66-M750, D67-M750, G68-M750, Q69-M750, Y70-M750, C71-M750, M72-M750, A73-M750, T74-M750, G75-M750, T76-M750, Y77-M750, K78-M750, P79-M750, Q80-M750, I81-M750, H82-M750, V83-M750, Y84-M750, E85-M750, F86-M750, A87-M750, N88-M750, L89-M750, S90-M750, L91-M750, K92-M750, F93-M750, D94-M750, R95-M750, H96-M750, T97-M750, N98-M750, V99-M750, E100-M750, N101-M750, I102-M750, D103-M750, F104-M750, L105-M750, I106-M750, L107-M750, S108-M750, N109-M750, D110-M750, W111-M750, T112-M750, K113-M750, S114-M750, V115-M750, H116-M750, L117-M750, Q118-M750, C119-M750, D120-M750, R121-M750, S122-M750, I123-M750, E124-M750, F125-M750, Q126-M750, T127-M750, A128-M750, G129-M750, G130-M750, V131-M750, H132-M750, Y133-M750, R134-M750, T135-M750, R136-M750, I137-M750, P138-M750, K139-M750, F140-M750, G141-M750, R142-M750, C143-M750, L144-M750, T145-M750, Y146-M750, N147-M750, P148-M750, I149-M750, N150-M750, C151-M750, D152-M750, L153-M750, I154-M750, V155-M750, G156-M750, S157-M750, S158-M750, S159-M750, D160-M750, E161-M750, L162-M750, Y163-M750, R164-M750, L165-M750, N166-M750, L167-M750, D168-M750, Q169-M750, G170-M750, R171-M750, F172-M750, L173-M750, S174-M750, P175-M750, L176-M750, K177-M750, L178-M750, D179-M750, M180-M750, T181-M750, D182-M750, G183-M750, G184-M750, N185-M750, I186-M750, D187-M750, S188-M750, G189-M750, C190-M750, N191-M750, A192-M750, V193-M750, D194-M750, I195-M750, N196-M750, S197-M750, M198-M750, H199-M750, G200-M750, L201-M750, I202-M750, S203-M750, A204-M750, G205-M750, L206-M750, D207-M750, D208-M750, G209-M750, T210-M750, V211-M750, E212-M750, F213-M750, W214-M750, D215-M750, P216-M750, R217-M750, S218-M750, K219-M750, Q220-M750, R221-M750, A222-M750, G223-M750, K224-M750, L225-M750, F226-M750, V227-M750, S228-M750, D229-M750, Q230-M750, L231-M750, I232-M750, N233-M750, S234-M750, T235-M750, N236-M750, N237-M750, T238-M750, E239-M750, Q240-M750, S241-M750, S242-M750, C243-M750, G244-M750, I245-M750, T246-M750, S247-M750, L248-M750, A249-M750, F250-M750, R251-M750, P252-M750, Q253-M750, D254-M750, A255-M750, L256-M750, N257-M750, F258-M750, A259-M750, C260-M750, G261-M750, T262-M750, S263-M750, N264-M750, G265-M750, Q266-M750, T267-M750, L268-M750, L269-M750, Y270-M750, D271-M750, L272-M750, R273-M750, A274-M750, S275-M750, E276-M750, P277-M750, Y278-M750, Q279-M750, I280-M750, K281-M750, D282-M750, Q283-M750, G284-M750, Y285-M750, G286-M750, Y287-M750, D288-M750, I289-M750, K290-M750, K291-M750, I292-M750, I293-M750, W294-M750, C295-M750, Q296-M750, D297-M750, S298-M750, L299-M750, K300-M750, P301-M750, E302-M750, M303-M750, I304-M750, L305-M750, T306-M750, S307-M750, D308-M750, K309-M750, R310-M750, I311-M750, V312-M750, K313-M750, I314-M750, W315-M750, D316-M750, H317-M750, T318-M750, N319-M750, G320-M750, K321-M750, S322-M750, F323-M750, A324-M750, S325-M750, M326-M750, E327-M750, P328-M750, T329-M750, V330-M750, D331-M750, I332-M750, N333-M750, D334-M750, I335-M750, C336-M750, H337-M750, I338-M750, P339-M750, Q340-M750, S341-M750, G342-M750, M343-M750, F344-M750, F345-M750, M346-M750, A347-M750, N348-M750, E349-M750, G350-M750, M351-M750, P352-M750, M353-M750, H354-M750, T355-M750, Y356-M750, Y357-M750, 1358-M750, P359-M750, N360-M750, L361-M750, G362-M750, S363-M750, A364-M750, P365-M750, N366-M750, W367-M750, C368-M750, S369-M750, F370-M750, L371-M750, D372-M750, N373-M750, V374-M750, T375-M750, E376-M750, E377-M750, L378-M750, E379-M750, E380-M750, K381-M750, P382-M750, S383-M750, N384-M750, S385-M750, I386-M750, Y387-M750, P388-M750, T389-M750, F390-M750, K391-M750, F392-M750, I393-M750, T394-M750, R395-M750, D396-M750, E397-M750, M398-M750, V399-M750, K400-M750, L401-M750, N402-M750, L403-M750, T404-M750, H405-M750, L406-M750, I407-M750, G408-M750, I409-M750, K410-M750, V411-M750, L412-M750, R413-M750, S414-M750, Y415-M750, M416-M750, H417-M750, G418-M750, F419-M750, F420-M750, I421-M750, N422-M750, T423-M750, E424-M750, L425-M750, Y426-M750, D427-M750, K428-M750, V429-M750, N430-M750, L431-M750, I432-M750, S433-M750, N434-M750, P435-M750, N436-M750, S437-M750, I438-M750, Y439-M750, D440-M750, Q441-M750, R442-M750, K443-M750, R444-M750, E445-M750, I446-M750, A447-M750, N448-M750, K449-M750, I450-M750, N451-M750, E452-M750, E453-M750, R454-M750, K455-M750, S456-M750, R457-M750, I458-M750, L459-M750, T460-M750, S461-M750, S462-M750, N463-M750, G464-M750, N465-M750, D466-M750, L467-M750, P468-M750, T469-M750, K470-M750, I471-M750, K472-M750, V473-M750, N474-M750, K475-M750, D476-M750, L477-M750, V478-M750, N479-M750, K480-M750, L481-M750, Q482-M750, T483-M750, K484-M750, F485-M750, A486-M750, E487-M750, N488-M750, G489-M750, T490-M750, P491-M750, D492-M750, G493-M750, N494-M750, A495-M750, N496-M750, G497-M750, A498-M750, T499-M750, D500-M750, Y501-M750, V502-M750, E503-M750, S504-M750, I505-M750, V506-M750, N507-M750, D508-M750, D509-M750, R510-M750, F511-M750, R512-M750, E513-M750, M514-M750, F515-M750, E516-M750, N517-M750, P518-M750, D519-M750, F520-M750, E521-M750, I522-M750, D523-M750, E524-M750, E525-M750, S526-M750, H527-M750, E528-M750, Y529-M750, K530-M750, Q531-M750, L532-M750, N533-M750, P534-M750, V535-M750, K536-M750, S537-M750, T538-M750, K539-M750, D540-M750, I541-M750, T542-M750, T543-M750, T544-M750, N545-M750, T546-M750, G547-M750, T548-M750, T549-M750, N550-M750, S551-M750, R552-M750, G553-M750, R554-M750, G555-M750, L556-M750, T557-M750, A558-M750, A559-M750, E560-M750, E561-M750, S562-M750, D563-M750, E564-M750, E565-M750, R566-M750, L567-M750, N568-M750, M569-M750, K570-M750, D571-M750, S572-M750, H573-M750, H574-M750, T575-M750, G576-M750, L577-M750, D578-M750, S579-M750, D580-M750, E581-M750, S582-M750, D583-M750, E584-M750, E585-M750, S586-M750, D587-M750, S588-M750, E589-M750, S590-M750, E591-M750, E592-M750, Q593-M750, S594-M750, E595-M750, D596-M750, E597-M750, A598-M750, K599-M750, S600-M750, A601-M750, E602-M750, T603-M750, R604-M750, E605-M750, R606-M750, V607-M750, G608-M750, K609-M750, E610-M750, L611-M750, N612-M750, K613-M750, I614-M750, R615-M750, Q616-M750, S617-M750, K618-M750, Q619-M750, K620-M750, Q621-M750, Q622-M750, Q623-M750, Q624-M750, D625-M750, S626-M750, K627-M750, K628-M750, F629-M750, Q630-M750, N631-M750, E632-M750, M633-M750, K634-M750, I635-M750, L636-M750, S637-M750, Q638-M750, Q639-M750, S640-M750, S641-M750, S642-M750, S643-M750, S644-M750, S645-M750, S646-M750, L647-M750, A648-M750, N649-M750, T650-M750, E651-M750, K652-M750, A653-M750, S654-M750, V655-M750, S656-M750, F657-M750, G658-M750, S659-M750, Q660-M750, V661-M750, N662-M750, K663-M750, L664-M750, N665-M750, K666-M750, I667-M750, S668-M750, K669-M750, Q670-M750, N671-M750, K672-M750, N673-M750, N674-M750, N675-M750, S676-M750, I677-M750, S678-M750, N679-M750, A680-M750, K681-M750, D682-M750, A683-M750, R684-M750, L685-M750, R686-M750, R687-M750, H688-M750, A689-M750, R690-M750, G691-M750, E692-M750, A693-M750, E694-M750, L695-M750, T696-M750, F697-M750, V698-M750, P699-M750, Q700-M750, K701-M750, S702-M750, K703-M750, S704-M750, K705-M750, S706-M750, T707-M750, K708-M750, L709-M750, K710-M750, F711-M750, N712-M750, N713-M750, N714-M750, H715-M750, S716-M750, D717-M750, D718-M750, E719-M750, K720-M750, L721-M750, D722-M750, S723-M750, G724-M750, K725-M750, T726-M750, K727-M750, D728-M750, S729-M750, G730-M750, R731-M750, T732-M750, K733-M750, Q734-M750, R735-M750, F736-M750, E737-M750, G738-M750, R739-M750, R740-M750, I741-M750, A742-M750, S743-M750, and/or K744-M750 of SEQ ID NO:17. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CaYGR145w deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CaYGR145w deletion polypeptides are encompassed by the present invention: M1-M750, M1-G749, M1-R748, M1-F747, M1-K746, M1-N745, M1-K744, M1-S743, M1-A742, M1-I741, M1-R740, M1-R739, M1-G738, M1-E737, M1-F736, M1-R735, M1-Q734, M1-K733, M1-T732, M1-R731, M1-G730, M1-S729, M1-D728, M1-K727, M1-T726, M1-K725, M1-G724, M1-S723, M1-D722, M1-L721, M1-K720, M1-E719, M1-D718, M1-D717, M1-S716, M1-H715, M1-N714, M1-N713, M1-N712, M1-F711, M1-K710, M1-L709, M1-K708, M1-T707, M1-S706, M1-K705, M1-S704, M1-K703, M1-S702, M1-K701, M1-Q700, M1-P699, M1-V698, M1-F697, M1-T696, M1-L695, M1-E694, M1-A693, M1-E692, M1-G691, M1-R690, M1-A689, M1-H688, M1-R687, M1-R686, M1-L685, M1-R684, M1-A683, M1-D682, M1-K681, M1-A680, M1-N679, M1-S678, M1-I677, M1-S676, M1-N675, M1-N674, M1-N673, M1-K672, M1-N671, M1-Q670, M1-K669, M1-S668, M1-I667, M1-K666, M1-N665, M1-L664, M1-K663, M1-N662, M1-V661, M1-Q660, M1-S659, M1-G658, M1-F657, M1-S656, M1-V655, M1-S654, M1-A653, M1-K652, M1-E651, M1-T650, M1-N649, M1-A648, M1-L647, M1-S646, M1-S645, M1-S644, M1-S643, M1-S642, M1-S641, M1-S640, M1-Q639, M1-Q638, M1-S637, M1-L636, M1-I635, M1-K634, M1-M633, M1-E632, M1-N631, M1-Q630, M1-F629, M1-K628, M1-K627, M1-S626, M1-D625, M1-Q624, M1-Q623, M1-Q622, M1-Q621, M1-K620, M1-Q619, M1-K618, M1-S617, M1-Q616, M1-R615, M1-I614, M1-K613, M1-N612, M1-L611, M1-E610, M1-K609, M1-G608, M1-V607, M1-R606, M1-E605, M1-R604, M1-T603, M1-E602, M1-A601, M1-S600, M1-K599, M1-A598, M1-E597, M1-D596, M1-E595, M1-S594, M1-Q593, M1-E592, M1-E591, M1-S590, M1-E589, M1-S588, M1-D587, M1-S586, M1-E585, M1-E584, M1-D583, M1-S582, M1-E581, M1-D580, M1-S579, M1-D578, M1-L577, M1-G576, M1-T575, M1-H574, M1-H573, M1-S572, M1-D571, M1-K570, M1-M569, M1-N568, M1-L567, M1-R566, M1-E565, M1-E564, M1-D563, M1-S562, M1-E561, M1-E560, M1-A559, M1-A558, M1-T557, M1-L556, M1-G555, M1-R554, M1-G553, M1-R552, M1-S551, M1-N550, M1-T549, M1-T548, M1-G547, M1-T546, M1-N545, M1-T544, M1-T543, M1-T542, M1-I541, M1-D540, M1-K539, M1-T538, M1-S537, M1-K536, M1-V535, M1-P534, M1-N533, M1-L532, M1-Q531, M1-K530, M1-Y529, M1-E528, M1-H527, M1-S526, M1-E525, M1-E524, M1-D523, M1-I522, M1-E521, M1-F520, M1-D519, M1-P518, M1-N517, M1-E516, M1-F515, M1-M514, M1-E513, M1-R512, M1-F511, M1-R510, M1-D509, M1-D508, M1-N507, M1-V506, M1-I505, M1-S504, M1-E503, M1-V502, M1-Y501, M1-D500, M1-T499, M1-A498, M1-G497, M1-N496, M1-A495, M1-N494, M1-G493, M1-D492, M1-P491, M1-T490, M1-G489, M1-N488, M1-E487, M1-A486, M1-F485, M1-K484, M1-T483, M1-Q482, M1-L481, M1-K480, M1-N479, M1-V478, M1-L477, M1-D476, M1-K475, M1-N474, M1-V473, M1-K472, M1-I471, M1-K470, M1-T469, M1-P468, M1-L467, M1-D466, M1-N465, M1-G464, M1-N463, M1-S462, M1-S461, M1-T460, M1-L459, M1-I458, M1-R457, M1-S456, M1-K455, M1-R454, M1-E453, M1-E452, M1-N451, M1-I450, M1-K449, M1-N448, M1-A447, M1-1446, M1-E445, M1-R444, M1-K443, M1-R442, M1-Q441, M1-D440, M1-Y439, M1-I438, M1-S437, M1-N436, M1-P435, M1-N434, M1-S433, M1-I432, M1-L431, M1-N430, M1-V429, M1-K428, M1-D427, M1-Y426, M1-L425, M1-E424, M1-T423, M1-N422, M1-I421, M1-F420, M1-F419, M1-G418, M1-H417, M1-M416, M1-Y415, M1-S414, M1-R413, M1-L412, M1-V411, M1-K410, M1-I409, M1-G408, M1-I407, M1-L406, M1-H405, M1-T404, M1-L403, M1-N402, M1-L401, M1-K400, M1-V399, M1-M398, M1-E397, M1-D396, M1-R395, M1-T394, M1-I393, M1-F392, M1-K391, M1-F390, M1-T389, M1-P388, M1-Y387, M1-I386, M1-S385, M1-N384, M1-S383, M1-P382, M1-K381, M1-E380, M1-E379, M1-L378, M1-E377, M1-E376, M1-T375, M1-V374, M1-N373, M1-D372, M1-L371, M1-F370, M1-S369, M1-C368, M1-W367, M1-N366, M1-P365, M1-A364, M1-S363, M1-G362, M1-L361, M1-N360, M1-P359, M1-I358, M1-Y357, M1-Y356, M1-T355, M1-H354, M1-M353, M1-P352, M1-M351, M1-G350, M1-E349, M1-N348, M1-A347, M1-M346, M1-F345, M1-F344, M1-M343, M1-G342, M1-S341, M1-Q340, M1-P339, M1-I338, M1-H337, M1-C336, M1-I335, M1-D334, M1-N333, M1-I332, M1-D331, M1-V330, M1-T329, M1-P328, M1-E327, M1-M326, M1-S325, M1-A324, M1-F323, M1-S322, M1-K321, M1-G320, M1-N319, M1-T318, M1-H317, M1-D316, M1-W315, M1-I314, M1-K313, M1-V312, M1-I311, M1-R310, M1-K309, M1-D308, M1-S307, M1-T306, M1-L305, M1-I304, M1-M303, M1-E302, M1-P301, M1-K300, M1-L299, M1-S298, M1-D297, M1-Q296, M1-C295, M1-W294, M1-I293, M1-I292, M1-K291, M1-K290, M1-I289, M1-D288, M1-Y287, M1-G286, M1-Y285, M1-G284, M1-Q283, M1-D282, M1-K281, M1-I280, M1-Q279, M1-Y278, M1-P277, M1-E276, M1-S275, M1-A274, M1-R273, M1-L272, M1-D271, M1-Y270, M1-L269, M1-L268, M1-T267, M1-Q266, M1-G265, M1-N264, M1-S263, M1-T262, M1-G261, M1-C260, M1-A259, M1-F258, M1-N257, M1-L256, M1-A255, M1-D254, M1-Q253, M1-P252, M1-R251, M1-F250, M1-A249, M1-L248, M1-S247, M1-T246, M1-I245, M1-G244, M1-C243, M1-S242, M1-S241, M1-Q240, M1-E239, M1-T238, M1-N237, M1-N236, M1-T235, M1-S234, M1-N233, M1-I232, M1-L231, M1-Q230, M1-D229, M1-S228, M1-V227, M1-F226, M1-L225, M1-K224, M1-G223, M1-A222, M1-R221, M1-Q220, M1-K219, M1-S218, M1-R217, M1-P216, M1-D215, M1-W214, M1-F213, M1-E212, M1-V211, M1-T210, M1-G209, M1-D208, M1-D207, M1-L206, M1-G205, M1-A204, M1-S203, M1-I202, M1-L201, M1-G200, M1-H199, M1-M198, M1-S197, M1-N196, M1-I195, M1-D194, M1-V193, M1-A192, M1-N191, M1-C190, M1-G189, M1-S188, M1-D187, M1-I186, M1-N185, M1-G184, M1-G183, M1-D182, M1-T181, M1-M180, M1-D179, M1-L178, M1-K177, M1-L176, M1-P175, M1-S174, M1-L173, M1-F172, M1-R171, M1-G170, M1-Q169, M1-D168, M1-L167, M1-N166, M1-L165, M1-R164, M1-Y163, M1-L162, M1-E161, M1-D160, M1-S159, M1-S158, M1-S157, M1-G156, M1-V155, M1-I154, M1-L153, M1-D152, M1-C151, M1-N150, M1-I149, M1-P148, M1-N147, M1-Y146, M1-T145, M1-L144, M1-C143, M1-R142, M1-G141, M1-F140, M1-K139, M1-P138, M1-I137, M1-R136, M1-T135, M1-R134, M1-Y133, M1-H132, M1-V131, M1-G130, M1-G129, M1-A128, M1-T127, M1-Q126, M1-F125, M1-E124, M1-I123, M1-S122, M1-R121, M1-D120, M1-C119, M1-Q118, M1-L117, M1-H116, M1-V115, M1-S114, M1-K113, M1-T112, M1-W111, M1-D110, M1-N109, M1-S108, M1-L107, M1-I106, M1-L105, M1-F104, M1-D103, M1-I102, M1-N101, M1-E100, M1-V99, M1-N98, M1-T97, M1-H96, M1-R95, M1-D94, M1-F93, M1-K92, M1-L91, M1-S90, M1-L89, M1-N88, M1-A87, M1-F86, M1-E85, M1-Y84, M1-V83, M1-H82, M1-I81, M1-Q80, M1-P79, M1-K78, M1-Y77, M1-T76, M1-G75, M1-T74, M1-A73, M1-M72, M1-C71, M1-Y70, M1-Q69, M1-G68, M1-D67, M1-N66, M1-T65, M1-V64, M1-K63, M1-I62, M1-K61, M1-N60, M1-S59, M1-A58, M1-E57, M1-S56, M1-F55, M1-E54, M1-F53, M1-D52, M1-Q51, M1-I50, M1-L49, M1-E48, M1-I47, M1-R46, M1-N45, M1-Q44, M1-Y43, M1-E42, M1-L41, M1-D40, M1-H39, M1-K38, M1-L37, M1-A36, M1-R35, M1-K34, M1-R33, M1-K32, M1-K31, M1-D30, M1-I29, M1-W28, M1-D27, M1-P26, M1-L25, M1-S24, M1-R23, M1-S22, M1-V21, M1-N20, M1-T19, M1-G18, M1-S17, M1-V16, M1-Q15, M1-Y14, M1-V13, M1-S12, M1-V11, M1-N10, M1-G9, M1-A8, and/or M1-T7 of SEQ ID NO:17. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CaYGR145w deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses polynucleotides sharing at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polynucleotide sequence provided in SEQ ID NO:6, and in particular to the coding region of the polynucleotide sequence provided in SEQ ID NO:6. Preferably such polynucleotides encode polypeptides that have biological activity.
The present invention also encompasses polypeptides sharing at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polypeptide sequence provided in SEQ ID NO:17.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:6 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 2236 of SEQ ID NO:6, b is an integer between 15 to 2250, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:6, and where b is greater than or equal to a+14.
Features of the Polypeptide Encoded by Polynucleotide No:7
The polynucleotide sequence (SEQ ID NO:7) and deduced amino acid sequence (SEQ ID NO:18) of the novel fungal essential gene, CaYDR412w (also referred to as FCG13), of the present invention. The CaYDR412w polypeptide (SEQ ID NO:18) is encoded by nucleotides 1 to 804 of SEQ ID NO:7 and has a predicted molecular weight of 31.3 kDa.
In preferred embodiments, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of CaYDR412w. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 4 thru 2250 of SEQ ID NO:7, and the polypeptide corresponding to amino acids 2 thru 268 of SEQ ID NO:18. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.
The invention also encompasses N- and/or C-terminal deletions of the CaYDR412w polypeptide of the present invention.
In preferred embodiments, the following N-terminal CaYDR412w deletion polypeptides are encompassed by the present invention: M1-K268, A2-K268, G3-K268, F4-K268, K5-K268, K6-K268, N7-K268, R8-K268, E9-K268, I10-K268, L11-K268, T12-K268, G13-K268, G14-K268, K15-K268, K16-K268, Y17-K268, I18-K268, Q19-K268, Q20-K268, K21-K268, Q22-K268, K23-K268, K24-K268, H25-K268, L26-K268, V27-K268, D28-K268, E29-K268, V30-K268, V31-K268, F32-K268, D33-K268, K34-K268, E35-K268, S36-K268, R37-K268, H38-K268, E39-K268, Y40-K268, L41-K268, T42-K268, G43-K268, F44-K268, H45-K268, K46-K268, R47-K268, K48-K268, L49-K268, Q50-K268, R51-K268, Q52-K268, K53-K268, K54-K268, A55-K268, Q56-K268, E57-K268, F58-K268, H59-K268, K60-K268, E61-K268, Q62-K268, E63-K268, R64-K268, L65-K268, A66-K268, K67-K268, I68-K268, E69-K268, E70-K268, R71-K268, K72-K268, Q73-K268, L74-K268, K75-K268, Q76-K268, E77-K268, R78-K268, E79-K268, R80-K268, D81-K268, L82-K268, Q83-K268, N84-K268, Q85-K268, L86-K268, Q87-K268, Q88-K268, F89-K268, K90-K268, K91-K268, T92-K268, A93-K268, Q94-K268, E95-K268, I96-K268, A97-K268, A98-K268, I99-K268, N100-K268, N101-K268, D102-K268, I103-K268, G104-K268, F105-K268, D106-K268, Q107-K268, S108-K268, D109-K268, D 110-K268, N111-K268, N112-K268, D113-K268, N114-K268, D115-K268, N116-K268, E117-K268, N118-K268, E119-K268, E120-K268, W121-K268, S122-K268, G123-K268, F124-K268, Q125-K268, E126-K268, D127-K268, E128-K268, E129-K268, G130-K268, E131-K268, G132-K268, E133-K268, E134-K268, V135-K268, T136-K268, D137-K268, E138-K268, D139-K268, D140-K268, E141-K268, D142-K268, K143-K268, E144-K268, K145-K268, P146-K268, L147-K268, K148-K268, G149-K268, I150-K268, L151-K268, H152-K268, H153-K268, T154-K268, E155-K268, I156-K268, Y157-K268, K158-K268, Q159-K268, D160-K268, P161-K268, S162-K268, L163-K268, S164-K268, N165-K268, I166-K268, T167-K268, N168-K268, N169-K268, G170-K268, A171-K268, I172-K268, I173-K268, D174-K268, D175-K268, E176-K268, T177-K268, T178-K268, V179-K268, V180-K268, V181-K268, E182-K268, S183-K268, L184-K268, D185-K268, N186-K268, P187-K268, N188-K268, A189-K268, V190-K268, D191-K268, T192-K268, E193-K268, E194-K268, K195-K268, L196-K268, Q197-K268, Q198-K268, L199-K268, A200-K268, K201-K268, L202-K268, N203-K268, N204-K268, V205-K268, N206-K268, L207-K268, D208-K268, K209-K268, S210-K268, D211-K268, Q212-K268, I213-K268, L214-K268, E215-K268, K216-K268, S217-K268, I218-K268, E219-K268, R220-K268, A221-K268, K222-K268, N223-K268, Y224-K268, A225-K268, V226-K268, I227-K268, C228-K268, G229-K268, V230-K268, A231-K268, K232-K268, P233-K268, N234-K268, P235-K268, I236-K268, K237-K268, Q238-K268, K239-K268, K240-K268, K241-K268, K242-K268, F243-K268, R244-K268, Y245-K268, L246-K268, T247-K268, K248-K268, A249-K268, E250-K268, R251-K268, R252-K268, E253-K268, N254-K268, V255-K268, R256-K268, K257-K268, E258-K268, K259-K268, S260-K268, K261-K268, and/or S262-K268 of SEQ ID NO:18. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CaYDR412w deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CaYDR412w deletion polypeptides are encompassed by the present invention: M1-K268, M1-K267, M1-G266, M1-K265, M1-S264, M1-K263, M1-S262, M1-K261, M1-S260, M1-K259, M1-E258, M1-K257, M1-R256, M1-V255, M1-N254, M1-E253, M1-R252, M1-R251, M1-E250, M1-A249, M1-K248, M1-T247, M1-L246, M1-Y245, M1-R244, M1-F243, M1-K242, M1-K241, M1-K240, M1-K239, M1-Q238, M1-K237, M1-I236, M1-P235, M1-N234, M1-P233, M1-K232, M1-A231, M1-V230, M1-G229, M1-C228, M1-I227, M1-V226, M1-A225, M1-Y224, M1-N223, M1-K222, M1-A221, M1-R220, M1-E219, M1-I218, M1-S217, M1-K216, M1-E215, M1-L214, M1-I213, M1-Q212, M1-D211, M1-S210, M1-K209, M1-D208, M1-L207, M1-N206, M1-V205, M1-N204, M1-N203, M1-L202, M1-K201, M1-A200, M1-L199, M1-Q198, M1-Q197, M1-L196, M1-K195, M1-E194, M1-E193, M1-T192, M1-D191, M1-V190, M1-A189, M1-N188, M1-P187, M1-N186, M1-D185, M1-L184, M1-S183, M1-E182, M1-V181, M1-V180, M1-V179, M1-T178, M1-T177, M1-E176, M1-D175, M1-D174, M1-I173, M1-I172, M1-A171, M1-G170, M1-N169, M1-N168, M1-T167, M1-I166, M1-N165, M1-S164, M1-L163, M1-S162, M1-P161, M1-D160, M1-Q159, M1-K158, M1-Y157, M1-I156, M1-E155, M1-T154, M1-H153, M1-H152, M1-L151, M1-I150, M1-G149, M1-K148, M1-L147, M1-P146, M1-K145, M1-E144, M1-K143, M1-D142, M1-E141, M1-D140, M1-D139, M1-E138, M1-D137, M1-T136, M1-V135, M1-E134, M1-E133, M1-G132, M1-E131, M1-G130, M1-E129, M1-E128, M1-D127, M1-E126, M1-Q125, M1-F124, M1-G123, M1-S122, M1-W121, M1-E120, M1-E119, M1-N118, M1-E117, M1-N116, M1-D115, M1-N114, M1-D113, M1-N112, M1-N111, M1-D110, M1-D109, M1-S108, M1-Q107, M1-D106, M1-F105, M1-G104, M1-I103, M1-D102, M1-N101, M1-N100, M1-I99, M1-A98, M1-A97, M1-I96, M1-E95, M1-Q94, M1-A93, M1-T92, M1-K91, M1-K90, M1-F89, M1-Q88, M1-Q87, M1-L86, M1-Q85, M1-N84, M1-Q83, M1-L82, M1-D81, M1-R80, M1-E79, M1-R78, M1-E77, M1-Q76, M1-K75, M1-L74, M1-Q73, M1-K72, M1-R71, M1-E70, M1-E69, M1-I68, M1-K67, M1-A66, M1-L65, M1-R64, M1-E63, M1-Q62, M1-E61, M1-K60, M1-H59, M1-F58, M1-E57, M1-Q56, M1-A55, M1-K54, M1-K53, M1-Q52, M1-R51, M1-Q50, M1-L49, M1-K48, M1-R47, M1-K46, M1-H45, M1-F44, M1-G43, M1-T42, M1-L41, M1-Y40, M1-E39, M1-H38, M1-R37, M1-S36, M1-E35, M1-K34, M1-D33, M1-F32, M1-V31, M1-V30, M1-E29, M1-D28, M1-V27, M1-L26, M1-H25, M1-K24, M1-K23, M1-Q22, M1-K21, M1-Q20, M1-Q19, M1-I18, M1-Y17, M1-K16, M1-K15, M1-G14, M1-G13, M1-T12, M1-L11, M1-I10, M1-E9, M1-R8, and/or M1-N7 of SEQ ID NO:18. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CaYDR412w deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses polynucleotides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polynucleotide sequence provided in SEQ ID NO:7, and in particular to the coding region of the polynucleotide sequence provided in SEQ ID NO:7. Preferably such polynucleotides encode polypeptides that have biological activity.
The present invention also encompasses polypeptides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polypeptide sequence provided in SEQ ID NO:18.
Most preferred are polypeptides that share at least about 99.4% identity with the polypeptide sequence provided in SEQ ID NO: 18.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:7 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 790 of SEQ ID NO:7, b is an integer between 15 to 804, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:7, and where b is greater than or equal to a+14.
Features of the Polypeptide Encoded by Polynucleotide No:8
The polynucleotide sequence (SEQ ID NO:8) and deduced amino acid sequence (SEQ ID NO:19) of the novel fungal essential gene, CaYOL010w (also referred to as FCG14), of the present invention. The CaYOL010w polypeptide (SEQ ID NO:19) is encoded by nucleotides 1 to 1113 of SEQ ID NO:8 and has a predicted molecular weight of 40.6 kDa.
In preferred embodiments, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of CaYOL010w. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 4 thru 1113 of SEQ ID NO:8, and the polypeptide corresponding to amino acids 2 thru 371 of SEQ ID NO:19. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.
The invention also encompasses N- and/or C-terminal deletions of the CaYOL010w polypeptide of the present invention.
In preferred embodiments, the following N-terminal CaYOL010w deletion polypeptides are encompassed by the present invention: M1-A371, S2-A371, S3-A371, V4-A371, A5-A371, S6-A371, K7-A371, K8-A371, I9-A371, I10-A371, T11-A371, F12-A371, E13-A371, G14-A371, H15-A371, R16-A371, N17-A371, F18-A371, R19-A371, L20-A371, R21-A371, L22-A371, V23-A371, L24-A371, A25-A371, T26-A371, L27-A371, S28-A371, G29-A371, K30-A371, P31-A371, I32-A371, K33-A371, I34-A371, T35-A371, K36-A371, I37-A371, R38-A371, S39-A371, Q40-A371, D41-A371, L42-A371, N43-A371, P44-A371, G45-A371, L46-A371, K47-A371, D48-A371, H49-A371, E50-A371, V51-A371, S52-A371, F53-A371, L54-A371, R55-A371, L56-A371, L57-A371, E58-A371, A59-A371, V60-A371, T61-A371, N62-A371, G63-A371, S64-A371, H65-A371, I66-A371, E67-A371, I68-A371, S69-A371, Y70-A371, T71-A371, G72-A371, T73-A371, T74-A371, I75-A371, I76-A371, Y77-A371, R78-A371, P79-A371, G80-A371, I81-A371, I82-A371, I83-A371, G84-A371, G85-A371, D86-A371, L87-A371, T88-A371, H89-A371, N90-A371, C91-A371, P92-A371, D93-A371, T94-A371, K95-A371, S96-A371, I97-A371, G98-A371, Y99-A371, F100-A371, I101-A371, E102-A371, P103-A371, M104-A371, L105-A371, M106-A371, F107-A371, P108-A371, L109-A371, F110-A371, S111-A371, K112-A371, K113-A371, K114-A371, F115-A371, S116-A371, I117-A371, I118-A371, F119-A371, K120-A371, G121-A371, L122-A371, T123-A371, N124-A371, I125-A371, A126-A371, G127-A371, N128-A371, D129-A371, T130-A371, G131-A371, V132-A371, D133-A371, A134-A371, I135-A371, K136-A371, W137-A371, G138-A371, L139-A371, L140-A371, P141-A371, V142-A371, M143-A371, E144-A371, K145-A371, F146-A371, G147-A371, V148-A371, R149-A371, E150-A371, V151-A371, S152-A371, L153-A371, H154-A371, I155-A371, L156-A371, K157-A371, R158-A371, G159-A371, S160-A371, A161-A371, P162-A371, L163-A371, G164-A371, G165-A371, G166-A371, E167-A371, V168-A371, H169-A371, L170-A371, L171-A371, C172-A371, S173-A371, S174-A371, L175-A371, I176-A371, P177-A371, Q178-A371, P179-A371, L180-A371, T181-A371, I182-A371, H183-A371, A184-A371, L185-A371, D186-A371, I187-A371, P188-A371, K189-A371, F190-A371, S191-A371, A192-A371, I193-A371, R194-A371, G195-A371, V196-A371, A197-A371, Y198-A371, C199-A371, T200-A371, R201-A371, V202-A371, S203-A371, P204-A371, S205-A371, I206-A371, V207-A371, N208-A371, R209-A371, M210-A371, I211-A371, D212-A371, S213-A371, A214-A371, R215-A371, A216-A371, V217-A371, L218-A371, K219-A371, P220-A371, T221-A371, G222-A371, C223-A371, E224-A371, V225-A371, N226-A371, I227-A371, T228-A371, A229-A371, D230-A371, V231-A371, W232-A371, R233-A371, G234-A371, E235-A371, N236-A371, S237-A371, G238-A371, K239-A371, S240-A371, P241-A371, G242-A371, F243-A371, G244-A371, I245-A371, T246-A371, L247-A371, V248-A371, A249-A371, E250-A371, L251-A371, K252-A371, R253-A371, G254-A371, W255-A371, R256-A371, I257-A371, V258-A371, T259-A371, E260-A371, N261-A371, V262-A371, G263-A371, S264-A371, A265-A371, G266-A371, S267-A371, L268-A371, P269-A371, E270-A371, D271-A371, S272-A371, G273-A371, E274-A371, L275-A371, T276-A371, A277-A371, Y278-A371, Q279-A371, L280-A371, L281-A371, E282-A371, E283-A371, I284-A371, S285-A371, N286-A371, S287-A371, G288-A371, V289-A371, V290-A371, G291-A371, R292-A371, Y293-A371, Q294-A371, L295-A371, P296-A371, L297-A371, A298-A371, L299-A371, V300-A371, Y301-A371, M302-A371, T303-A371, I304-A371, G305-A371, K306-A371, E307-A371, D308-A371, I309-A371, G310-A371, R311-A371, L312-A371, K313-A371, L314-A371, Q315-A371, K316-A371, S317-A371, E318-A371, I319-A371, D320-A371, E321-A371, N322-A371, L323-A371, V324-A371, S325-A371, V326-A371, L327-A371, R328-A371, D329-A371, I330-A371, Q331-A371, E332-A371, V333-A371, F334-A371, G335-A371, T336-A371, E337-A371, A338-A371, F339-A371, F340-A371, K341-A371, D342-A371, D343-A371, A344-A371, E345-A371, E346-A371, L347-A371, D348-A371, S349-A371, D350-A371, D351-A371, K352-A371, F353-A371, M354-A371, T355-A371, V356-A371, S357-A371, I358-A371, K359-A371, G360-A371, V361-A371, G362-A371, F363-A371, T364-A371, and/or N365-A371 of SEQ ID NO:19. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CaYOL010w deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CaYOL010w deletion polypeptides are encompassed by the present invention: M1-A371, M1-I370, M1-K369, M1-K368, M1-S367, M1-V366, M1-N365, M1-T364, M1-F363, M1-G362, M1-V361, M1-G360, M1-K359, M1-I358, M1-S357, M1-V356, M1-T355, M1-M354, M1-F353, M1-K352, M1-D351, M1-D350, M1-S349, M1-D348, M1-L347, M1-E346, M1-E345, M1-A344, M1-D343, M1-D342, M1-K341, M1-F340, M1-F339, M1-A338, M1-E337, M1-T336, M1-G335, M1-F334, M1-V333, M1-E332, M1-Q331, M1-I330, M1-D329, M1-R328, M1-L327, M1-V326, M1-S325, M1-V324, M1-L323, M1-N322, M1-E321, M1-D320, M1-I319, M1-E318, M1-S317, M1-K316, M1-Q315, M1-L314, M1-K313, M1-L312, M1-R311, M1-G310, M1-I309, M1-D308, M1-E307, M1-K306, M1-G305, M1-I304, M1-T303, M1-M302, M1-Y301, M1-V300, M1-L299, M1-A298, M1-L297, M1-P296, M1-L295, M1-Q294, M1-Y293, M1-R292, M1-G291, M1-V290, M1-V289, M1-G288, M1-S287, M1-N286, M1-S285, M1-I284, M1-E283, M1-E282, M1-L281, M1-L280, M1-Q279, M1-Y278, M1-A277, M1-T276, M1-L275, M1-E274, M1-G273, M1-S272, M1-D271, M1-E270, M1-P269, M1-L268, M1-S267, M1-G266, M1-A265, M1-S264, M1-G263, M1-V262, M1-N261, M1-E260, M1-T259, M1-V258, M1-I257, M1-R256, M1-W255, M1-G254, M1-R253, M1-K252, M1-L251, M1-E250, M1-A249, M1-V248, M1-L247, M1-T246, M1-I245, M1-G244, M1-F243, M1-G242, M1-P241, M1-S240, M1-K239, M1-G238, M1-S237, M1-N236, M1-E235, M1-G234, M1-R233, M1-W232, M1-V231, M1-D230, M1-A229, M1-T228, M1-I227, M1-N226, M1-V225, M1-E224, M1-C223, M1-G222, M1-T221, M1-P220, M1-K219, M1-L218, M1-V217, M1-A216, M1-R215, M1-A214, M1-S213, M1-D212, M1-I211, M1-M210, M1-R209, M1-N208, M1-V207, M1-I206, M1-S205, M1-P204, M1-S203, M1-V202, M1-R201, M1-T200, M1-C199, M1-Y198, M1-A197, M1-V196, M1-G195, M1-R194, M1-I193, M1-A192, M1-S191, M1-F190, M1-K189, M1-P188, M1-I187, M1-D186, M1-L185, M1-A184, M1-H183, M1-I182, M1-T181, M1-L180, M1-P179, M1-Q178, M1-P177, M1-I176, M1-L175, M1-S174, M1-S173, M1-C172, M1-L171, M1-L170, M1-H169, M1-V168, M1-E167, M1-G166, M1-G165, M1-G164, M1-L163, M1-P162, M1-A161, M1-S160, M1-G159, M1-R158, M1-K157, M1-L156, M1-I155, M1-H154, M1-L153, M1-S152, M1-V151, M1-E150, M1-R149, M1-V148, M1-G147, M1-F146, M1-K145, M1-E144, M1-M143, M1-V142, M1-P141, M1-L140, M1-L139, M1-G138, M1-W137, M1-K136, M1-I135, M1-A134, M1-D133, M1-V132, M1-G131, M1-T130, M1-D129, M1-N128, M1-G127, M1-A126, M1-I125, M1-N124, M1-T123, M1-L122, M1-G121, M1-K120, M1-F119, M1-I118, M1-I117, M1-S116, M1-F115, M1-K114, M1-K113, M1-K112, M1-S111, M1-F110, M1-L109, M1-P108, M1-F107, M1-M106, M1-L105, M1-M104, M1-P103, M1-E102, M1-I101, M1-F100, M1-Y99, M1-G98, M1-I97, M1-S96, M1-K95, M1-T94, M1-D93, M1-P92, M1-C91, M1-N90, M1-H89, M1-T88, M1-L87, M1-D86, M1-G85, M1-G84, M1-I83, M1-I82, M1-I81, M1-G80, M1-P79, M1-R78, M1-Y77, M1-I76, M1-I75, M1-T74, M1-T73, M1-G72, M1-T71, M1-Y70, M1-S69, M1-168, M1-E67, M1-I66, M1-H65, M1-S64, M1-G63, M1-N62, M1-T61, M1-V60, M1-A59, M1-E58, M1-L57, M1-L56, M1-R55, M1-L54, M1-F53, M1-S52, M1-V51, M1-E50, M1-H49, M1-D48, M1-K47, M1-L46, M1-G45, M1-P44, M1-N43, M1-L42, M1-D41, M1-Q40, M1-S39, M1-R38, M1-I37, M1-K36, M1-T35, M1-I34, M1-K33, M1-I32, M1-P31, M1-K30, M1-G29, M1-S28, M1-L27, M1-T26, M1-A25, M1-L24, M1-V23, M1-L22, M1-R21, M1-L20, M1-R19, M1-F18, M1-N17, M1-R16, M1-H15, M1-G14, M1-E13, M1-F12, M1-T11, M1-I10, M1-I9, M1-K8, and/or M1-K7 of SEQ ID NO:19. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CaYOL010w deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses polynucleotides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polynucleotide sequence provided in SEQ ID NO:8, and in particular to the coding region of the polynucleotide sequence provided in SEQ ID NO:8. Preferably such polynucleotides encode polypeptides that have biological activity, particularly RNA 3′-terminal phosphate cyclase activity.
Most preferred are polynucleotides that share at least about 99.5% identity with the polynucleotide sequence provided in SEQ ID NO:8.
The present invention also encompasses polypeptides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polypeptide sequence provided in SEQ ID NO:19.
Most preferred are polypeptides that share at least about 99.0% identity with the polypeptide sequence provided in SEQ ID NO:19.
The present invention is also directed to a homology model detailing the three-dimensional structure of the CaYOL010w polypeptide (SEQ ID NO:19) of the present invention.
Protein threading and molecular modeling of CaYOL010w suggest that CaYOL010w has a three dimensional fold similar to that of the RNA 3′-terminal phosphate cyclase from E. coli (Palm et. al., 1999), Protein Data Bank (PDB, Bernstein et. al., 1977 & Berman et. al., 2000) (Protein Data Bank entry 1QMH; Genbank Accession No. gi|12644279; SEQ ID NO:253). Based on sequence, structure, motifs and known cyclase signature sequences, CaYOL010w contains a novel RNA cyclase domain.
The polypeptide CaYOL010w contains a distinct structural domain similar to the RNA cyclase domains that are a family of RNA modifying enzymes conserved in eucarya, bacteria and archaea.
The Three Dimensional Crystallographic Structure for Escherichia coli 
RNA 3′-terminal phosphate cyclase has been reported and was deposited into the Protein Data Bank (Palm et. al., 1999, Bernstein et. al., 1977, Berman et. al., 2000).
The structure (Protein Data Bank, PDB entry 1QMH) of RNA 3′-terminal phosphate cyclase Escherichia coli is a novel fold that consists on known structural elements connected in a unique manner. The structure of cyclase consists of two structurally distinct domains. The larger domain is composed of the N-terminal 184 amino acids (1-184) and the C-terminal 59 amino acids (280-339). The domain contains three repeats of a folding unit comprising two α-helices and a four-stranded β-sheet. The smaller domain of the cyclase, residues 185-279, comprises the same 4 stranded sheet covered by two α-helices but the connection topology is different. The active site of the of RNA 3′-terminal phosphate cyclase contains a histidine (H309) at the active site that was identified by labeling and lysines in the active site have been reported as part of the catalytic mechanism of nucleotidyl transferases including cyclases. The H309 lies at the bottom of a deep cleft surrounded by 5 loops containing conserved residues (from across the orthologs of cyclases). A RNA 3′-terminal phosphate cyclase signature sequence is found in the N-terminal region of the larger domain. This motif H/R-G-X-P-G-G-G-X-V (SEQ ID NO:256) is similar to the glycine rich loops known to contact ATP, GTP and other nucleotides at binding sites in other enzymes. The histidine or arginine residue is thought to bind to the nucleotide. For the E. coli RNA 3′-terminal phosphate cyclase H158 through V168 comprises the cyclase functional signature.
This structure-based information and sequence information from novel genes can be used to identify other protein family members that share this same fold.
The CaYOL010w three dimensional model provides for a specific description of the distinct domain and functional/active sites in the RNA 3′-terminal phosphate cyclase, CaYOL010w polypeptide.
The structural domain and functional/active sites are defined by atomic coordinates (Table 10). Based on this data, the inventors have ascribed the CaYOL010w polypeptide as having RNA cyclase activity(s) and cellular and systemic regulatory function(s).
The invention also relates to in silico screening methods including in silico docking and methods of structure based drug design which utilize the three dimensional coordinates of CaYOL010w (Table 10). Also provided are methods of identifying modulators of CaYOL010w that include modulator building or searching utilizing computer programs and algorithms. In an embodiment of the invention a method is provided for designing potential modulators of CaYOL010w comprising any combination of steps which utilize said three dimensional structure to design or select potential modulators.
Homology models are useful when there is no experimental information available on the protein of interest. A three dimensional model can be constructed on the basis of the known structure of a homologous protein (Greer et. al., 1991, Lesk, et. al., 1992, Levitt, 1992, Cardozo, et. al., 1995, Sali, et. al., 1995).
Those of skill in the art will understand that a homology model is constructed on the basis of first identifying a template, or, protein of known structure which is similar to the protein without known structure. This can be accomplished by through pairwise alignment of sequences using such programs as FASTA (Pearson, et. al. 1990) and BLAST (Altschul, et. al., 1990). In cases where sequence similarity is high (greater than 30%) these pairwise comparison methods may be adequate. Likewise, multiple sequence alignments or profile-based methods can be used to align a query sequence to an alignment of multiple (structurally and biochemically) related proteins. When the sequence similarity is low, more advanced techniques are used such as fold recognition (protein threading; Hendlich, et. al., 1990, Koppensteiner et. al. 2000, Sippl & Weitckus 1992, Sippl 1993), where the compatibility of a particular sequence with the three dimensional fold of a potential template protein is gauged on the basis of a knowledge-based potential. Following the initial sequence alignment, the query template can be optimally aligned by manual manipulation or by incorporation of other features (motifs, secondary structure predictions, and allowed sequence conservation). Next, structurally conserved regions can be identified and are used to construct the core secondary structure (Levitt, 1992, Sali, et. al., 1995) elements in the three dimensional model. Variable regions, called “unconserved regions” and loops can be added using knowledge-based techniques. The complete model with variable regions and loops can be refined performing forcefield calculations (Sali, et. al., 1995, Cardozo, et. al., 1995).
For CaYOL010w a pairwise alignment generated by protein threading (Hendlich, et. al., 1990, Koppensteiner et. al. 2000, Sippl & Weitckus 1992, Sippl 1993) was used to align the sequence of CaYOL010w with the sequence the RNA 3′-terminal phosphate cyclase, Escherichia coli (Palm et. al., 1999), (Protein Data Bank code 1QMH). The alignment of CaYOL010w with PDB entry 1QMH chain A is set forth in FIG. 32. In this invention, the homology model of CaYOL010w was derived from the sequence alignment set forth in FIG. 32. An overall atomic model including plausible sidechain orientations was generated using the program LOOK (Levitt, 1992). The three dimensional model for CaYOL010w is defined by the set of structure coordinates as set forth in Table 10 and is shown in FIG. 33 rendered by backbone secondary structures.
In order to recognize errors in three-dimensional structures knowledge based mean fields can be used to judge the quality of protein folds (Sippl 1993). The methods can be used to recognize misfolded structures as well as faulty parts of structural models. The technique generates an energy graph where the energy distribution for a given protein fold is displayed on the y-axis and residue position in the protein fold is displayed on the x-axis. The knowledge based mean fields compose a force field derived from a set of globular protein structures taken as a subset from the Protein Data Bank (Bernstein et. al. 1977). To analyze the quality of a model the energy distribution is plotted and compared to the energy distribution of the template from which the model was generated. FIG. 34 shows the energy graph for the CaYOL010w model (dotted line) and the template (RNA 3′-terminal phosphate cyclase) from which the model was generated. The model has virtually an identical energy plot when compared to RNA 3′-terminal phosphate cyclase template demonstrating that CaYOL010w has similar structural characteristics and suggest the overall three-dimensional fold is “native-like”. This graph supports the motif and sequence alignments in confirming that the three dimensional structure coordinates of CaYOL010w are an accurate and useful representation for the polypeptide.
The term “structure coordinates” refers to Cartesian coordinates generated from the building of a homology model.
Those of skill in the art will understand that a set of structure coordinates for a protein is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates, as emanate from generation of similar homology models using different alignment templates (i.e., other than the structure coordinates of 1QMH), and/or using different methods in generating the homology model, will have minor effects on the overall shape. Variations in coordinates may also be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in Table 10 could be manipulated by fractionalization of the structure coordinates; integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
Various computational analyses are therefore necessary to determine whether a molecule or a portion thereof is sufficiently similar to all or parts of CaYOL010w described above as to be considered the same. Such analyses may be carried out in current software applications, such as INSIGHTII (Accelrys Inc., San Diego, Calif.) version 2000 as described in the User's Guide, online or software applications available in the SYBYL software suite (Tripos Inc., St. Louis, Mo.).
Using the superimposition tool in the program INSIGHTII comparisons can be made between different structures and different conformations of the same structure. The procedure used in INSIGHTII to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); the second structure (i.e., moving structure) is identified as the source structure. Since atom equivalency within INSIGHTII is defined by user input, for the purpose of this invention we will define equivalent atoms as protein backbone atoms (N, Cα, C and O) for all conserved residues between the two structures being compared. We will also consider only rigid fitting operations. When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by INSIGHTII.
For the purpose of this invention, any homology model of a CaYOL010w that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) of less than 2.0 A when superimposed on the relevant backbone atoms described by structure coordinates listed in Table 10 are considered identical. More preferably, the root mean square deviation is less than 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 Angstroms.
The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the “root mean square deviation” defines the variation in the backbone of a protein from the relevant portion of the backbone of CaYOL010w as defined by the structure coordinates described herein.
This invention as embodied by the three-dimensional model enables the structure-based design of modulators of the biological function of CaYOL010w, as well as mutants with altered biological function and/or specificity.
The sequence alignment (FIG. 32) used as a template for creating the three-dimensional model of CaYOL010w RNA 3′-terminal phosphate cyclase domain shows 24% sequence identity between catalytic domain of CaYOL010w and yeast RNA 3′-terminal phosphate cyclase, PDB code 1QMH. For the RNA 3′-terminal phosphate cyclases there are at least two functional regions that are critical. In the N-terminal region of the enzyme the cyclase signature motif is thought to contain the nucleotide binding site has been shown to be highly conserved among cyclases. FIG. 32 shows this region highlighted by (*) and corresponds to R158-V168 in the three dimensional model for YOL010w (Table 10). The nucleotide binding site and surrounding sequence is completely conserved at the sequence and structure level. The second functional site is the region corresponding to RNA 3′-terminal phosphate cyclase H309. FIG. 32 shows that this region is not conserved in the model but there are several lysines and arginines nearby in the sequence alignment that suggest that this protein utilizes a basic residue like the DNA or RNA ligases. The nucleotidyl group is transferred and a covalent lysyl-NMP intermediate is formed. The conservation of the amino acids in the functional sites and the overall 24% sequence identity emphasize the significance of the three-dimensional model. The conserved residues are located in the functional sites at the cyclase signature sequence which is the presumed nucleotide binding site and region of the cyclase active site presenting a well structured catalytic domain. These functional site residues play critical roles in the mechanism of catalysis, substrate specificity and RNA processing and modification.
The structure coordinates of a CaYOL010w homology model, and portions thereof, are stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery and target prioritization and validation.
Accordingly, in one embodiment of this invention is provided a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Table 10.
For the first time, the present invention permits the use, through homology modeling based upon the sequence of CaYOL010w (FIGS. 18 and 33) of structure-based or rational drug design techniques to design, select, and synthesizes chemical entities that are capable of modulating the biological function of CaYOL010w. Comparison of the CaYOL010w homology model with the structures of other the RNA cyclases enable the use of rational or structure based drug design methods to design, select or synthesize specific chemical modulators of CaYOL010w.
Accordingly, the present invention is also directed to the entire sequence in FIG. 18 or any portion thereof for the purpose of generating a homology model for the purpose of three dimensional structure-based drug designs.
For purposes of this invention, we include mutants or homologues of the sequence in FIG. 18 or any portion thereof. In a preferred embodiment, the mutants or homologues have at least 25% identity, more preferably 50% identity, more preferably 75% identity, and most preferably 90% identity to the amino acid residues in FIG. 18.
The three-dimensional model structure of the CaYOL010w will also provide methods for identifying modulators of biological function. Various methods or combination thereof can be used to identify these compounds.
Structure coordinates of the active site region defined above can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential CaYOL010w modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic interactions, and dipole interaction. Alternatively, or in conjunction, the three-dimensional structural model can be employed to design or select compounds as potential CaYOL010w modulators. Compounds identified as potential CaYOL010w modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the CaYOL010w, or in characterizing CaYOL010w deactivation in the presence of a small molecule. Examples of assays useful in screening of potential CaYOL010w modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids from CaYOL010w according to Table 10.
However, as will be understood by those of skill in the art upon this disclosure, other structure based design methods can be used. Various computational structure based design methods have been disclosed in the art.
For example, a number of computer modeling systems are available in which the sequence of the CaYOL010w and the CaYOL010w structure (i.e., atomic coordinates of CaYOL010w and/or the atomic coordinates of the active site region as provided in Table 10) can be input. The computer system then generates the structural details of one or more these regions in which a potential CaYOL010w modulator binds so that complementary structural details of the potential modulators can be determined. Design in these modeling systems is generally based upon the compound being capable of physically and structurally associating with CaYOL010w. In addition, the compound must be able to assume a conformation that allows it to associate with CaYOL010w. Some modeling systems estimate the potential inhibitory or binding effect of a potential CaYOL010w modulator prior to actual synthesis and testing.
Methods for screening chemical entities or fragments for their ability to associate with a given protein target are well known. Often these methods begin by visual inspection of the binding site on the computer screen. Selected fragments or chemical entities are then positioned in one or more positions and orientations within the active site region in CaYOL010w. Molecular docking is accomplished using software such as INSIGHTII, ICM (Molsoft LLC, La Jolla, Calif.), and SYBYL, following by energy minimization and molecular dynamics with standard molecular mechanic forcefields such as CHARMM and MMFF. Examples of computer programs which assist in the selection of chemical fragment or chemical entities useful in the present invention include, but are not limited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntz et. al. 1982).
Alternatively, compounds may be designed de novo using either an empty active site or optionally including some portion of a known inhibitor. Methods of this type of design include, but are not limited to LUDI (Bohm 1992), LeapFrog (Tripos Associates, St. Louis Mo.) and DOCK (Kuntz et. al., 1982). Programs such as DOCK (Kuntz et. al. 1982) can be used with the atomic coordinates from the homology model to identify potential ligands from databases or virtual databases which potentially bind the in the active site region, and which may therefore be suitable candidates for synthesis and testing. The computer programs may utilize a combination of the following steps:
1) Selection of fragments or chemical entities from a database and then positioning the chemical entity in one or more orientations within the CaYOL010w catalytic domain defined by Table 10.
2) Characterization of the structural and chemical features of the chemical entity and active site including van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interactions
3) Search databases for molecular fragments which can be joined to or replace the docked chemical entity and spatially fit into regions defined by the said CaYOL010w catalytic domain or catalytic domain functional sites
4) Evaluate the docked chemical entity and fragments using a combination of scoring schemes which account for van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic interactions
Databases that may be used include ACD (Molecular Designs Limited), Aldrich (Aldrich Chemical Company), NCI (National Cancer Institute), Maybridge (Maybridge Chemical Company Ltd), CCDC (Cambridge Crystallographic Data Center), CAST (Chemical Abstract Service), Derwent (Derwent Information Limited).
Upon selection of preferred chemical entities or fragments, their relationship to each other and CaYOL010w can be visualized and then assembled into a single potential modulator. Programs useful in assembling the individual chemical entities include, but are not limited to SYBYL and LeapFrog (Tripos Associates, St. Louis Mo.), LUDI (Bohm 1992) as well as 3D Database systems (Martin 1992).
Additionally, the three-dimensional homology model of CaYOL010w will aid in the design of mutants with altered biological activity. Site directed mutagenesis can be used to generate proteins with similar or varying degrees of biological activity compared to native CaYOL010w. This invention also relates to the generation of mutants or homologs of CaYOL010w. It is clear that molecular modeling using the three dimensional structure coordinates set forth in Table 10 and visualization of the CaYOL010w model, FIG. 34 can be utilized to design homologs or mutant polypeptides of CaYOL010w that have similar or altered biological activities, function or reactivities.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:8 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1099 of SEQ ID NO:8, b is an integer between 15 to 1113, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:8, and where b is greater than or equal to a+14.
Features of the Polypeptide Encoded by Polynucleotide No:9
The polynucleotide sequence (SEQ ID NO:9) and deduced amino acid sequence (SEQ ID NO:20) of the novel fungal essential gene, CaYOR004w (also referred to as FCG 15), of the present invention. The CaYOR004w polypeptide (SEQ ID NO:20) is encoded by nucleotides 1 to 771 of SEQ ID NO:9 and has a predicted molecular weight of 29.5 kDa.
In preferred embodiments, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of CaYOR004w. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 4 thru 771 of SEQ ID NO:9, and the polypeptide corresponding to amino acids 2 thru 257 of SEQ ID NO:20. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.
The invention also encompasses N- and/or C-terminal deletions of the CaYOR004w polypeptide of the present invention.
In preferred embodiments, the following N-terminal CaYOR004w deletion polypeptides are encompassed by the present invention: M1-N257, R2-N257, Q3-N257, K4-N257, R5-N257, A6-N257, K7-N257, A8-N257, Y9-N257, K10-N257, K11-N257, Q12-N257, M13-N257, S14-N257, V15-N257, Y16-N257, V17-N257, H18-N257, A19-N257, F20-N257, K21-N257, F22-N257, R23-N257, E24-N257, P25-N257, Y26-N257, Q27-N257, I28-N257, I29-N257, V30-N257, D31-N257, N32-N257, E33-N257, L34-N257, I35-N257, T36-N257, T37-N257, C38-N257, Q39-N257, S40-N257, A41-N257, S42-N257, F43-N257, D44-N257, I45-N257, N46-N257, K47-N257, G48-N257, F49-N257, T50-N257, R51-N257, T52-N257, I53-N257, Q54-N257, A55-N257, E56-N257, N57-N257, K58-N257, P59-N257, M60-N257, I61-N257, T62-N257, Q63-N257, C64-N257, C65-N257, I66-N257, Q67-N257, A68-N257, L69-N257, Y70-N257, D71-N257, T72-N257, K73-N257, N74-N257, Q75-N257, P76-N257, A77-N257, I78-N257, D79-N257, I80-N257, A81-N257, K82-N257, S83-N257, F84-N257, E85-N257, R86-N257, R87-N257, K88-N257, C89-N257, N90-N257, H91-N257, R92-N257, E93-N257, A94-N257, I95-N257, D96-N257, P97-N257, S98-N257, Q99-N257, C100-N257, I101-N257, E102-N257, S103-N257, I104-N257, V105-N257, N106-N257, I107-N257, K108-N257, G109-N257, Q110-N257, N111-N257, K112-N257, H113-N257, R114-N257, Y115-N257, I116-N257, V117-N257, A118-N257, S119-N257, Q120-N257, D121-N257, L122-N257, Q123-N257, L124-N257, R125-N257, K126-N257, K127-N257, L128-N257, R129-N257, K130-N257, I131-N257, P132-N257, G133-N257, V134-N257, P135-N257, L136-N257, I137-N257, Y138-N257, M139-N257, N140-N257, R141-N257, S142-N257, V143-N257, M144-N257 V145-N257, M146-N257, E147-N257, P148-N257, I149-N257, S150-N257, D151-N257, V152-N257, S153-N257, N154-N257, Q155-N257, Y156-N257, N157-N257, M158-N257, N159-N257, Y160-N257, E161-N257, S162-N257, K163-N257, K164-N257, L165-N257, T166-N257, G167-N257, G168-N257, L169-N257, N170-N257, D171-N257, I172-N257, E173-N257, A174-N257, G175-N257, K176-N257, L177-N257, E178-N257, K179-N257, Q180-N257, N181-N257, E182-N257, G183-N257, E184-N257, D185-N257, G186-N257, D187-N257, G188-N257, D189-N257, E190-N257, L191-N257, E192-N257, V193-N257, K194-N257, K195-N257, K196-N257, K197-N257, R198-N257, K199-N257, G200-N257, P201-N257, K202-N257, E203-N257, P204-N257, N205-N257, P206-N257, L207-N257, S208-N257, V209-N257, K210-N257, K211-N257, K212-N257, K213-N257, T214-N257, D215-N257, N216-N257, A217-N257, T218-N257, A219-N257, A220-N257, S221-N257, T222-N257, N223-N257, Q224-N257, E225-N257, Q226-N257, K227-N257, K228-N257, K229-N257, P230-N257, N231-N257, R232-N257, R233-N257, K234-N257, R235-N257, H236-N257, A237-N257, Q238-N257, V239-N257, K240-N257, S241-N257, R242-N257, R243-N257, E244-N257, G245-N257, R246-N257, P247-N257, R248-N257, T249-N257, G250-N257, and/or A251-N257 of SEQ ID NO:20. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CaYOR004w deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CaYOR004w deletion polypeptides are encompassed by the present invention: M1-N257, M1-N256, M1-S255, M1-R254, M1-E253, M1-S252, M1-A251, M1-G250, M1-T249, M1-R248, M1-P247, M1-R246, M1-G245, M1-E244, M1-R243, M1-R242, M1-S241, M1-K240, M1-V239, M1-Q238, M1-A237, M1-H236, M1-R235, M1-K234, M1-R233, M1-R232, M1-N231, M1-P230, M1-K229, M1-K228, M1-K227, M1-Q226, M1-E225, M1-Q224, M1-N223, M1-T222, M1-S221, M1-A220, M1-A219, M1-T218, M1-A217, M1-N216, M1-D215, M1-T214, M1-K213, M1-K212, M1-K211, M1-K210, M1-V209, M1-S208, M1-L207, M1-P206, M1-N205, M1-P204, M1-E203, M1-K202, M1-P201, M1-G200, M1-K199, M1-R198, M1-K197, M1-K196, M1-K195, M1-K194, M1-V193, M1-E192, M1-L191, M1-E190, M1-D189, M1-G188, M1-D187, M1-G186, M1-D185, M1-E184, M1-G183, M1-E182, M1-N181, M1-Q180, M1-K179, M1-E178, M1-L177, M1-K176, M1-G175, M1-A174, M1-E173, M1-I172, M1-D171, M1-N170, M1-L169, M1-G168, M1-G167, M1-T166, M1-L165, M1-K164, M1-K163, M1-S162, M1-E161, M1-Y160, M1-N159, M1-M158, M1-N157, M1-Y156, M1-Q155, M1-N154, M1-S153, M1-V152, M1-D151, M1-S150, M1-I149, M1-P148, M1-E147, M1-M146, M1-V145, M1-M144, M1-V143, M1-S142, M1-R141, M1-N140, M1-M139, M1-Y138, M1-I137, M1-L136, M1-P135, M1-V134, M1-G133, M1-P132, M1-I131, M1-K130, M1-R129, M1-L128, M1-K127, M1-K126, M1-R125, M1-L124, M1-Q123, M1-L122, M1-D121, M1-Q120, M1-S119, M1-A118, M1-V117, M1-I116, M1-Y115, M1-R114, M1-H113, M1-K112, M1-N111, M1-Q110, M1-G109, M1-K108, M1-I107, M1-N106, M1-V105, M1-I104, M1-S103, M1-E102, M1-I101, M1-C100, M1-Q99, M1-S98, M1-P97, M1-D96, M1-I95, M1-A94, M1-E93, M1-R92, M1-H91, M1-N90, M1-C89, M1-K88, M1-R87, M1-R86, M1-E85, M1-F84, M1-S83, M1-K82, M1-A81, M1-I80, M1-D79, M1-I78, M1-A77, M1-P76, M1-Q75, M1-N74, M1-K73, M1-T72, M1-D71, M1-Y70, M1-L69, M1-A68, M1-Q67, M1-I66, M1-C65, M1-C64, M1-Q63, M1-T62, M1-I61, M1-M60, M1-P59, M1-K58, M1-N57, M1-E56, M1-A55, M1-Q54, M1-I53, M1-T52, M1-R51, M1-T50, M1-F49, M1-G48, M1-K47, M1-N46, M1-I45, M1-D44, M1-F43, M1-S42, M1-A41, M1-S40, M1-Q39, M1-C38, M1-T37, M1-T36, M1-I35, M1-L34, M1-E33, M1-N32, M1-D31, M1-V30, M1-I29, M1-I28, M1-Q27, M1-Y26, M1-P25, M1-E24, M1-R23, M1-F22, M1-K21, M1-F20, M1-A19, M1-H18, M1-V17, M1-Y16, M1-V15, M1-S14, M1-M13, M1-Q12, M1-K11, M1-K10, M1-Y9, M1-A8, and/or M1-K7 of SEQ ID NO:20. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CaYOR004w deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses polynucleotides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polynucleotide sequence provided in SEQ ID NO:9, and in particular to the coding region of the polynucleotide sequence provided in SEQ ID NO:9. Preferably such polynucleotides encode polypeptides that have biological activity.
Most preferred are polynucleotides that share at least about 88.9% identity with the polynucleotide sequence provided in SEQ ID NO:9.
The present invention also encompasses polypeptides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polypeptide sequence provided in SEQ ID NO:20.
Most preferred are polypeptides that share at least about 95.4% identity with the polypeptide sequence provided in SEQ ID NO:20.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:9 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 757 of SEQ ID NO:9, b is an integer between 15 to 771, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:9, and where b is greater than or equal to a+14.
Features of the Polypeptide Encoded by Polynucleotide No:10
The polynucleotide sequence (SEQ ID NO:10) and deduced amino acid sequence (SEQ ID NO:21) of the novel fungal essential gene, CaYOR056c (also referred to as FCG16), of the present invention. The CaYOR056c polypeptide (SEQ ID NO:21) is encoded by nucleotides 1 to 1398 of SEQ ID NO:10 and has a predicted molecular weight of 52.6 kDa.
In preferred embodiments, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of CaYOR056c. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 4 thru 1398 of SEQ ID NO:10, and the polypeptide corresponding to amino acids 2 thru 466 of SEQ ID NO:21. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.
The invention also encompasses N- and/or C-terminal deletions of the CaYOR056c polypeptide of the present invention.
In preferred embodiments, the following N-terminal CaYOR056c deletion polypeptides are encompassed by the present invention: M1-K466, S2-K466, E3-K466, T4-K466, K5-K466, N6-K466, I7-K466, E8-K466, S9-K466, L10-K466, I11-K466, S12-K466, D13-K466, A14-K466, G15-K466, P16-K466, L17-K466, I18-K466, T19-K466, Q20-K466, P21-K466, A22-K466, T23-K466, T24-K466, L25-K466, Q26-K466, Q27-K466, Y28-K466, A29-K466, T30-K466, A31-K466, Y32-K466, Y33-K466, T34-K466, T35-K466, P36-K466, G37-K466, V38-K466, H39-K466, S40-K466, E41-K466, L42-K466, K43-K466, D44-K466, E45-K466, Y46-K466, A47-K466, R48-K466, Q49-K466, Q50-K466, L51-K466, A52-K466, I53-K466, W54-K466, G55-K466, D56-K466, S57-K466, L58-K466, K59-K466, I60-K466, K61-K466, Q62-K466, P63-K466, K64-K466, Q65-K466, E66-K466, Y67-K466, I68-K466, D69-K466, R70-K466, V71-K466, V72-K466, K73-K466, F74-K466, A75-K466, K76-K466, L77-K466, T78-K466, G79-K466, D80-K466, Y81-K466, S82-K466, V83-K466, L84-K466, S85-K466, V86-K466, N87-K466, D88-K466, L89-K466, H90-K466, I91-K466, V92-K466, A93-K466, L94-K466, A95-K466, Y96-K466, E97-K466, L98-K466, E99-K466, C100-K466, L101-K466, N102-K466, N103-K466, G104-K466, E105-K466, D106-K466, N107-K466, L108-K466, R109-K466, S110-K466, F111-K466, P112-K466, G113-K466, E114-K466, V115-K466, L116-K466, K117-K466, N118-K466, Q119-K466, Q120-K466, A121-K466, E122-K466, N123-K466, E124-K466, N125-K466, G126-K466, S127-K466, N128-K466, K129-K466, M130-K466, S13′-K466, N132-K466, I133-K466, I134-K466, G135-K466, D136-K466, D137-K466, D138-K466, G139-K466, F140-K466, V141-K466, V142-K466, A143-K466, T144-K466, K145-K466, R146-K466, R147-K466, G148-K466, G149-K466, R150-K466, R151-K466, Q152-K466, R153-K466, E154-K466, K155-K466, A156-K466, E157-K466, L158-K466, R159-K466, K160-K466, K161-K466, G162-K466, L163-K466, L164-K466, P165-K466, T166-K466, F167-K466, S168-K466, P169-K466, K170-K466, P171-K466, K172-K466, G173-K466, G174-K466, L175-K466, E176-K466, T177-K466, E178-K466, E179-K466, P180-K466, N181-K466, E182-K466, L183-K466, S184-K466, N185-K466, D186-K466, K187-K466, T188-K466, I189-K466, D190-K466, E191-K466, T192-K466, P193-K466, Q194-K466, T195-K466, D196-K466, L197-K466, I198-K466, K199-K466, G200-K466, V201-K466, D202-K466, V203-K466, Q204-K466, E205-K466, Q206-K466, E207-K466, S208-K466, Q209-K466, E210-K466, E211-K466, P212-K466, V213-K466, S214-K466, E215-K466, S216-K466, N217-K466, T218-K466, V219-K466, G220-K466, L221-K466, D222-K466, E223-K466, I224-K466, T225-K466, E226-K466, E227-K466, Y228-K466, N229-K466, E230-K466, D231-K466, D232-K466, D233-K466, D234-K466, G235-K466, E236-K466, W237-K466, I238-K466, T239-K466, P240-K466, E241-K466, N242-K466, L243-K466, Q244-K466, E245-K466, E246-K466, I247-K466, I248-K466, K249-K466, D250-K466, K251-K466, N252-K466, E253-K466, Q254-K466, V255-K466, Q256-K466, E257-K466, S258-K466, N259-K466, T260-K466, N261-K466, G262-K466, P263-K466, L264-K466, I265-K466, K266-K466, V267-K466, A268-K466, L269-K466, A270-K466, T271-K466, G272-K466, D273-K466, F274-K466, A275-K466, C276-K466, Q277-K466, N278-K466, V279-K466, A280-K466, M281-K466, Q282-K466, I283-K466, G284-K466, I285-K466, K286-K466, L287-K466, L288-K466, N289-K466, A290-K466, M291-K466, S292-K466, G293-K466, K294-K466, Q295-K466, I296-K466, T297-K466, R298-K466, V299-K466, R300-K466, N301-K466, Y302-K466, M303-K466, Y304-K466, R305-K466, C306-K466, H307-K466, A308-K466, C309-K466, F310-K466, R311-K466, L312-K466, T313-K466, P314-K466, M315-K466, S316-K466, K317-K466, D318-K466, G319-K466, R320-K466, P321-K466, K322-K466, H323-K466, F324-K466, C325-K466, P326-K466, K327-K466, C328-K466, G329-K466, G330-K466, N331-K466, T332-K466, L333-K466, L334-K466, R335-K466, C336-K466, A337-K466, V338-K466, S339-K466, V340-K466, D341-K466, N342-K466, K343-K466, T344-K466, G345-K466, K346-K466, I347-K466, T348-K466, P349-K466, H350-K466, L351-K466, K352-K466, Q353-K466, N354-K466, F355-K466, Q356-K466, W357-K466, I358-K466, R359-K466, R360-K466, G361-K466, E362-K466, R363-K466, Y364-K466, S365-K466, L366-K466, P367-K466, S368-K466, P369-K466, L370-K466, S371-K466, K372-K466, N373-K466, Q374-K466, K375-K466, K376-K466, L377-K466, Q378-K466, G379-K466, N380-K466, G381-K466, G382-K466, Y383-K466, Q384-K466, H385-K466, N386-K466, K387-K466, E388-K466, N389-K466, R390-K466, H391-K466, K392-K466, S393-K466, L394-K466, Q395-K466, T396-K466, P397-K466, L398-K466, I399-K466, L400-K466, N401-K466, E402-K466, D403-K466, Q404-K466, K405-K466, E406-K466, Y407-K466, Q408-K466, R409-K466, A410-K466, L411-K466, K412-K466, N413-K466, D414-K466, E415-K466, W416-K466, E417-K466, R418-K466, K419-K466, Q420-K466, Q421-K466, D422-K466, K423-K466, M424-K466, L425-K466, Q426-K466, E427-K466, W428-K466, I429-K466, G430-K466, G431-K466, G432-K466, S433-K466, A434-K466, D435-K466, N436-K466, F437-K466, V438-K466, S439-K466, P440-K466, F441-K466, G442-K466, N443-K466, T444-K466, I445-K466, R446-K466, N447-K466, S448-K466, G449-K466, V450-K466, K451-K466, V452-K466, G453-K466, R454-K466, G455-K466, R456-K466, Y457-K466, A458-K466, N459-K466, and/or S460-K466 of SEQ ID NO:21. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CaYOR056c deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CaYOR056c deletion polypeptides are encompassed by the present invention: M1-K466, M1-R465, M1-K464, M1-K463, M1-K462, M1-S461, M1-S460, M1-N459, M1-A458, M1-Y457, M1-R456, M1-G455, M1-R454, M1-G453, M1-V452, M1-K451, M1-V450, M1-G449, M1-S448, M1-N447, M1-R446, M1-I445, M1-T444, M1-N443, M1-G442, M1-F441, M1-P440, M1-S439, M1-V438, M1-F437, M1-N436, M1-D435, M1-A434, M1-S433, M1-G432, M1-G431, M1-G430, M1-I429, M1-W428, M1-E427, M1-Q426, M1-L425, M1-M424, M1-K423, M1-D422, M1-Q421, M1-Q420, M1-K419, M1-R418, M1-E417, M1-W416, M1-E415, M1-D414, M1-N413, M1-K412, M1-L411, M1-A410, M1-R409, M1-Q408, M1-Y407, M1-E406, M1-K405, M1-Q404, M1-D403, M1-E402, M1-N401, M1-L400, M1-I399, M1-L398, M1-P397, M1-T396, M1-Q395, M1-L394, M1-S393, M1-K392, M1-H391, M1-R390, M1-N389, M1-E388, M1-K387, M1-N386, M1-H385, M1-Q384, M1-Y383, M1-G382, M1-G381, M1-N380, M1-G379, M1-Q378, M1-L377, M1-K376, M1-K375, M1-Q374, M1-N373, M1-K372, M1-S371, M1-L370, M1-P369, M1-S368, M1-P367, M1-L366, M1-S365, M1-Y364, M1-R363, M1-E362, M1-G361, M1-R360, M1-R359, M1-I358, M1-W357, M1-Q356, M1-F355, M1-N354, M1-Q353, M1-K352, M1-L351, M1-H350, M1-P349, M1-T348, M1-I347, M1-K346, M1-G345, M1-T344, M1-K343, M1-N342, M1-D341, M1-V340, M1-S339, M1-V338, M1-A337, M1-C336, M1-R335, M1-L334, M1-L333, M1-T332, M1-N331, M1-G330, M1-G329, M1-C328, M1-K327, M1-P326, M1-C325, M1-F324, M1-H323, M1-K322, M1-P321, M1-R320, M1-G319, M1-D318, M1-K317, M1-S316, M1-M315, M1-P314, M1-T313, M1-L312, M1-R311, M1-F310, M1-C309, M1-A308, M1-H307, M1-C306, M1-R305, M1-Y304, M1-M303, M1-Y302, M1-N301, M1-R300, M1-V299, M1-R298, M1-T297, M1-I296, M1-Q295, M1-K294, M1-G293, M1-S292, M1-M291, M1-A290, M1-N289, M1-L288, M1-L287, M1-K286, M1-I285, M1-G284, M1-I283, M1-Q282, M1-M281, M1-A280, M1-V279, M1-N278, M1-Q277, M1-C276, M1-A275, M1-F274, M1-D273, M1-G272, M1-T271, M1-A270, M1-L269, M1-A268, M1-V267, M1-K266, M1-I265, M1-L264, M1-P263, M1-G262, M1-N261, M1-T260, M1-N259, M1-S258, M1-E257, M1-Q256, M1-V255, M1-Q254, M1-E253, M1-N252, M1-K251, M1-D250, M1-K249, M1-I248, M1-I247, M1-E246, M1-E245, M1-Q244, M1-L243, M1-N242, M1-E241, M1-P240, M1-T239, M1-I238, M1-W237, M1-E236, M1-G235, M1-D234, M1-D233, M1-D232, M1-D231, M1-E230, M1-N229, M1-Y228, M1-E227, M1-E226, M1-T225, M1-I224, M1-E223, M1-D222, M1-L221, M1-G220, M1-V219, M1-T218, M1-N217, M1-S216, M1-E215, M1-S214, M1-V213, M1-P212, M1-E211, M1-E210, M1-Q209, M1-S208, M1-E207, M1-Q206, M1-E205, M1-Q204, M1-V203, M1-D202, M1-V201, M1-G200, M1-K199, M1-I198, M1-L197, M1-D196, M1-T195, M1-Q194, M1-P193, M1-T192, M1-E191, M1-D190, M1-I189, M1-T188, M1-K187, M1-D186, M1-N185, M1-S184, M1-L183, M1-E182, M1-N181, M1-P180, M1-E179, M1-E178, M1-T177, M1-E176, M1-L175, M1-G174, M1-G173, M1-K172, M1-P171, M1-K170, M1-P169, M1-S168, M1-F167, M1-T166, M1-P165, M1-L164, M1-L163, M1-G162, M1-K161, M1-K160, M1-R159, M1-L158, M1-E157, M1-A156, M1-K155, M1-E154, M1-R153, M1-Q152, M1-R151, M1-R150, M1-G149, M1-G148, M1-R147, M1-R146, M1-K145, M1-T144, M1-A143, M1-V142, M1-V141, M1-F140, M1-G139, M1-D138, M1-D137, M1-D136, M1-G135, M1-I134, M1-I133, M1-N132, M1-S131, M1-M130, M1-K129, M1-N128, M1-S127, M1-G126, M1-N125, M1-E124, M1-N123, M1-E122, M1-A121, M1-Q120, M1-Q19, M1-N118, M1-K117, M1-L116, M1-V115, M1-E114, M1-G113, M1-P112, M1-F111, M1-S110, M1-R109, M1-L108, M1-N107, M1-D106, M1-E105, M1-G104, M1-N103, M1-N102, M1-L101M1-C100, M1-E99, M1-L98, M1-E97, M1-Y96, M1-A95, M1-L94, M1-A93, M1-V92, M1-I91, M1-H90, M1-L89, M1-D88, M1-N87, M1-V86, M1-S85, M1-L84, M1-V83, M1-S82, M1-Y81, M1-D80, M1-G79, M1-T78, M1-L77, M1-K76, M1-A75, M1-F74, M1-K73, M1-V72, M1-V71, M1-R70, M1-D69, M1-I68, M1-Y67, M1-E66, M1-Q65, M1-K64, M1-P63, M1-Q62, M1-K61, M1-I60, M1-K59, M1-L58, M1-S57, M1-D56, M1-G55, M1-W54, M1-I53, M1-A52, M1-L51, M1-Q50, M1-Q49, M1-R48, M1-A47, M1-Y46, M1-E45, M1-D44, M1-K43, M1-L42, M1-E41, M1-S40, M1-H39, M1-V38, M1-G37, M1-P36, M1-T35, M1-T34, M1-Y33, M1-Y32, M1-A31, M1-T30, M1-A29, M1-Y28, M1-Q27, M1-Q26, M1-L25, M1-T24, M1-T23, M1-A22, M1-P21, M1-Q20, M1-T19, M1-I18, M1-L17, M1-P16, M1-G15, M1-A14, M1-D13, M1-S12, M1-I11, M1-L10, M1-S9, M1-E8, and/or M1-17 of SEQ ID NO:21. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CaYOR056c deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses polynucleotides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polynucleotide sequence provided in SEQ ID NO:10, and in particular to the coding region of the polynucleotide sequence provided in SEQ ID NO:10. Preferably such polynucleotides encode polypeptides that have biological activity.
The present invention also encompasses polypeptides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polypeptide sequence provided in SEQ ID NO:21.
Most preferred are polypeptides that share at least about 99.5% identity with the polypeptide sequence provided in SEQ ID NO:21.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:10 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 1384 of SEQ ID NO:10, b is an integer between 15 to 1398, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:10, and where b is greater than or equal to a+14.
Features of the Polypeptide Encoded by Polynucleotide No:11
The polynucleotide sequence (SEQ ID NO:11) and deduced amino acid sequence (SEQ ID NO:22) of the novel fungal essential gene, CaYLR009w (also referred to as FCG17), of the present invention. The CaYLR009w polypeptide (SEQ ID NO:22) is encoded by nucleotides 1 to 585 of SEQ ID NO:11 and has a predicted molecular weight of 23.1 kDa.
In preferred embodiments, the present invention encompasses a polynucleotide lacking the initiating start codon, in addition to, the resulting encoded polypeptide of CaYLR009w. Specifically, the present invention encompasses the polynucleotide corresponding to nucleotides 4 thru 585 of SEQ ID NO:11, and the polypeptide corresponding to amino acids 2 thru 195 of SEQ ID NO:22. Also encompassed are recombinant vectors comprising said encoding sequence, and host cells comprising said vector.
The invention also encompasses N- and/or C-terminal deletions of the CaYLR009w polypeptide of the present invention.
In preferred embodiments, the following N-terminal CaYLR009w deletion polypeptides are encompassed by the present invention: M1-C195, R2-C195, I3-C195, Y4-C195, Q5-C195, C6-C195, H7-C195, F8-C195, C9-C195, S10-C195, S11-C195, P12-C195, V13-C195, Y14-C195, P15-C195, L16-C195, H17-C195, G18-C195, I19-C195, T20-C195, F21-C195, V22-C195, R23-C195, N24-C195, D25-C195, A26-C195, K27-C195, E28-C195, F29-C195, R30-C195, F31-C195, C32-C195, R33-C195, S34-C195, K35-C195, C36-C195, H37-C195, K38-C195, A39-C195, F40-C195, K41-C195, Q42-C195, R43-C195, R44-C195, N45-C195, P46-C195, R47-C195, K48-C195, L49, C195, R50-C195, W51-C195, T52-C195, K53-C195, A54-C195, F55-C195, R56-C195, K57-C195, A58-C195, A59-C195, G60-C195, K61-C195, E62-C195, L63-C195, V64-C195, V65-C195, D66-C195, S67-C195, T68-C195, L69-C195, T70-C195, F71-C195, A72-C195, A73-C195, R74-C195, R75-C195, N76-C195, V77-C195, P78-C195, V79-C195, R80-C195, Y81-C195, N82-C195, R83-C195, D84-C195, L85-C195, V86-C195, A87-C195, T88-C195, T89-C195, L90-C195, K91-C195, G92-C195, M93-C195, S94-C195, R95-C195, I96-C195, E97-C195, E98-C195, I99-C195, R100-C195, Q101-C195, R102-C195, R103-C195, E104-C195, R105-C195, A106-C195, F107-C195, Y108-C195, K109-C195, N110-C195, R111′-C195, M112-C195, K113-C195, G114-C195, N115-C195, K116-C195, E117-C195, R118-C195, Q119-C195, L120-C195, A121-C195, A122-C195, D123-C195, R124-C195, K125-C195, L126-C195, V127-C195, A128-C195, D129-C195, N130-C195, P131-C195, E132-C195, L133-C195, L134-C195, R135-C195, L136-C195, R137-C195, E138-C195, V139-C195, E140-C195, L141-C195, R142-C195, R143-C195, K144-C195, A145-C195, E146-C195, K147-C195, L148-C195, A149-C195, A150-C195, K151-C195, E152-C195, N153-C195, A154-C195, M155-C195, E156-C195, E157-C195, D158-C195, E159-C195, E160-C195, T161-C195, E162-C195, V163-C195, E164-C195, E165-C195, E166-C195, G167-C195, E168-C195, G169-C195, D170-C195, E171-C195, E172-C195, M173-C195, I174-C195, S175-C195, G176-C195, E177-C195, E178-C195, E179-C195, W180-C195, E181-C195, S182-C195, E183-C195, D184-C195, E185-C195, S186-C195, E187-C195, R188-C195, and/or E189-C195 of SEQ ID NO:22. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal CaYLR009w deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal CaYLR009w deletion polypeptides are encompassed by the present invention: M1-C195, M1-T194, M1-K193, M1-T192, M1-D191, M1-S190, M1-E189, M1-R188, M1-E187, M1-S186, M1-E185, M1-D184, M1-E183, M1-S182, M1-E181, M1-W180, M1-E179, M1-E178, M1-E177, M1-G176, M1-S175, M1-I174, M1-M173, M1-E172, M1-E171, M1-D170, M1-G169, M1-E168, M1-G167, M1-E166, M1-E165, M1-E164, M1-V163, M1-E162, M1-T161, M1-E160, M1-E159, M1-D158, M1-E157, M1-E156, M1-M155, M1-A154, M1-N153, M1-E152, M1-K151, M1-A150, M1-A149, M1-L148, M1-K147, M1-E146, M1-A145, M1-K144, M1-R143, M1-R142, M1-L141, M1-E140, M1-V139, M1-E138, M1-R137, M1-L136, M1-R135, M1-L134, M1-L133, M1-E132, M1-P131, M1-N130, M1-D129, M1-A128, M1-V127, M1-L126, M1-K125, M1-R124, M1-D123, M1-A122, M1-A121, M1-L120, M1-Q119, M1-R118, M1-E117, M1-K116, M1-N115, M1-G114, M1-K113, M1-M112, M1-R111, M1-N110, M1-K109, M1-Y108, M1-F107, M1-A106, M1-R105, M1-E104, M1-R103, M1-R102, M1-Q101, M1-R100, M1-I99, M1-E98, M1-E97, M1-I96, M1-R95, M1-S94, M1-M93, M1-G92, M1-K91, M1-L90, M1-T89, M1-T88, M1-A87, M1-V86, M1-L85, M1-D84, M1-R83, M1-N82, M1-Y81, M1-R80, M1-V79, M1-P78, M1-V77, M1-N76, M1-R75, M1-R74, M1-A73, M1-A72, M1-F71, M1-T70, M1-L69, M1-T68, M1-S67, M1-D66, M1-V65, M1-V64, M1-L63, M1-E62, M1-K61, M1-G60, M1-A59, M1-A58, M1-K57, M1-R56, M1-F55, M1-A54, M1-K53, M1-T52, M1-W51, M1-R50, M1-L49, M1-K48, M1-R47, M1-P46, M1-N45, M1-R44, M1-R43, M1-Q42, M1-K41, M1-F40, M1-A39, M1-K38, M1-H37, M1-C36, M1-K35, M1-S34, M1-R33, M1-C32, M1-F31, M1-R30, M1-F29, M1-E28, M1-K27, M1-A26, M1-D25, M1-N24, M1-R23, M1-V22, M1-F21, M1-T20, M1-I19, M1-G18, M1-H17, M1-L16, M1-P15, M1-Y14, M1-V13, M1-P12, M1-S11, M1-S10, M1-C9, M1-F8, and/or M1-H7 of SEQ ID NO:22. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal CaYLR009w deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses polynucleotides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polynucleotide sequence provided in SEQ ID NO:11, and in particular to the coding region of the polynucleotide sequence provided in SEQ ID NO:11. Preferably such polynucleotides encode polypeptides that have biological activity.
Most preferred are polynucleotides that share at least about 91.8% identity with the polynucleotide sequence provided in SEQ ID NO:22.
The present invention also encompasses polypeptides sharing at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a the polypeptide sequence provided in SEQ ID NO:22.
Most preferred are polypeptides that share at least about 88.6% identity with the polypeptide sequence provided in SEQ ID NO:22.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:11 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a−b, where a is any integer between 1 to 571 of SEQ ID NO:11, b is an integer between 15 to 585, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:11, and where b is greater than or equal to a+14.
Homology Models
One embodiment of the homology models of the present invention utilizes System 10 as disclosed in WO 98/11134, the disclosure of which is incorporated herein by reference in its entirety. Briefly, one version of these embodiments comprises a computer comprising a central processing unit (“CPU”), a working memory which may be, e.g., RAM (random-access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals, one or more keyboards, one or more input lines, and one or more output lines, all of which are interconnected by a conventional bi-directional system bus.
Input hardware, coupled to the computer by input lines, may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives. In conjunction with a display terminal, keyboard may also be used as an input device.
Output hardware, coupled to the computer by output lines, may similarly be implemented by conventional devices. By way of example, output hardware may include a CRT display terminal for displaying a graphical representation of a region or domain of the present invention using a program such as QUANTA as described herein. Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.
In operation, the CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage, and accesses to and from the working memory, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system are included as appropriate throughout the following description of the data storage medium.
For the purpose of the present invention, any magnetic data storage medium which can be encoded with machine-readable data would be sufficient for carrying out the storage requirements of the system. The medium could be a conventional floppy diskette or hard disk, having a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, on one or both sides, containing magnetic domains whose polarity or orientation could be altered magnetically, for example. The medium may also have an opening for receiving the spindle of a disk drive or other data storage device.
The magnetic domains of the coating of a medium may be polarized or oriented so as to encode in a manner which may be conventional, machine readable data such as that described herein, for execution by a system such as the system described herein.
Another example of a suitable storage medium which could also be encoded with such machine-readable data, or set of instructions, which could be carried out by a system such as the system described herein, could be an optically-readable data storage medium. The medium could be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk which is optically readable and magneto-optically writable. The medium preferably has a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, usually of one side of substrate.
In the case of a CD-ROM, as is well known, the coating is reflective and is impressed with a plurality of pits to encode the machine-readable data. The arrangement of pits is read by reflecting laser light off the surface of the coating. A protective coating, which preferably is substantially transparent, is provided on top of the reflective coating.
In the case of a magneto-optical disk, as is well known, the coating has no pits, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser. The orientation of the domains can be read by measuring the polarization of laser light reflected from the coating. The arrangement of the domains encodes the data as described above.
Recombinants and Expression
The present invention provides recombinant DNA molecules containing polynucleotide sequences encoding essential polynucleotide polypeptides. “Recombinant DNA molecules” include both cloning and expression vectors.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding essential polynucleotide. For example, in one embodiment, routine cloning, subcloning, and propagation of polynucleotide sequences encoding essential polynucleotides can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or PSPORT plasmid (Life Technologies). Ligation of sequences encoding essential polynucleotide into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S. M. Schuster J. Biol 264: 5503-5509 (1989)).
In a preferred embodiment, the present invention provides expression vectors containing a polynucleotide that encodes essential polynucleotide polypeptides. Preferably, the expression vectors of the present invention comprise polynucleotides that encode polypeptides including the amino acid residue sequences of SEQ ID NO: 12 through SEQ ID NO: 24.
An “expression vector” refers to an assembly which is capable of directing the expression of desired proteins. The vector must include regulatory sequences which are operably linked to a gene(s) of interest. The vector may be composed of either DNA or RNA or a combination of the two. Optionally, the vector may include a polyadenylation sequence, one or more restriction sites, as well as one or more selectable markers such as neomycin phosphotransferase or hygromycin phosphotransferase. Additionally, depending on the host cell chosen and the vector employed, other genetic elements such as an origin of replication, additional nucleic acid restriction sites, enhancers, sequences conferring inducibility of transcription and selectable markers, may also be incorporated in the vectors described herein.
The invention provides expression vectors including a polynucleotide disclosed herein operatively linked to a regulatory sequence. “Regulatory sequences” include enhancers and promoters. Preferably, the expression vectors of the invention comprise polynucleotide operatively linked to a prokaryotic promoter. More preferably, the expression vectors of the present invention comprise a polynucleotide operatively linked to a eukaryotic promoter, and the expression vectors further comprise a polyadenylation signal that is positioned 3′ of the carboxy-terminal amino acid and within a transcriptional unit of the encoded polypeptide.
A promoter is a region of a DNA molecule typically within about 100 nucleotide pairs in front of (upstream of) the point at which transcription begins (i.e., a transcription start site). That region typically contains several types of DNA sequence elements that are located in similar relative positions in different polynucleotides. As used herein, the term “promoter” includes what is referred to in the art as an upstream promoter region, a promoter region or a promoter of a generalized eukaryotic RNA Polymerase II transcription unit.
Another type of regulatory sequence is an enhancer. An enhancer provides specificity of time, location and expression level for a particular encoding region (e.g., gene). A major function of an enhancer is to increase the level of transcription of a coding sequence in a cell that contains one or more transcription factors that bind to that enhancer. Unlike a promoter, an enhancer can function when located at variable distances from transcription start sites so long as a promoter is present.
In one aspect of the invention, the enhancer and/or promoter is operatively linked to a coding sequence that encodes at least one polynucleotide product. As used herein, the phrase “operatively linked” means that a regulatory sequence is connected to a coding sequence in such a way that the transcription of that coding sequence is controlled and regulated by that enhancer and/or promoter. Means for operatively linking an enhancer and/or promoter to a coding sequence are well-known in the art. As is also well-known in the art, the precise orientation and location relative to a coding sequence whose transcription is controlled, is dependent upon the specific nature of the regulatory sequence. Thus, a TATA box minimal promoter is typically located from about 25 to about 30 base pairs upstream of a transcription initiation site and an upstream promoter element is typically located from about 100 to about 200 base pairs upstream of a transcription initiation site. In contrast, an enhancer can be located downstream from the initiation site and can be at a considerable distance from that site.
Microbial promoters most commonly used in recombinant DNA construction include the beta-lactamase (penicillinase) and lactose promoter systems and a tryptophan (TRP) promoter system (EPO Appl. Publ. No. 0036776). While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to introduce functional promoters into plasmid vectors.
Suitable promoter sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these polynucleotides are also introduced into the expression vector downstream from the sequences to be expressed to provide polyadenylation of the mRNA and termination. Other promoters, which have the additional advantage of transcription controlled by growth conditions, are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter, origin or replication and termination sequences is suitable.
In one embodiment, a regulatable promoter is used. By “regulatable promoter” is meant a promoter other than the native promoter for the essential polynucleotide which can be regulated by the addition and/or removal of specific materials or for example by other environmental changes.
Some examples of regulatable promoters for use in organisms include GAL I for use in S. cerevisiae (repressed by glucose induced by galactose); NMT1 for use in S. pombe (repressed by thiamine); for use in C. albicans: MALI (repressed by glucose, induced by maltose, sucrose); for use in E. coli: araB (repressed by glucose, induced by arabinose); for use in Gram-positive bacteria such as Staphylococci, Enterococci, Streptococci and Bacilli: xylA/xylR (from S. xylosus) (repressed by glucose, induced by xylose); for use in E. coli and B. subtilis pSPAC (an artificial promoter derived from E. coli lac, regulated by IPTG, see Vagner et al. Microbiology, 144, 3097-3104 (1998)); and for all of the above organisms plus further unspecified fungi, bacteria and mammalian cell lines: tetA/tetR (from various bacterial tetracycline resistance cassettes) this system exists in various versions, see Gossen et al. Current Opin. Biotechnol. 5, pp 516-520 (1994), that are repressible or inducible by various tetracycline analogues.
Other conditional regulatable promoters include, but are not limited to, those such as MET25, MAL2, PHO5,5 GAL I; STE2, or STE3.
A preferred regulatable promoter for use in some embodiments includes the MET3 promoter (repressible by methionine, cysteine or both).
For use in mammalian cells, the control functions on the expression vectors are often derived from viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, Cytomegalovirus and most frequently Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments can also be used, provided there is included the approximately 250 bp sequence extending from the HindIII site toward the BglI site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired polynucleotide sequence, provided such control sequences are compatible with the host cell systems.
An origin of replication can be provided constructing the vector to include an exogenous origin, such as can be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV, CMV) source, or can be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
A coding sequence of an expression vector is operatively linked to a transcription terminating region. RNA polymerase transcribes an encoding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to terminate transcription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA). Transcription-terminating regions are well-known in the art.
The invention provides an expression vector having a polynucleotide that encodes an essential polynucleotide polypeptide. Such a polypeptide is meant to include a sequence of nucleotide bases encoding an essential polynucleotide polypeptide sufficient in length to distinguish said fragment from a polynucleotide fragment encoding a non-essential polynucleotide polypeptide. A polypeptide of the invention can also encode biologically functional polypeptides or peptides which have variant amino acid sequences, such as with changes selected based on considerations such as the relative hydropathic score of the amino acids being exchanged. These variant sequences are those isolated from natural sources or induced in the sequences disclosed herein using a mutagenic procedure such as site-directed mutapolynucleotidesis.
The present invention also provides recombinant vectors that may be used to integrate exogenously provided sequences into the genome of a host cell. The recombinant integration vectors of the present invention include a polynucleotide that encodes a selectable marker and polynucleotide sequences of the invention or fragment thereof. The integration vectors are used to integrate the essential polynucleotide sequences into a target polynucleotide sequence that resides within the fungal host genome (e.g., endogenous sequence), thereby disrupting the function of the target polynucleotide sequence within the fungal cells. These integration vectors may be used in a polynucleotide disruption assay to screen candidate sequences in order to identify the candidate sequences that encode a polynucleotide product that is required for fungal cell viability.
Accordingly, these recombinant integration vectors include candidate sequences such as homologues of SEQ ID NO: 1 through to SEQ ID NO: 11, SEQ ID NO: 1 through to SEQ ID NO. 11, SEQ ID NO: 12 or a fragment thereof to determine if the candidate sequences encode a polynucleotide product that is required for cell viability. The candidate sequences that is included as part of the recombinant integration vector is the “exogenous” candidate sequence that is employed as the “disrupting” sequence in a polynucleotide disruption assay. The candidate sequence that resides within the host genome is the “endogenous” or target candidate sequence.
The integration event rarely occurs, for example, by non-homologous recombination in which a recombinant vector, that includes the exogenous candidate sequence, inserts the exogenous candidate sequence into a random location within the host genome.
In a more preferred embodiment, the integration event inserts the exogenous candidate sequence into a specific target site within the host genome. The targeted integration event can involve homologous recombination in which the integration vector, that includes the exogenous candidate sequence, inserts the exogenous candidate sequence into its homologous target candidate sequence that resides within the host's genome (e.g., the endogenous candidate sequence). The exogenous candidate sequences can result in disruption of the function of the endogenous candidate sequence. For example, disrupting the function of the endogenous sequence may result in the loss of fungal cell viability.
Host Cells and Host Organisms
In yet another embodiment, the present invention provides recombinant host cells transformed or transfected with a polynucleotide that encodes essential polynucleotide polypeptides, as well as transgenic cells derived from those transformed or transfected cells. Preferably, the recombinant host cells of the present invention are transfected with the polynucleotide of SEQ ID NO: 1 to SEQ ID NO: 11, or a variant or fragment thereof.
A variety of cells are amenable to the method of the invention involving polypeptide expression, for instance, yeast cells, human cell lines, and other eukaryotic cell lines well-known to those of skill in the art.
Means of transforming or transfecting cells with an exogenous polynucleotide such as DNA molecules are well-known in the art and include techniques such as calcium-phosphate- or DEAE-dextran-mediated transfection, protoplast fusion, electroporation, liposome mediated transfection, direct microinjection and adenovirus infection (Sambrook, et al., supra).
The most widely used method is transfection mediated by either calcium phosphate or DEAE-dextran. Although the mechanism remains obscure, it is believed that the transfected DNA enters the cytoplasm of the cell by endocytosis and is transported to the nucleus. Depending on the cell type, up to 90% of a population of cultured cells can be transfected at any one time. Because of its high efficiency, transfection mediated by calcium phosphate or DEAE-dextran is the method of choice for experiments that require transient expression of foreign DNA in large numbers of cells. Calcium phosphate-mediated transfection is also used to establish cell lines that integrate copies of foreign DNA, which are usually arranged in head-to-tail tandem arrays into the host cell genome.
In the protoplast fusion method, protoplasts derived from bacteria carrying high numbers of copies of a plasmid of interest are mixed directly with cultured mammalian cells. After fusion of the cell membranes (usually with polyethylene glycol), the contents of the bacteria are delivered into the cytoplasm of the mammalian cells and the plasmid DNA is transported to the nucleus. Protoplast fusion frequently yields multiple copies of the plasmid DNA tandemly integrated into the host chromosome.
The application of brief, high-voltage electric pulses to a variety of mammalian cells leads to the formation of nanometer-sized pores in the plasma membrane. DNA is taken directly into the cell cytoplasm either through these pores or as a consequence of the redistribution of membrane components that accompanies closure of the pores. Electroporation can be extremely efficient and can be used both for transient expression of cloned polynucleotides and for establishment of cell lines that carry integrated copies of the polynucleotide of interest. Electroporation, in contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few, integrated copies of the foreign DNA.
Liposome transfection involves encapsulation of DNA and RNA within liposomes, followed by fusion of the liposomes with the cell membrane. The mechanism of how DNA is delivered into the cell is unclear but transfection efficiencies can be as high as 90%.
Direct microinjection of a DNA molecule into nuclei has the advantage of not exposing DNA to cellular compartments such as low-pH endosomes. Microinjection is therefore used primarily as a method to establish lines of cells that carry integrated copies of the DNA of interest.
The use of adenovirus as a vector for cell transfection is well-known in the art. Adenovirus vector-mediated cell transfection has been reported for various cells (Stratford-Perricaudet, et al., J Clin Invest. 90(2):626-630).
A transfected cell can be prokaryotic or eukaryotic. In one embodiment, the host cells of the invention are eukaryotic host cells.
When the recombinant host cells of the present invention are prokaryotic host cells Escherichia coli bacterial cells are preferred. In general, prokaryotes are preferred for the initial cloning of DNA sequences and constructing the vectors useful in the invention. For example, E. coli K12 strains can be particularly useful. Another microbial strain which can be used includes E. coli X1776 (ATCC No. 31537). These examples are, of course, intended to be illustrative rather than limiting.
Prokaryotes can also be used for expression. The aforementioned strains, as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325), bacilli such as Bacillus subtilis, or other enterobacteriaceae such as Salmonella typhimurium or Serratus marcesens, and various Pseudomonas species can be used.
In addition to prokaryotes, eukaryotic microbes such as yeast can also be used. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, is used. This plasmid already contains the trpl polynucleotide which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076. The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
In addition to microorganisms, cultures of cells derived from multicellular organisms can also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. Examples of such useful host cell lines are AtT-20, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COSM6, COS-7, 293 and MDCK cell lines.
Following transfection, the cell is maintained under culture conditions for a period of time sufficient for expression of an essential polynucleotide polypeptide. Culture conditions are well-known in the art and include ionic composition and concentration, temperature, pH and the like. Typically, transfected cells are maintained under culture conditions in a culture medium. Suitable medium for various cell types are well-known in the art. In a preferred embodiment, temperature is from about 20° C. to about 50° C., more preferably from about 30° C. to about 40° C. and, even more preferably, about 37° C. pH is preferably from about a value of 6.0 to a value of about 8.0, more preferably from about a value of about 6.8 to a value of about 7.8 and, most preferably, about 7.4. Osmolality is preferably from about 200 milliosmols per liter (mosm/L) to about 400 mosm/L and, more preferably, from about 290 mosm/L to about 310 mosm/L. Other biological conditions needed for transfection and expression of an encoded protein are well-known in the art.
Transfected cells are maintained for a period of time sufficient for expression of an essential polynucleotide polypeptide. A suitable time depends upon the cell type used and is readily determinable by a skilled artisan. Typically, maintenance time is from about 2 to about 14 days. Recombinant essential polynucleotide polypeptide is recovered or collected either from the transfected cells or the medium in which those cells are cultured. Recovery comprises isolating and purifying the essential polynucleotide polypeptide. Isolation and purification techniques for polypeptides are well-known in the art and include such procedures as precipitation, filtration, chromatography, electrophoresis and the like.
Fungal Strains of the Invention
The invention also provides Candida albicans strains which may be used for drug screening. According to the invention, one copy of an essential polynucleotide of the invention is eliminated (such as those encoding an essential polypeptide having the amino acid sequence of SEQ ID NO:12 through to SEQ ID NO 22), while the second allele is placed under the control of a regulatable promoter.
In a preferred embodiment, precise replacement of one copy of a target polynucleotide is facilitated by using a PCR-based polynucleotide disruption tool (see, e.g., Wilson et al., J. Bacteriol. 181:1868-1874 (1999), herein incorporated by reference). Genes are disrupted by using PCR to generate a selectable marker (for example, URA3) surrounded by the 5′ and 3′ sequences of the polynucleotide to be disrupted. The marker sequences are part of a plasmid (see, e.g., Example 4). Disruption cassettes are synthesized by PCR using primers containing both short flanking homology (SFH) regions and regions which anneal to the marker in the plasmid. The SHF regions are about 25 to about 60 bp long. The disruption cassettes are generated in one-step PCR synthesis and homologous recombination transplaces the recombinant null construct into the genome, generating an allele such as yfg1::URA3 (yfg1 is “your favorite gene”).
Where the polynucleotide is essential, elimination of both alleles will be lethal or severely crippling for growth. Therefore, in the present invention, a regulatable promoter is used to provide a range of levels of expression of the second allele. Depending on the conditions, the second allele can be non-expressing, underexpressing, or expressing at a normal level relative to that when the allele is linked to its native promoter.
Regulatable promoters include, but are not limited to, those such as MET25, MAL2, PHO5,5 GAL I; STE2, or STE3. A preferred regulatable promoter is the MET3 promoter.
Preferably, replacement of the promoter of the second copy with the MET3 promoter is accomplished by the use of a promoter swapping cassette. The “PCR-based promoter swapping cassette” as used herein refers to a cassette comprised of a regulatable promoter, a selectable marker, and short flanking regions to on the 5′ and 3′ ends of the cassette which are homologous to the native promoter and the start of the polynucleotide coding region, respectively.
This promoter swapping cassette works analogously to the PCR based polynucleotide disruption cassette described above. A promoter of ‘YFG’ is disrupted by using PCR to generate a selectable marker (for example, URA3 or ARG4) containing a sequence of the promoter region to be disrupted. Disruption cassettes are synthesized by PCR, using short flanking homology (SFH) regions to the promoter of interest. SHF regions are about 25 to about 60 bp long. When the disruption cassette is transformed into yeast cells, the promoter of YFG is displaced by homologous recombination.
In order to replace the endogenous promoter of YFG, the PCR-based promoter swapping cassette contains a selective marker amplified from a plasmid using a pair of primers designed so that the forward primer contains about 50 to about 60 bp of flanking sequences that are derived from sequences 500-100 bp upstream of the ATG codon of YFG to ensure it will anneal upstream or on the boundary of the endogenous promoter and this portion of the forward primer is attached to the 5′-end of the forward common promoter primer. The forward common promoter primer region is common to the plasmid.
The reverse primer has 20 to 60 or preferably about 50 to about 60 bp of flanking sequences, which are derived from the start codon region of the polynucleotide or ORF including ATG attached to the 3′ end of the reverse common primer. The resulting PCR product contains the promoter cassette that is flanked by about 50 to about 60 bp of sequences, on either end, homologous to the upstream promoter region and to the coding region of the polynucleotide of interest, respectively. Once introduced into the cells heterozygous for the polynucleotide of interest obtained via the regular PCR-based polynucleotide disruption approach, the PCR-based promoter swapping cassette would replace the endogenous promoter of the remaining allele via homologous recombination.
The MET3 promoter for use in the construction of the C. albicans strains as described above is a homologue of S. cervisiae MET3. The cloning of this promoter is described in Care et al., supra). The MET3 polynucleotide of S. cervisiae encodes ATP sulphurylase (ATP: sulphate adenyltransferase, E.C. 2.7.7.4) which catalyses the production of adenosine 5′-phosphosulphate (APS) from inorganic sulphate and ATP, the first step in the assimilation of inorganic sulphate. Expression of MET3 is repressed by exogenous methionine and S-adenysl methionine (SAM). Methionine is converted into SAM, which is thought to be the true repressor. Sulphur assimilation is also required for the biosynthesis of cysteine. According to an alternative view, cysteine is the true repressor of the enzymes of sulphur metabolism, the action of methionine and SAM being dependent on the interconversion of sulphur-containing amino acids through transulphurylation pathways (Care et al. supra, herein incorporated by reference).
In the methods of the invention, the MET3 promoter may be completely or partially repressed using cysteine, methionine or both amino acids.
In particular, the present invention encompasses strains of Candida albicans cells in which both alleles of a polynucleotide are modified. A first copy of a polynucleotide comprising a nucleotide sequence selected from the group consisting of one of SEQ ID NO 1 to 11 is inactive and a second copy of the polynucleotide is under the control of a regulatable promoter.
Additionally, the invention also provides a strain of Candida albicans having a nucleic acid molecule comprising a nucleotide sequence selected from one of SEQ ID NO: 1 to 11 under the control of a regulatable promoter.
Target Evaluation in Animal Model Systems
In one embodiment, the essential strains provided by the invention are used in animal studies to examine the effect of polynucleotide inactivation by conditional expression. Animal studies, using mice, for example, may be inoculated with one or more of the strains of the invention. In a more desirable embodiment of the invention, the effect on mice injected with a lethal inoculum of one of the essential strains could be determined depending on whether the mice were provided with an appropriate concentration of promoter repressor to inactivate expression of a drug target polynucleotide. The lack of expression of a polynucleotide demonstrated to be essential under laboratory conditions can thus be correlated with prevention of a terminal C. albicans infection. In this type of experiment, only mice “treated” with promoter repressor supplements are predicted to survive infection because inactivation of the target polynucleotide has killed the essential strain pathogen within the host.
Identification of Essential Genes
Also provided herein are methods to identify essential polynucleotides. In order to determine essentially, a strain is constructed as described above and then the strain is cultured under conditions wherein the second modified allele of the polynucleotide which is under conditional expression is substantially underexpressed or not expressed. A “promoter repressor”, i.e., a substance that inhibits the ability of a regulatable promoter to cause expression in the second allele is added to the culture medium. The preferred promoter repressor used herein may be methionine and cysteine or a combination thereof when the MET3 promoter is used in the compositions and methods of the invention. The preferred regulatable promoter is MET3. The viability and/or growth of the strain is compared with that of control cells cultured without the addition of promoter supressor. A loss or reduction of viability or growth in the cells cultured with promoter suppressor indicates that the polynucleotide is essential to the survival of the fungus.
The fungal strains and cells used to identify essential polynucleotides with the method of the invention, include but are not limited to Absidia corymbigera, Aspergillus flavis, Aspergillus fumigatus, Aspergillus niger, Botrytis cinerea, Candida dublinensis, Candida glabrata, Candida krusei, Candia parapsilopsis, Candia tropicalis, Coccidioides immitis, Cryptococcus neoformans, Erysiphe graminis, Exophalia dermatiditis, Fusarium osysproum, Histoplasma capsulatum, Magnaporthe grisea, Mucor rouxii, Pneumocystis carinii, Puccinia graminis, Puccinia recodita, Rhizomucor pusillus, Puccinia striiformis, Rhizopus arrhizus, Septoria avenae, Septoria nodorum, Septoria triticii, Tilletia controversa, Tilletia tritici, Trichospoon beigelii and Ustilago maydis. Preferably, Candida albicans strains are used.
Haploid or diploid strains may used to identify essential polynucleotides by the method of the invention. In the case of haploid strains, the promoter of the polynucleotide of interest is replaced with a regulatable promoter and tested for essentiality as described herein. Since there is no diploidy, the first step using a PCR based disruption cassette is not required.
The nucleotide sequences encoding candidate polynucleotides which are assayed using the method of the invention to determine essentiality are preferably conserved polynucleotides. A polynucleotide can be identified as belonging to a repertoire of conserved polynucleotides using several methods. For example, an isolated polynucleotide may be used as a hybridization probe under low stringency conditions to detect other members of the repertoire of conserved polynucleotides present in genomic DNA using the methods described by Southern, J. Mol. Biol., 98:503 (1975). Additionally, conserved polynucleotides can be identified using a concordance analysis such as that described herein.
Strains Hypersensitive to Drugs and Titration of Gene Products
Also, provided herein are methods to create strains which are hypersensitive to potential antifungal drugs. Current cell based assays used to identify or to characterize compounds for drug discovery and development frequently depend on detecting the ability of a test compound to modulate the activity of a target molecule located within a cell or located on the surface of a cell. Most often such target molecules are proteins such as enzymes, receptors and the like. However, target molecules also include other molecules such as DNAs, lipids, carbohydrates and RNAs including messenger RNAs, ribosomal RNAs, tRNAs and the like. A number of highly sensitive cell-based assay methods are available to those of skill in the art to detect binding and interaction of test compounds with specific target molecules. However, these methods are generally not highly effective when the test compounds binds to or otherwise interacts with its target molecule with moderate or low affinity. Thus, current cell-based assay methods are limited in that they are not effective in identifying or characterizing compounds that interact with their targets with moderate to low affinity or compounds that interact with targets that are not readily accessible.
The methods of the invention to create cells which are hypersensitive to potential antifungal compounds may be used to overcome these limitations. The sensitizing assays of the present invention are capable of detecting compounds exhibiting low or moderate potency against the target molecule of interest because such compounds are substantially more potent on sensitized cells than on non-sensitized cells. The effect may be such that a test compound may be two to several times more potent, at least 10 times more potent, at least 20 times more potent, at least 50 times more potent, at least 100 times more potent, at least 1000 times more potent, or even more than 1000 times more potent when tested on the sensitized cells as compared to the non-sensitized cells.
Such assays are useful to identify compounds that previously would not have been readily identified. A target which expresses a significant amount of product may not result in a particular effect by a particular compound. However, when the amount of product is reduced, the compound may be revealed to in fact have an effect on the polynucleotide product. An initial hit compound which exhibits moderate or even low potency may be able to be developed into a drug. For example, once a hit compound is identified with low or moderate potency, a combinatorial chemical library consisting of compounds with structures related to the hit compound but containing systematic variations including additions, subtractions and substitutions of various structural features may be included. When tested for activity against the target molecule, structural features may be identified that either alone or in combination with other features enhance or reduce activity. This information may be used to design subsequent directed libraries containing compounds with enhanced activity against the target molecule. After one or several iterations of this process, compounds with substantially increased activity against target molecules are identified and may be further developed as drugs. This process is facilitated by the use of the sensitized strains of the present invention since compounds acting at the selected targets exhibit increased potency in such cell-based assays, thus, more compounds can now be characterized providing more useful information than would be obtained otherwise.
The method of sensitizing a cell entails selecting an essential polynucleotide such as those identified in the present invention. The next step is to obtain a cell in which the level or activity of the target can be reduced to a level where it is rate limiting for viability. For example, the cell may be a strain of the present invention in which the selected polynucleotide is under the control of a MET3 promoter. The amount of RNA transcribed from the selected polynucleotide is limited by varying the concentration of methionine, cysteine or both, which acts on the MET3 promoter, thereby varying the activity of the promoter driving transcription of the RNA. Thus, cells are sensitized by exposing them to a repressor concentration that results in an RNA level such that the function of the selected polynucleotide product becomes rate limiting for fungal growth, survival or proliferation.
In one embodiment of the present invention, a Candida strain is created by inactivating one copy of a polynucleotide by the insertion of a nucleotide sequence encoding a selectable marker and the second polynucleotide copy has been modified by recombination with a promoter swapping cassette, to place the second copy under the controlled expression of a MET3 promoter. The strain is then grown under a first set of conditions where the MET3 promoter is expressed at a relatively low level and the extent of growth is determined. This measurement may be carried out using any appropriate standard known to those skilled in the art, including optical density, wet weight of pelleted cells, total cell count, viable count, DNA content and the like. This experiment is repeated in the presence of a test compound and a second measurement of growth is obtained. The estimate of growth in the presence and in the absence of the test compound, which can conveniently be expressed in terms of indicator values, are then compared. A dissimilarity in the extent of growth or indicator values provides an indication that the test compound may interact with the target essential polynucleotide product.
To gain additional information, additional experiments may be performed in various embodiments. For example, using a second set of non-repressing growth conditions where the second polynucleotide copy, under the control of the MET3 promoter, is expressed at various levels higher that in the rest set of conditions described above. The extent of growth or indicator values is determined in the presence and absence of the test compound under this second set of conditions. The extent of growth or indicator values in the presence and in the absence of the test compound are then compared. A dissimilarity in the extent of growth or indicator values provides an indication that the test compounds may interact with the target essential polynucleotide product.
Furthermore, the extent of growth in the first and in the second set of growth conditions can also be compared. If the extent of growth is essentially the same, the data suggest that the test compound does not inhibit the polynucleotide product encoded by the modified allelic polynucleotide pair carried by the strain tested. However, if the extent of growth is substantially different, the data indicate that the level of expression of the subject polynucleotide product may determine the degree of inhibition by the test compound and therefore it is likely that the subject polynucleotide product is the target of that test compound.
In one embodiment, the strains of the invention in which the sequence required for fungal growth, survival or proliferation of Candida described herein is under the control of MET3, and grown in the presence of a concentration of promoter repressor which causes the function of the polynucleotide products encoded by these sequences to be rate limiting for fungal growth. To achieve that goal, a growth inhibition dose curve is calculated by plotting various doses of repressor against corresponding growth inhibition caused by the limited levels of the polynucleotide product required for fungal proliferation. From this dose-response curve, conditions providing various growth rates for 1 to 100%, as compared to repressor-free growth, can be determined. For example, the diploid fungal strains of the invention are grown in medium comprising a range of methionine concentrations to obtain the growth inhibitory response curve for each strain. First, seed cultures of the diploid fungal strains of the invention are grown in the appropriate medium. Subsequently, aliquots of the seed cultures are diluted into medium containing varying concentrations of methionine. For example, the strains may be grown in duplicate cultures containing two-fold serial dilutions of methionine Additionally, control cells are grown in duplicate without methionine. The control cultures are started from equal amounts of cells derived from the same initial seed culture of the strain of interest. The cells are grown for an appropriate period of time and the extent of growth is determined using any appropriate technique For example, the extent of growth may be determined by measuring the optical density of the cultures. When the control culture reaches mid-log phase the percent growth (relative to the control culture) for each of the methionine containing cultures is plotted against the log concentrations of methionine to produce a growth inhibitory dose response curve for methionine. The concentration of methionine that inhibits cell growth at 50% (IC50) as compared to the 0 mM methionine control (0% growth inhibition) is then calculated from the curve. Alternative methods of measuring growth are also contemplated. Examples of these methods include measurements of protein, the expression of which is engineered of the cells being tested and can readily be measured.
Thus, in one embodiment, the method described above may be used to titrate the amount of essential polynucleotide product expressed in a diploid fungal cell.
In another embodiment, a homologue of the essential polynucleotide sequences of the present invention that are identified in a haploid organism may similarly be used as the basis for detection of an antifungal or therapeutic agent. In this embodiment, the test organism (e.g., Aspergillus fumigatus or Cryptococcus neoformans) or any other haploid organism in a strain constructed by modifying the single allele of the target polynucleotide in one step recombination with a promoter swapping cassette such that the expression of the polynucleotide is conditionally regulated by the promoter. Like individual diploid strains of the invention, sensitized haploid cells may be similarly used in whole cell-based assay methods to identify compounds displaying a preferential activity against the affected target.
In various embodiments, the modified strain is grown under a first set of conditions where the regulatable promoter is expressed at a relatively low level and the extent of growth determined. This experiment is repeated in the presence of a test compound and a second measurement of growth obtained. The extent of growth in the presence and in the absence of the test compounds are then compared to provide a first indicator value. Two further experiments are performed using non-repressing growth conditions where the target polynucleotide is expressed at substantially higher levels than in the first set of conditions. Extent of growth is determined in the presence and absence of the test compound under the second set of conditions to obtain a second indicator value. The first and second indicator values are then compared. If the indicator values are essentially the same, the data suggest that the test compound does not inhibit the test target. However, if the two indicator values are substantially different, the data indicate that the level of expression of the target polynucleotide product may determine the degree of inhibition by the test compounds and therefore it is likely that the polynucleotide product is the target of that test compound. Whole-cell assays comprising collections or subsets of multiple sensitized strains may be screened, for example, in a series of 96 well, 384 well or even 1586 well microtiter plates.
Cells to be assayed are exposed to the above-determined concentrations of methionine or other promoter repressor. The presence of the repressor at this sub-lethal concentration reduces the amount of the proliferation-required polynucleotide product to the lowest amount in the cell that will support growth. Cells grown in the presence of this concentration of repressor are more sensitive to inhibitors of the proliferation-required protein or RNA of interest as well as to inhibitors of proteins or RNAs in the same biological pathway as the proliferation-required protein or RNA of interest but not specifically more sensitive to inhibitors of unrelated proteins or RNAs.
Cells pretreated with sub-inhibitory concentrations of repressors which therefore contain a reduced amount of proliferation-required target polynucleotide product are used to screen for compounds that reduce cell growth. The sub-lethal concentration of repressor may be any concentration consistent with the intended use of the assay to identify candidate compounds to which the cells are more sensitive than are control cells in which this polynucleotide product is not rate-limiting. For example, the sub-lethal concentration of the repressor may be such that growth inhibition is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least 80%, at least 90%, at least 95% or more than 96%. Cells which are pre-sensitized using the preceding method are more sensitive to inhibitors of the target protein because these cells contain less target protein to inhibit than wild-type cells. Cells are then contacted with a candidate compound and growth of the cells in the methionine containing medium is compared to growth of the control cells in medium which lacks methionine to determine whether the candidate compound inhibits growth of the sensitized cells (i.e., cells grown in the presence of methionine) to a greater extent than the candidate compound inhibits the growth of cells grown in the absence of methionine. For example, if a significant difference in growth is observed between the sensitized cells and the non-sensitized cells, the candidate compound may be used to inhibit the proliferation of the organism or may be further optimized to identify compounds which have an even greater ability to inhibit the growth survival or proliferation of the organism.
When screening for antimicrobial agents against a polynucleotide product required for fungal growth, survival or proliferation or growth inhibition of cells containing a limiting amount of that polynucleotide product can be assayed. Growth inhibition can be measured by directly comparing the amount of growth measured by the optical density of the culture relative to uninoculated growth medium between and experimental sample and a control sample. Alternative methods for assaying cell proliferation include measuring green fluorescent protein report construct emissions, various enzymatic activity assays and other methods well-known in the art.
It will be appreciated that the above cell-based assays may be used to identify compounds which inhibit the activity of polynucleotide products from organisms other than Candida albicans which are homologous to the Candida albicans nucleotide sequences encoding essential polypeptides described herein. For example, the nucleotide sequences encoding polypeptides may be from animal fungal pathogens such as Aspergillus fumigatus, Aspergillus niger, Aspergillus flavis, Candida tropicalis, Candida parapsilopsis, Candida krusei, Cryptococcus neofomras, Coccidioides immitis, Exophalia dermatiditis, Fusarium oxygporum, Histoplasma capsulaturm, Pneumocystis carinii, Trichosporan beigelii, Rhizopus arrhizus, Mucor rouxii, Rhizomucor pusillus or Absidia corymbigera or the plant fungal pathogens such as Botrytis cinerea, Erysiphe graminis, Magnaporthe grisea, Puccinia recodita, Spetoria triticii, Tilletia controversa, Ustilago maydis or any species falling with the genera of any of the above species. In some embodiments, the essential polynucleotides are from an organism other than Saccharomyces cerevisiae. 
Protein Based Assays
The present invention also provides methods for identifying an antimycotic compound comprising screening a plurality of compounds to identify a compound that modulates the activity or level of a polynucleotide product (mRNA or protein), said polynucleotide product being encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11, or a nucleotide sequence that is the homologue of a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11.
Binding assays may be used to identify antimycotic compounds. These assays involve preparing a reaction mixture comprising the target polynucleotide product and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which is removed and/or detected within the reaction mixture. These assays may be conducted in a variety of ways. For example, one method involves anchoring a target polynucleotide product or the test substance onto a solid phase and detecting target polynucleotide product/test compound complexes anchored, via the intermolecular binding reaction to the solid phase at the end of the reaction. In one embodiment, the target polynucleotide product is anchored onto a solid surface and the test compound which is not anchored is labeled either directly or indirectly.
Microtiter plates may be utilized as the solid phase. The anchored component is immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished by simply coating the solid surface with a solution of the protein and drying the coated surface. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized is used to anchor the protein to the solid surface.
Alternatively, a reaction is conducted in a liquid phase, the reaction products are separated from unreacted components, and complexes are detected; e.g., using an immobilized antibody specific for the target polynucleotide product or for the test compound, to anchor complexes formed in solution, and a second labeled antibody, specific for the other component of the complex to allow detection of anchored complexes.
In another aspect of the invention, methods are employed to for detecting protein-protein interactions for identifying novel target protein-cellular or extracellular protein interactions. Any suitable method may be used.
The target polynucleotide products of the invention may interact, in vivo, with one or more cellular or extracellular macromolecules, such as proteins. Such macromolecules include, but are not limited to, nucleic acid molecules and proteins identified via methods such as those described above. For purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as “binding partners.” Compounds that disrupt such interactions can be useful in regulating the activity of the target polynucleotide protein, especially mutant target polynucleotide proteins. Such compounds include, but are not limited to molecules such as antibodies, peptides, and the like.
The basic principle of the assay systems used to identify compounds that interfere with the interaction between the target polynucleotide product and its cellular or extracellular binding partner or partners involves preparing a reaction mixture containing the target polynucleotide product and the binding partner under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound is initially included in the reaction mixture, or added at a time subsequent to the addition of target polynucleotide product and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound. The formation of complexes between the target polynucleotide protein and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target polynucleotide protein and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target polynucleotide protein can also be compared to complex formation within reaction mixtures containing the test compound and a mutant target polynucleotide protein. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt intermolecular interactions involving mutant but not normal target polynucleotide proteins.
The assay for compounds that interfere with the interaction of the target polynucleotide products and binding partners is conducted in either a heterogeneous or a homogeneous format.
Heterogeneous assays involve anchoring either the target polynucleotide product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants is varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target polynucleotide products and the binding partners, e.g., by competition, are identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the target polynucleotide protein and an interacting cellular or extracellular binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, are tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.
In a heterogeneous assay system, either the target polynucleotide protein or the interactive cellular or extracellular binding partner, is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized. The anchored species is immobilized either by non-covalent or covalent attachment.
Non-covalent attachment is accomplished simply by coating the solid surface with a solution of the target polynucleotide product or binding partner and drying the coated surface. Alternatively, an immobilized antibody specific for the species to be anchored is used to anchor the species to the solid surface. The surfaces can be prepared in advance and stored.
In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface is accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, is directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes are detected.
Alternatively, the reaction is conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a second, labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex or which disrupt preformed complexes are identified.
In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the target polynucleotide protein and the interacting cellular or extracellular binding partner is prepared in which either the target polynucleotide product or its binding partner is labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex results in the generation of a signal above background. In this way, test substances which disrupt target polynucleotide protein/cellular or extracellular binding partner interaction are identified.
In a particular embodiment the target polynucleotide product is prepared for immobilization using recombinant DNA techniques described above. For example, the target polynucleotide coding region is fused to a glutathione-5-transferase (GST) polynucleotide using a fusion vector, such as pGEX-5X-1, in such a manner that its binding activity is maintained in the resulting fusion protein. The interactive cellular or extracellular binding partner is purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and as described below. This antibody is labeled with the radioactive isotope “ ”, for example, by methods routinely practiced in the art. In a heterogeneous assay, e.g., the GST-target polynucleotide fusion protein is anchored to glutathione-agarose beads. The interactive cellular or extracellular binding partner is then added in the presence or absence of the test compound in a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody is added to the system and allowed to bind to the complexed components. The interaction between the target polynucleotide protein and the interactive cellular or extracellular binding partner is detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound results in a decrease in measured radioactivity.
Alternatively, the GST-target polynucleotide fusion protein and the interactive cellular or extracellular binding partner are mixed together in liquid in the absence of the solid glutathione-agarose beads. The test compound is added either during or after the species are allowed to interact. This mixture is added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the target polynucleotide product/binding partner interaction is detected by adding the labeled antibody and measuring the radioactivity associated with the beads.
In another embodiment of the invention, these same techniques are employed using peptide fragments that correspond to the binding domains of the target polynucleotide product and/or the interactive cellular or extracellular binding partner (in cases where the binding partner is a protein), in place of one or both of the full length proteins. Any number of methods routinely practiced in the art are used to identify and isolate the binding sites. These methods include, but are not limited to, mutapolynucleotidesis of the polynucleotide encoding one of the proteins and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the polynucleotide encoding the second species in the complex are then selected. Sequence analysis of the polynucleotides encoding the respective proteins reveals the mutations that correspond to the region of the protein involved in interactive binding. Alternatively, one protein is anchored to a solid surface using methods described above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain remains associated with the solid material, and can be isolated and identified by amino acid sequencing. Also, once the polynucleotide coding for the cellular or extracellular binding partner is obtained, short polynucleotide segments are engineered to express peptide fragments of the protein, which are tested for binding activity and purified or synthesized.
For example, and not by way of limitation, a target polynucleotide product is anchored to a solid material as described, above, by making a GST-target polynucleotide fusion protein and allowing it to bind to glutathione agarose beads. The interactive cellular or extracellular binding partner is labeled with a radioactive isotope, and cleaved with a proteolytic enzyme such as trypsin. Cleavage products are added to the anchored GST-target polynucleotide fusion protein and allowed to bind. After washing away unbound peptides, labeled bound material, representing the cellular or extracellular binding partner binding domain, is eluted, purified, and analyzed for amino acid sequence by well-known methods. Peptides so identified are produced synthetically or fused to appropriate facilitative proteins using well-known recombinant DNA technology.
In one embodiment of the present invention, the proteins encoded by the fungal polynucleotides identified using the methods of the present invention are isolated and expressed. These recombinant proteins are then used as targets in assays to screen libraries of compounds for potential drug candidates. The generation of chemical libraries is well-known in the art. For example, combinatorial chemistry is used to generate a library of compounds to be screened in the assays described herein. A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical “building block” reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining amino acids in every possible combination to yield peptides of a given length. Millions of chemical compounds theoretically can be synthesized through such combinatorial mixings of chemical building blocks. For example, one commentator observed that the systematic, combinatorial mixing of interchangeable chemical building blocks results in the theoretical synthesis of million tetrameric compounds or billion pentameric compounds. (Gallop et al., “Applications of Combinatorial Technologies to Drug Discovery, Background and Peptide Combinatorial Libraries,” Journal of Medicinal Chemistry, Vol. 37, No. 9, 1233-1250 (1994). Other chemical libraries known to those in the art may also be used, including natural product libraries.
Once generated, combinatorial libraries are screened for compounds that possess desirable biological properties. For example, compounds which may be useful as drugs or to develop drugs would likely have the ability to bind to the target protein identified, expressed and purified as discussed above. Further, if the identified target protein is an enzyme, candidate compounds would likely interfere with the enzymatic properties of the target protein. For example, the enzymatic function of a target protein may be to serve as a protease, nuclease, phosphatase, dehydrogenase, transporter protein, transcriptional enzyme, replication component, and any other type of enzyme known or unknown. Thus, the present invention contemplates using the protein products described above to screen combinatorial chemical libraries.
In some embodiments of the present invention, the biochemical activity of the protein, as well as the chemical structure of a substrate on which the protein acts is known. In other embodiments of the present invention, the biochemical activity of the target protein is unknown and the target protein has no known substrates.
In some embodiments of the present invention, libraries of compounds are to identify compounds that function as inhibitors of the target polynucleotide product. First, a library of small molecules is generated using methods of combinatorial library formation well-known in the art. U.S. Pat. Nos. 5,463,564 and 5,574, 656, to Agraflotis, et al., entitled “System and Method of Automatically Generating Chemical Compounds with Desired Properties”, the disclosures of which are incorporated herein by reference in their entireties, are two such teachings. Then the library compounds are screened to identify those compounds that possess desired structural and functional properties. U.S. Pat. No. 5,684,711, the disclosure of which is incorporated herein by reference in its entirety, also discusses a method for screening libraries.
To illustrate the screening process, the target polynucleotide product, an enzyme, and chemical compounds of the library are combined and permitted to interact with one another. A labeled substrate is added to the incubation. The label on the substrate is such that a detectable signal is emitted from metabolized substrate molecules. The emission of this signal permits one to measure the effect of the combinatorial library compounds on the enzymatic activity of target enzymes by comparing it to the signal emitted in the absence of combinatorial library compounds. The characteristics of each library compound are encoded so that compounds demonstrating activity against the enzyme can be analyzed and features common to the various compounds identified can be isolated and combined into future iterations of libraries.
Once a library of compounds is screened, subsequent libraries are generated using those chemical building blocks that possess the features shown in the first round of screen to have activity against the target enzyme. Using this method, subsequent iterations of candidate compounds will possess more and more of those structural and functional features required to inhibit the function of the target enzyme, until a group of enzyme inhibitors with high specificity or the enzyme can be found. These compounds can then be further tested for their safety and efficacy as antibiotics for use in mammals.
It will be readily appreciated that this particular screening methodology is exemplary only. Other methods are well-known to those skilled in the art. For example, a wide variety of screening techniques are known for a large number of naturally-occurring targets when the biochemical function of the target protein is known. For example, some techniques involve the generation and use of small peptides to probe and analyze target proteins both biochemically and genetically in order to identify and develop drug leads. Such techniques include the methods described in PCT Publications Nos. WO9935494 and WO9819162.
Drug Screening
The fungal essential polypeptides and/or peptides of the present invention, or immunogenic fragments or oligopeptides thereof, can be used for screening therapeutic drugs or compounds in a variety of drug screening techniques. The fragment employed in such a screening assay may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The reduction or abolition of activity of the formation of binding complexes between the ion channel protein and the agent being tested can be measured. Thus, the present invention provides a method for screening or assessing a plurality of compounds for their specific binding affinity with a fungal essential polypeptide, or a bindable peptide fragment, of this invention, comprising providing a plurality of compounds, combining the fungal essential polypeptide, or a bindable peptide fragment, with each of a plurality of compounds for a time sufficient to allow binding under suitable conditions and detecting binding of the fungal essential polypeptide or peptide to each of the plurality of test compounds, thereby identifying the compounds that specifically bind to the fungal essential polypeptide or peptide.
Methods of identifying compounds that modulate the activity of the novel fungal essential polypeptides and/or peptides are provided by the present invention and comprise combining a potential or candidate compound or drug modulator of biological activity with an fungal essential polypeptide or peptide, for example, the fungal essential amino acid sequence as set forth in SEQ ID NO:2, and measuring an effect of the candidate compound or drug modulator on the biological activity of the fungal essential polypeptide or peptide. Such measurable effects include, for example, physical binding interaction; the ability to cleave a suitable substrate; effects on native and cloned fungal essential-expressing cell line; and effects of modulators or other-mediated physiological measures.
Another method of identifying compounds that modulate the biological activity of the novel fungal essential polypeptides of the present invention comprises combining a potential or candidate compound or drug modulator of a biological activity with a host cell that expresses the fungal essential polypeptide and measuring an effect of the candidate compound or drug modulator on the biological activity of the fungal essential polypeptide. The host cell can also be capable of being induced to express the fungal essential polypeptide, e.g., via inducible expression. Physiological effects of a given modulator candidate on the fungal essential polypeptide can also be measured. Thus, cellular assays for particular modulators may be either direct measurement or quantification of the physical biological activity of the fungal essential polypeptide, or they may be measurement or quantification of a physiological effect. Such methods preferably employ a fungal essential polypeptide as described herein, or an overexpressed recombinant fungal essential polypeptide in suitable host cells containing an expression vector as described herein, wherein the fungal essential polypeptide is expressed, overexpressed, or undergoes upregulated expression.
Another aspect of the present invention embraces a method of screening for a compound that is capable of modulating the biological activity of a fungal essential polypeptide, comprising providing a host cell containing an expression vector harboring a nucleic acid sequence encoding a fungal essential polypeptide, or a functional peptide or portion thereof (e.g., SEQ ID NOS:2); determining the biological activity of the expressed fungal essential polypeptide in the absence of a modulator compound; contacting the cell with the modulator compound and determining the biological activity of the expressed fungal essential polypeptide in the presence of the modulator compound. In such a method, a difference between the activity of the fungal essential polypeptide in the presence of the modulator compound and in the absence of the modulator compound indicates a modulating effect of the compound.
Essentially any chemical compound can be employed as a potential modulator or ligand in the assays according to the present invention. Compounds tested as modulators can be any small chemical compound, or biological entity (e.g., protein, sugar, nucleic acid, lipid). Test compounds will typically be small chemical molecules and peptides. Generally, the compounds used as potential modulators can be dissolved in aqueous or organic (e.g., DMSO-based) solutions. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source. Assays are typically run in parallel, for example, in microtiter formats on microtiter plates in robotic assays. There are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland), for example. Also, compounds may be synthesized by methods known in the art.
High throughput screening methodologies are particularly envisioned for the detection of modulators of the novel fungal essential polynucleotides and polypeptides described herein. Such high throughput screening methods typically involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (e.g., ligand or modulator compounds). Such combinatorial chemical libraries or ligand libraries are then screened in one or more assays to identify those library members (e.g., particular chemical species or subclasses) that display a desired characteristic activity. The compounds so identified can serve as conventional lead compounds, or can themselves be used as potential or actual therapeutics.
A combinatorial chemical library is a collection of diverse chemical compounds generated either by chemical synthesis or biological synthesis, by combining a number of chemical building blocks (i.e., reagents such as amino acids). As an example, a linear combinatorial library, e.g., a polypeptide or peptide library, is formed by combining a set of chemical building blocks in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide or peptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
The preparation and screening of combinatorial chemical libraries is well known to those having skill in the pertinent art. Combinatorial libraries include, without limitation, peptide libraries (e.g. U.S. Pat. No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; and Houghton et al., 1991, Nature, 354:84-88). Other chemistries for generating chemical diversity libraries can also be used. Nonlimiting examples of chemical diversity library chemistries include, peptoids (PCT Publication No. WO 91/019735), encoded peptides (PCT Publication No. WO 93/20242), random bio-oligomers (PCT Publication No. WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc. Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagihara et al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J. Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of small compound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116:2661), oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidyl phosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries (e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) and PCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996, Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organic molecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993, page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; and the like).
Devices for the preparation of combinatorial libraries are commercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, a large number of combinatorial libraries are commercially available (e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, Russia; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., and the like).
In one embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the cell or tissue expressing an ion channel is attached to a solid phase substrate. In such high throughput assays, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to perform a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 96 modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; thus, for example, assay screens for up to about 6,000-20,000 different compounds are possible using the described integrated systems.
In another of its aspects, the present invention encompasses screening and small molecule (e.g., drug) detection assays which involve the detection or identification of small molecules that can bind to a given protein, i.e., a fungal essential polypeptide or peptide. Particularly preferred are assays suitable for high throughput screening methodologies.
In such binding-based detection, identification, or screening assays, a functional assay is not typically required. All that is needed is a target protein, preferably substantially purified, and a library or panel of compounds (e.g., ligands, drugs, small molecules) or biological entities to be screened or assayed for binding to the protein target. Preferably, most small molecules that bind to the target protein will modulate activity in some manner, due to preferential, higher affinity binding to functional areas or sites on the protein.
An example of such an assay is the fluorescence based thermal shift assay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) as described in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano et al.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assay allows the detection of small molecules (e.g., drugs, ligands) that bind to expressed, and preferably purified, ion channel polypeptide based on affinity of binding determinations by analyzing thermal unfolding curves of protein-drug or ligand complexes. The drugs or binding molecules determined by this technique can be further assayed, if desired, by methods, such as those described herein, to determine if the molecules affect or modulate function or activity of the target protein.
To purify a fungal essential polypeptide or peptide to measure a biological binding or ligand binding activity, the source may be a whole cell lysate that can be prepared by successive freeze-thaw cycles (e.g., one to three) in the presence of standard protease inhibitors. The fungal essential polypeptide may be partially or completely purified by standard protein purification methods, e.g., affinity chromatography using specific antibody described infra, or by ligands specific for an epitope tag engineered into the recombinant fungal essential polypeptide molecule, also as described herein. Binding activity can then be measured as described.
Compounds which are identified according to the methods provided herein, and which modulate or regulate the biological activity or physiology of the fungal essential polypeptides according to the present invention are a preferred embodiment of this invention. It is contemplated that such modulatory compounds may be employed in treatment and therapeutic methods for treating a condition that is mediated by the novel fungal essential polypeptides by administering to an individual in need of such treatment a therapeutically effective amount of the compound identified by the methods described herein.
In addition, the present invention provides methods for treating an individual in need of such treatment for a disease, disorder, or condition that is mediated by the fungal essential polypeptides of the invention, comprising administering to the individual a therapeutically effective amount of the fungal essential-modulating compound identified by a method provided herein.
Antibodies
Described herein are methods for the production of antibodies capable of specifically recognizing epitopes of one or more of the essential polynucleotide polypeptides described above.
On one embodiment, the antibodies of the present invention are human antibodies capable of neutralizing a fungal pathogen in a human host, such that the human host can effectively combat the invading pathogen, and thus treat or ameliorate the symptoms caused by the invading pathogen.
In the present invention, “epitopes” refer to polypeptide fragments having antigenic or immunogenic activity in an animal. A preferred embodiment of the present invention relates to a polypeptide fragment including an epitope, as well as the polynucleotide encoding this fragment. A region of a protein molecule to which an antibody can bind is defined as an “antigenic epitope”. In contrast, an “immunogenic epitope” is defined as a part of a protein that elicits an antibody response. (See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).) In the present invention, antigenic epitopes preferably contain a sequence of at least six, more preferably at least nine, and most preferably between about 15 to about 30 amino acids. Antigenic epitopes are useful to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe, J. G. et al., Science 219:660-666 (1983)). Similarly, immunogenic epitopes can be used to induce antibodies according to methods well-known in the art. (See, for instance, Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985), both of which are herein incorporated by reference.) The immunogenic epitopes may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier.
Accordingly, the invention provides a method of eliciting an immune response in an animal, comprising introducing into the animal an immunogenic composition comprising an isolated polypeptide, the amino acid sequence of which comprises at least 6 consecutive residues of one of SEQ ID NO: 12 to SEQ ID NO: 22 or one of SEQ ID NO: 48 to SEQ ID NO: 73.
As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab′)2 fragments) which are capable of specifically binding to protein. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983).). Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library.
As described above, the antibodies are preferably monoclonal, but may also be polyclonal. Monoclonal antibodies may be produced by methods known in the art. These methods include the immunological method described by Kohler and Millstein in Nature vol. 256, pp 495-497 (1975) and by Campbell in “Monoclonal Antibody Technology, The Production And Characterization Of Rodent And Human Hybridomas,” in Burdon, et al. (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology, vol. 13, Elsebier Science Publishers, Amsterdam, Nebr. (1985); and Coligan, J. E., et al. (Eds.), Current Protocols in Immunology, Wiley Intersciences, Inc., New York, (1999); as well as the recombinant DNA method described by Huse, et al., Science, 246:1275-1281 (1989). The recombinant DNA method preferably comprises screening phage libraries for human antibody fragments.
In order to produce monoclonal antibodies, a host mammal is inoculated with an essential polynucleotide peptide or peptide fragment as described above, and then boosted. Spleens are collected from inoculated mammals a few days after the final boost. Cell suspensions from the spleens are fused with a target cell in accordance with the general method described by Kohler and Millstein, Nature, 256:495-497 (1975). In order to be useful, the peptide fragment must contain sufficient amino acid residues to define the epitope of the molecule being detected.
Antibodies directed against an essential polynucleotide polypeptide or fragment thereof can be used therapeutically to treat an infectious disease by preventing infection and/or inhibiting growth of the pathogen. Antibodies can also be used to modify a biological activity of an essential polynucleotide polypeptide. Antibodies to essential polynucleotide polypeptides can also be used to alleviate one or more symptoms associated with infection by the organism. To facilitate or enhance its therapeutic effect, an antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a toxin or fungicidal agent. Techniques for conjugating a therapeutic moiety to antibodies are well-known, see, e.g., Thorpe et al., Immunol. Rev., 62: 119-58 (1982).
Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ ID NO:12 to 22, and/or an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Moreover, the term “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules, as well as, antibody fragments (such as, for example, Fab and F(ab′)2 fragments) which are capable of specifically binding to protein. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation of the animal or plant, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies.
Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homologue of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologues of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M, 10-6M, 5×10-7 M, 107 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M, 10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M, 5×10-15 M, or 10-15 M.
The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.
The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).
Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionucleotides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.
The antibodies of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
The antibodies of the present invention may be generated by any suitable method known in the art.
The antibodies of the present invention may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2nd ed. (1988); and Current Protocols, Chapter 2; which are hereby incorporated herein by reference in its entirety). In a preferred method, a preparation of the fungal essential polynucleotide protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. The administration of the polypeptides of the present invention may entail one or more injections of an immunizing agent and, if desired, an adjuvant. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art. For the purposes of the invention, “immunizing agent” may be defined as a polypeptide of the invention, including fragments, variants, and/or derivatives thereof, in addition to fusions with heterologous polypeptides and other forms of the polypeptides described herein.
Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections, though they may also be given intramuscularly, and/or through IV). The immunizing agent may include polypeptides of the present invention or a fusion protein or variants thereof. Depending upon the nature of the polypeptides (i.e., percent hydrophobicity, percent hydrophilicity, stability, net charge, isoelectric point etc.), it may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Such conjugation includes either chemical conjugation by derivitizing active chemical functional groups to both the polypeptide of the present invention and the immunogenic protein such that a covalent bond is formed, or through fusion-protein based methodology, or other methods known to the skilled artisan. Examples of such immunogenic proteins include, but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Additional examples of adjuvants which may be employed includes the MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
The antibodies of the present invention may comprise monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2nd ed. (1988), by Hammerling, et al., Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, N.Y., pp. 563-681 (1981); Köhler et al., Eur. J. Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976), or other methods known to the artisan. Other examples of methods which may be employed for producing monoclonal antibodies includes, but are not limited to, the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
In a hybridoma method, a mouse, a humanized mouse, a mouse with a human immune system, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
The immunizing agent will typically include polypeptides of the present invention or a fusion protein thereof. Preferably, the immunizing agent consists of an fungal essential polynucleotide polypeptide or, more preferably, with a fungal essential polynucleotide polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56 degrees C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin. Generally, either peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pp. 59-103). Immortalized cell lines are usually, transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. More preferred are the parent myeloma cell line (SP20) as provided by the ATCC. As inferred throughout the specification, human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptides of the present invention. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or, enzyme-linked immunoabsorbant assay (ELISA). Such techniques are known in the art and within the skill of the artisan. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollart, Anal. Biochem., 107:220 (1980).
After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra, and/or according to Wands et al. (Gastroenterology 80:225-232 (1981)). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The skilled artisan would acknowledge that a variety of methods exist in the art for the production of monoclonal antibodies and thus, the invention is not limited to their sole production in hydridomas. For example, the monoclonal antibodies may be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. In this context, the term “monoclonal antibody” refers to an antibody derived from a single eukaryotic, phage, or prokaryotic clone. The DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to polynucleotides encoding the heavy and light chains of murine antibodies, or such chains from human, humanized, or other sources). The hydridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transformed into host cells such as Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al, supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples described herein. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.
For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage polynucleotide III or polynucleotide VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).
For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; Cabilly et al., Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possible some FR residues are substituted from analogous sites in rodent antibodies.
In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988)l and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).
Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. The techniques of cole et al., and Boerder et al., are also available for the preparation of human monoclonal antibodies (cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol., 147(1):86-95, (1991)).
Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin polynucleotides. For example, the human heavy and light chain immunoglobulin polynucleotide complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain polynucleotides. The mouse heavy and light chain immunoglobulin polynucleotides may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transpolynucleotides harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc. (Princeton, N.J.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin polynucleotides have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including polynucleotide rearrangement, assembly, and creation of an antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,106, and in the following scientific publications: Marks et al., Biotechnol., 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Fishwild et al., Nature Biotechnol., 14:845-51 (1996); Neuberger, Nature Biotechnol., 14:826 (1996); Lonberg and Huszer, Intern. Rev. Immunol., 13:65-93 (1995).
Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).
Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.
Such anti-idiotypic antibodies capable of binding to the fungal essential polynucleotide polypeptide can be produced in a two-step procedure. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody that binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones that produce an antibody whose ability to bind to the protein-specific antibody can be blocked by the polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein-specific antibody and can be used to immunize an animal to induce formation of further protein-specific antibodies.
The antibodies of the present invention may be bispecific antibodies. Bispecific antibodies are monoclonal, Preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present invention, one of the binding specificities may be directed towards a polypeptide of the present invention, the other may be for any other antigen, and preferably for a cell-surface protein, receptor, receptor subunit, tissue-specific antigen, virally derived protein, virally encoded envelope protein, bacterially derived protein, or bacterial surface protein, etc.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transformed into a suitable host organism. For further details of generating bispecific antibodies see, for example Suresh et al., Meth. In Enzym., 121:210 (1986).
Heteroconjugate antibodies are also contemplated by the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for the treatment of HIV infection (WO 91/00360; WO 92/20373; and EP03089). It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioester bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.
The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:12 to 22.
The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular polynucleotide sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutapolynucleotidesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing polynucleotides from a mouse antibody molecule of appropriate antigen specificity together with polynucleotides from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038-1041 (1988)).
More preferably, a clone encoding an antibody of the present invention may be obtained according to the method described in the Example section herein.
The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early polynucleotide promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target polynucleotide product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign polynucleotides. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric polynucleotide may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the polynucleotide product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and polynucleotide products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the polynucleotide product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) polynucleotides can be employed in tk−, hgprt− or aprt− cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following polynucleotides: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.
The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on polynucleotide amplification for the expression of cloned polynucleotides in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker polynucleotide. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.
The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341 (1992) (said references incorporated by reference in their entireties).
As discussed, supra, the polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:12 to 22 may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:12 to 22 may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP A 232,262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem . . . 270:9459-9471 (1995).
Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.
The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc.
Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologues thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).
Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.
An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
The present invention also encompasses the creation of synthetic antibodies directed against the polypeptides of the present invention. One example of synthetic antibodies is described in Radrizzani, M., et al., Medicina, (Aires), 59(6):753-8, (1999)). Recently, a new class of synthetic antibodies has been described and are referred to as molecularly imprinted polymers (MIPs) (Semorex, Inc.). Antibodies, peptides, and enzymes are often used as molecular recognition elements in chemical and biological sensors. However, their lack of stability and signal transduction mechanisms limits their use as sensing devices. Molecularly imprinted polymers (MIPs) are capable of mimicking the function of biological receptors but with less stability constraints. Such polymers provide high sensitivity and selectivity while maintaining excellent thermal and mechanical stability. MIPs have the ability to bind to small molecules and to target molecules such as organics and proteins' with equal or greater potency than that of natural antibodies. These “super” MIPs have higher affinities for their target and thus require lower concentrations for efficacious binding.
During synthesis, the MIPs are imprinted so as to have complementary size, shape, charge and functional groups of the selected target by using the target molecule itself (such as a polypeptide, antibody, etc.), or a substance having a very similar structure, as its “print” or “template.” MIPs can be derivatized with the same reagents afforded to antibodies. For example, fluorescent ‘super’ MIPs can be coated onto beads or wells for use in highly sensitive separations or assays, or for use in high throughput screening of proteins.
Moreover, MIPs based upon the structure of the polypeptide(s) of the present invention may be useful in screening for compounds that bind to the polypeptide(s) of the invention. Such a MIP would serve the role of a synthetic “receptor” by minimicking the native architecture of the polypeptide. In fact, the ability of a MIP to serve the role of a synthetic receptor has already been demonstrated for the estrogen receptor (Ye, L., Yu, Y., Mosbach, K, Analyst., 126(6):760-5, (2001); Dickert, F, L., Hayden, O., Halikias, K, P, Analyst., 126(6):766-71, (2001)). A synthetic receptor may either be mimicked in its entirety (e.g., as the entire protein), or mimicked as a series of short peptides corresponding to the protein (Rachkov, A., Minoura, N, Biochim, Biophys, Acta., 1544(1-2):255-66, (2001)). Such a synthetic receptor MIPs may be employed in any one or more of the screening methods described elsewhere herein.
MIPs have also been shown to be useful in “sensing” the presence of its mimicked molecule (Cheng, Z., Wang, E., Yang, X, Biosens, Bioelectron., 16(3):179-85, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst., 126(6):798-802, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst., 126(6):798-802, (2001)). For example, a MIP designed using a polypeptide of the present invention may be used in assays designed to identify, and potentially quantitate, the level of said polypeptide in a sample. Such a MIP may be used as a substitute for any component described in the assays, or kits, provided herein (e.g., ELISA, etc.).
A number of methods may be employed to create MIPs to a specific receptor, ligand, polypeptide, peptide, organic molecule. Several preferred methods are described by Esteban et al in J. Anal, Chem., 370(7):795-802, (2001), which is hereby incorporated herein by reference in its entirety in addition to any references cited therein. Additional methods are known in the art and are encompassed by the present invention, such as for example, Hart, B, R., Shea, K, J. J. Am. Chem, Soc., 123(9):2072-3, (2001); and Quaglia, M., Chenon, K., Hall, A, J., De, Lorenzi, E., Sellergren, B, J. Am. Chem, Soc., 123(10):2146-54, (2001); which are hereby incorporated by reference in their entirety herein.
The antibodies of the present invention have various utilities. For example, such antibodies may be used in diagnostic assays to detect the presence or quantification of the polypeptides of the invention in a sample. Such a diagnostic assay may be comprised of at least two steps. The first, subjecting a sample with the antibody, wherein the sample is a tissue (e.g., human, animal, etc.), biological fluid (e.g., blood, urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract (e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g., See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or a chromatography column, etc. And a second step involving the quantification of antibody bound to the substrate. Alternatively, the method may additionally involve a first step of attaching the antibody, either covalently, electrostatically, or reversibly, to a solid support, and a second step of subjecting the bound antibody to the sample, as defined above and elsewhere herein.
Various diagnostic assay techniques are known in the art, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., (1987), pp 147-158). The antibodies used in the diagnostic assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase, green fluorescent protein, or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem., 13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219 (1981); and Nygren, J. Histochem. And Cytochem., 30:407 (1982).
Antibodies directed against the polypeptides of the present invention are useful for the affinity purification of such polypeptides from recombinant cell culture or natural sources. In this process, the antibodies against a particular polypeptide are immobilized on a suitable support, such as a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample containing the polypeptides to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except for the desired polypeptides, which are bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the desired polypeptide from the antibody.
The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. The translation product of the polynucleotide of the present invention may be useful as a cell specific marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).
These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.
The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.
The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M, 10-6 M, 5×10-7 M, 10-7 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M, 10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M, 5×10-15 M, and 10-15 M.
Antibodies directed against polypeptides of the present invention are useful for inhibiting allergic reactions in animals. For example, by administering a therapeutically acceptable dose of an antibody, or antibodies, of the present invention, or a cocktail of the present antibodies, or in combination with other antibodies of varying sources, the animal may not elicit an allergic response to antigens.
Likewise, one could envision cloning the polynucleotide encoding an antibody directed against a polypeptide of the present invention, said polypeptide having the potential to elicit an allergic and/or immune response in an organism, and transforming the organism with said antibody polynucleotide such that it is expressed (e.g., constitutively, inducibly, etc.) in the organism. Thus, the organism would effectively become resistant to an allergic response resulting from the ingestion or presence of such an immune/allergic reactive polypeptide. Moreover, such a use of the antibodies of the present invention may have particular utility in preventing and/or ameliorating autoimmune diseases and/or disorders, as such conditions are typically a result of antibodies being directed against endogenous proteins. For example, in the instance where the polypeptide of the present invention is responsible for modulating the immune response to auto-antigens, transforming the organism and/or individual with a construct comprising any of the promoters disclosed herein or otherwise known in the art, in addition, to a polynucleotide encoding the antibody directed against the polypeptide of the present invention could effective inhibit the organisms immune system from eliciting an immune response to the auto-antigen(s). Detailed descriptions of therapeutic and/or polynucleotide therapy applications of the present invention are provided elsewhere herein.
Alternatively, antibodies of the present invention could be produced in a plant (e.g., cloning the polynucleotide of the antibody directed against a polypeptide of the present invention, and transforming a plant with a suitable vector comprising said polynucleotide for constitutive expression of the antibody within the plant), and the plant subsequently ingested by an animal, thereby conferring temporary immunity to the animal for the specific antigen the antibody is directed towards (See, for example, U.S. Pat. Nos. 5,914,123 and 6,034,298).
In another embodiment, antibodies of the present invention, preferably polyclonal antibodies, more preferably monoclonal antibodies, and most preferably single-chain antibodies, can be used as a means of inhibiting polynucleotide expression of a particular gene, or polynucleotides, in a human, mammal, and/or other organism. See, for example, International Publication Number WO 00/05391, published Feb. 3, 2000, to Dow Agrosciences LLC. The application of such methods for the antibodies of the present invention are known in the art, and are more particularly described elsewhere herein.
In yet another embodiment, antibodies of the present invention may be useful for multimerizing the polypeptides of the present invention. For example, certain proteins may confer enhanced biological activity when present in a multimeric state (i.e., such enhanced activity may be due to the increased effective concentration of such proteins whereby more protein is available in a localized location).
In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of polynucleotide therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.
Any of the methods for polynucleotide therapy available in the art can be used according to the present invention. Exemplary methods are described below.
For general reviews of the methods of polynucleotide therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).
In a preferred aspect, the compound comprises polynucleotides encoding an antibody, said polynucleotides being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such polynucleotides have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the polynucleotides include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.
Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo polynucleotide therapy.
In a specific embodiment, the polynucleotides are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a polynucleotide gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem . . . 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).
In a specific embodiment, viral vectors that contains polynucleotides encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The polynucleotides encoding the antibody to be used in polynucleotide therapy are cloned into one or more vectors, which facilitates delivery of the polynucleotide into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 polynucleotide to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in polynucleotide therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
Adenoviruses are other viral vectors that can be used in polynucleotide therapy. Adenoviruses are especially attractive vehicles for delivering polynucleotides to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based polynucleotide therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer polynucleotides to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in polynucleotide therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used.
Adeno-associated virus (AAV) has also been proposed for use in polynucleotide therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).
Another approach to polynucleotide therapy involves transferring a polynucleotide to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred polynucleotide. Those cells are then delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the polynucleotides, cell fusion, chromosome-mediated polynucleotide transfer, microcell-mediated polynucleotide transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign polynucleotides into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
Cells into which a nucleic acid can be introduced for purposes of polynucleotide therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
In a preferred embodiment, the cell used for polynucleotide therapy is autologous to the patient.
In an embodiment in which recombinant cells are used in polynucleotide therapy, polynucleotides encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).
In a specific embodiment, the nucleic acid to be introduced for purposes of polynucleotide therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. Demonstration of Therapeutic or Prophylactic Activity.
The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.
In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a polynucleotide gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of polynucleotide expression with a standard polynucleotide expression level, whereby an increase or decrease in the assayed polypeptide polynucleotide expression level compared to the standard expression level is indicative of aberrant expression.
The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of polynucleotide expression with a standard polynucleotide expression level, whereby an increase or decrease in the assayed polypeptide polynucleotide expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein polynucleotide expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99 mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).
Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.
In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, Mo.).
The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
Antisense Molecules
The invention also provides for the identification of compounds that modulate (e.g., activate or inhibit) the function of a polypeptide of the invention. Such compounds can provide lead-compounds for developing drugs for diagnosing and/or treating conditions associated with fungal infections. The modulator is a compound that may alter the function of a polypeptide of the invention including SEQ ID NO: 12 through to SEQ ID NO 22, such as activating or inhibiting the function of a polypeptide of the invention. For example, the compound can act as an agonist, antagonist, partial agonist, partial antagonist, cytotoxic agent, inhibitor of cell proliferation, and cell proliferation-promoting agents. The activity of the compound may be known, unknown or partially known.
In one embodiment, an antisense molecule is used as an antagonist of a polynucleotide product of the nucleic acid molecules of the invention. The present invention also provides the therapeutic or prophylactic use of nucleic acids of at least six nucleotides that are antisense to a target essential polynucleotide or a portion thereof. An “antisense” target nucleic acid as used herein refers to a nucleic acid capable of hybridizing to a portion of a target polynucleotide RNA or mRNA by virtue of some sequence complementarity. The invention further provides pharmaceutical compositions comprising an effective amount of the antisense nucleic acids of the invention in a pharmaceutical acceptable carrier as described below.
In another embodiment, the invention is directed to methods for inhibiting the expression of a target polynucleotide in an organism of interest, such as C. albicans either in vitro or in vivo comprising providing the cell with an effective amount of a composition comprising an antisense nucleic acid of the invention.
It is preferred that in vitro studies are first performed to quantitate the ability of the antisense molecule to inhibit polynucleotide expression. It is preferred that these studies utilize controls that distinguish between antisense polynucleotide inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide. It is preferred that the control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.
Antisense molecules of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Applied Biosystems, Palo Alto, Calif.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Steinet et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
Antisense nucleotides complementary to the coding region of a target polynucleotide may be used, as well as those complementary to the transcribed untranslated region.
Antisense oligonucleotides may be single or double stranded. Double stranded RNA's may be designed based upon the teachings of Paddison et al., Proc. Nat. Acad. Sci., 99:1443-1448 (2002); and International Publication Nos. WO 01/29058, and WO 99/32619; which are hereby incorporated herein by reference.
Pharmaceutical compositions of the invention comprising an effective amount of an antisense nucleic acid in a pharmaceutically acceptable carrier can be administered to a subject infected with the pathogen of interest.
The amount of antisense nucleic acid which will be effective in the treatment of a particular disease caused by the fungal pathogen will depend on the site of the infection or condition. Where possible, it is desirable to determine the antisense cytotoxicity of the fungus to be treated in vitro, and then in useful animal model systems prior to testing and use in humans.
A number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site in which the pathogens are residing, or modified antisense molecules, designed to target the desired cells (e.g., antisense molecule linked to peptides or antibodies that specifically bind receptors or antigens expressed on the pathogen's cell surface) can be administered systemically. Antisense molecules can be delivered to the desired cell population via a delivery complex. In a specific embodiment, pharmaceutical compositions comprising antisense nucleic acids of the target polynucleotides are administered via biopolymers (e.g., poly-β-1-4-N-acetylglucosamine polysaccharide), liposomes, microparticles, or microcapsules.
In various embodiments of the invention, it may be useful to use such compositions to achieve sustained release of the antisense nucleic acids. In a specific embodiment, it may be desirable to utilize liposomes targeted via antibodies to specific identifiable pathogen antigens (Leonetti et al., 1990, Proc. Nat. Acad. Sci. U.S.A. 87:2448-2451; Renneisen et al., 1990, J. Biol. Chem. 265:16337-16342).
Transcriptional Profiling
Gene expression profiling techniques are important tools for the identification of antifungal compounds. To carry out profiling, polynucleotide expression arrays and microarrays can be employed. Gene expression arrays are high density arrays of DNA samples deposited at specific locations on, for example, a glass surface or nylon membrane. Such arrays are used by researchers to quantify relative polynucleotide expression under different conditions. An example of this technology is found in U.S. Pat. No. 5,807,522, which is hereby incorporated by reference.
It is possible to study the expression of substantially all of the polynucleotides in the genome of a particular microbial organism using a single array. For example, the arrays may consist of 12×24 cm nylon filters containing a PCR product corresponding to ORFs from Candida albicans. 10 ng of each PCR product may be spotted for example every 1.5 mm on the filter. Single stranded labeled cDNAs are prepared for hybridization to the array and placed in contact with the filter. Thus, the labeled cDNAs are of “antisense” orientation. Quantitative analysis may be done using a phosphorimager.
Hybridization of cDNA made from a sample of total cellular mRNA to such an array followed by detection of binding by one or more of various techniques known to those in the art provides a signal at each location on the array to which cDNA is hybridized. The intensity of the hybridization signal obtained at each location in the array thus reflects the amount of mRNA for that specific polynucleotide that was present in the sample. Comparing the results obtained for mRNA isolated from cells grown under different conditions thus allows for a comparison of the relative amount of expression of each individual polynucleotides during growth under different conditions.
Gene expression arrays are use to analyze the total mRNA expression pattern at various time points after reduction in the level or activity of a polynucleotide product required for fungal proliferation. Reduction of the level or activity of the polynucleotide product is accomplished by growing a diploid strain of the invention under conditions in which the product of the nucleic acid linked to the MET3 promoter is rate limiting for fungal growth or survival or proliferation or by contacting the cells with an agent which reduces the level or activity of the target polynucleotide product. Analysis of the expression pattern indicated by hybridization to the array provides information on other polynucleotides whose expression is influenced by reduction in the level or activity of the polynucleotide product. For example, levels of other mRNAs may be observed to increase, decrease or stay the same following reduction in the level or activity of the polynucleotide product required for growth survival or proliferation. Thus, the mRNA expression pattern observed following reduction in the level or activity of a polynucleotide product required for growth, survival or proliferation identifies other nucleic acids required for expression patterns observed when the fungi are exposed to candidate drug compounds or known antibiotics are compared to those observed when the level or activity of a polynucleotide product required for fungal growth survival or proliferation is reduced. If the mRNA expression pattern observed with the candidate drug compound is similar to that observed with the level of the polynucleotide product is reduced, the drug compound is a promising therapeutic candidate. The assay is useful in assisting in the selection of promising candidate drug compounds for use in drug development.
In another embodiment, the present invention provides a method of quantitative analysis of the expressed protein complement of a diploid fungal cell: a first protein expression profile is developed for a control diploid fungus, which has two unmodified copies of the target polynucleotide. Mutants of the control strain, in which one copy of the target polynucleotide is inactivated, for example, one of the strains of the present invention, by insertion of disruption cassette is generated. The allele is modified such that expression of the allele is under the control of a MET3 promoter. A second protein expression profile is developed for this mutant fungus under conditions where the second allele is substantially overexpressed as compared to the expression of the two alleles of the polynucleotide in the control strain. Similarly, if desired, a third protein expression profile is developed under conditions where the second allele is substantially underexpressed as compared to the expression of the two alleles of the polynucleotide in the control strain. The first protein expression profile is then compared with the second expression profile and if applicable to a third, forth, fifth or sixth or more expression profile to identify an expressed protein detected a higher level in the second profile and if applicable at a lower level in the third profile, etc., as compared to the level in the first profile.
Accordingly, the invention provides a method for evaluating a compound against a target polynucleotide product encoded by a nucleotide sequence comprising one of SEQ ID NO 1 to 11 said method comprising the steps of (a) contacting wild type diploid fungal cells or control cells with the compound and generating a first protein expression profile; (b) determining the protein expression profile of mutant diploid fungal cells such as a strain of the invention which have been cultured under conditions wherein the second allele of the target polynucleotide is substantially underexpressed not expressed or overexpressed and generating a second protein expression profile for the cultured cells and comparing the first protein expression profile with the second protein expression profile to identify similarity in profiles. For comparisons, similarities of profiles can be expressed as an indicator value; and the higher the indicator value, the more desirable is the compound.
The pattern of expression of a set of proteins in a strain of the invention may be determined by methods well-known in the art for establishing a protein expression pattern such as two-dimensional gel electrophoresis. A plurality of protein expression patterns will be generated for a strain of the invention when the strain is cultured under different conditions and different levels of expression of one of the modified alleles.
Pharmaceutical Compositions and Uses Thereof
Compounds including nucleic acid molecules that are identified by the methods of the invention as described herein can be administered to a subject at therapeutically effective doses to treat or prevent infections by a fungal organism such as Candia albicans. A therapeutically effective dose refers to that amount of a compound (including nucleic acid molecules) sufficient to result in a healthful benefit in the treated subject. Typically, but not so limited, the compounds act by reducing the activity or level of polynucleotide product encoded by a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO 1 to SEQ ID NO: 11, or homologues thereof.
To treat a patient afflicted with a fungal infection it may be beneficial to deliver an essential polynucleotide polypeptide, polynucleotide or modulating agent to the intracellular space. Such targeting may be achieved using well-known techniques, such as through the use of polyethylene glycol or liposomes, as described in Turrens, Xenobiotica 21:103 3-1040 (1991), herein incorporated by reference.
For certain embodiments, it may be beneficial to also link a drug to a polypeptide or modulating agent. As used herein, the term “drug” refers to any bioactive agent intended for administration to a mammal to prevent or treat an undesirable condition.
To prepare a pharmaceutical composition, an effective amount of one or more polypeptides, polynucleotides and/or modulating agents is mixed with a suitable pharmaceutical carrier. Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical application can include, for example, a sterile diluent (such as water), saline solution, fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent; antimicrobial agents (such as benzyl alcohol and methylparabens); antioxidants (such as ascorbic acid and sodium bisulfite) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates and phosphates). If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol and mixtures thereof. In addition, other pharmaceutically active ingredients and/or suitable excipients such as salts, buffers and stabilizers may, but need not, be present within the composition.
A pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The number and degree of acceptable side effects depend upon the condition for which the composition is administered. For example, certain toxic and undesirable side effects that are tolerated when treating life-threatening illnesses, such as tumors, would not be tolerated when treating disorders of lesser consequence. The concentration of active component in the composition will depend on absorption, inactivation and excretion rates thereof, the dosage schedule and the amount administered, as well as other factors that may be readily determined by those of skill in the art.
A polypeptide, polynucleotide or modulating agent may be prepared with carriers that protect it against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others known to those of ordinary skill in the art. Such formulations may generally be prepared using well-known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain a polynucleotide, polypeptide or modulating agent dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Preferably the formulation provides a relatively constant level of modulating agent release. The amount of active component contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
Administration may be effected by incubation of cells ex vivo or in vivo, such as by topical treatment, delivery by specific carrier or by vascular supply. Appropriate dosages and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease and the method of administration. In general, an appropriate dosage and treatment regimen provides the polypeptide, polynucleotide and/or modulating agent(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (i.e., an amount that ameliorates the symptoms or treats or delays or prevents progression of the condition).
The precise dosage and duration of treatment is a function of the condition being treated and may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Dosages may also vary with the severity of the condition to be alleviated. The composition may be administered one time, or may be divided into a number of smaller doses to be administered at intervals of time. In general, the use of the minimum dosage that is sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art, and for any particular subject, specific dosage regimens may be adjusted over time according to the individual need.
For pharmaceutical compositions comprising polynucleotides, the polynucleotide may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid, bacterial and viral expression systems, and colloidal dispersion systems such as liposomes. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal, as described above). The DNA may also be “naked”, as described, for example, in Ulmer et al., Science 259:1745-1749 (1993).
Various viral vectors that can be used to introduce a nucleic acid sequence into the targeted patient's cells include, but are not limited to, vaccinia or other pox virus, herpes virus, retrovirus, or adenovirus. Techniques for incorporating DNA into such vectors are well-known to those of ordinary skill in the art. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus including, but not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
A retroviral vector may additionally transfer or incorporate a polynucleotide for a selectable marker (to aid in the identification or selection of transduced cells) and/or a polynucleotide that encodes the ligand for a receptor on a specific target cell (to render the vector target specific).
Viral vectors are typically non-pathogenic (defective), replication competent viruses, which require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids that encode all of the structural polynucleotides of the retrovirus under the control of regulatory sequences, but that are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsulation. Such helper cell lines include (but are not limited to) PA317 and PA12. A retroviral vector introduced into such cells can be packaged and vector virion produced. The vector virions produced by this method can then be used to infect a tissue cell line, such as NIH3T3 cells, to produce large quantities of chimeric retroviral virions.
Another targeted delivery system for polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). RNA. DNA and intact virions can be encapsulated within the aqueous interior and delivered to cells in a biologically active form. The preparation and use of liposomes is well-known to those of ordinary skill in the art.
Modulation of an essential gene-like function, either in vitro or in vivo, may generally be achieved by administering a modulating agent that inhibits essential polynucleotide transcription, translation or activity.
Routes and frequency of administration, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques.
In general, the pharmaceutical compositions may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. A suitable dose is an amount of a compound that, when administered as described above, is capable of causing modulation of an essential gene-like activity that leads to an improved clinical outcome (e.g., more frequent remissions, complete or partial or longer disease-free survival) in vaccinated patients as compared to non-vaccinated patients. In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients. In general, suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
Infectious Disease
A polypeptide or polynucleotide and/or agonist or antagonist of the present invention can be used to treat, prevent, and/or diagnose infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated, prevented, and/or diagnosed. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polypeptide or polynucleotide and/or agonist or antagonist of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention. Examples of viruses, include, but are not limited to Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia, polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines. In a further specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose AIDS.
Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, include, but not limited to, the following Gram-Negative and Gram-positive bacteria and bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, and Salmonella paratyphi), Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis, Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B), Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal, Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcus pneumoniae and Group B Streptococcus). These bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections. Polynucleotides or polypeptides, agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat, prevent, and/or diagnose: tetanus, Diptheria, botulism, and/or meningitis type B.
Moreover, parasitic agents causing disease or symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis, polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose malaria.
Preferably, treatment or prevention using a polypeptide or polynucleotide and/or agonist or antagonist of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.
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EXAMPLES
Example 1
Target Identification
A concordance or whole genome parallel comparison was performed as described by Bruccoleri, et al. Nucleic Acids Res 26:4482-4486 (1998) to find polynucleotides that are conserved in both S. cervisiae and C. albicans. To generate the dataset, all C. albicans open reading frames with an overall sequence similarity to S. cervisiae protein sequences greater than or equal to 40% were selected. The data were further required not to match bacterial (E. coli and B. subtilis) or human sequences at greater than 30% overall protein sequence similarity.
The sequences used herein were obtained from a variety of sources. These sources include the PathoGenomeTM system from Genome Therapeutics Corporation (Waltham, Mass.), GenBank, The Institute for Genomic Research (TIGR), the Yeast Proteome Database, Proteome, Inc. Beverly, Mass., Saccharomyces Genome database, Stanford University, Stanford, Calif., the Candida albicans Sequencing Project, Stanford Genome Technology Center, Stanford, Calif., and the Sanger Center of the Medical Research Council of the United Kingdom. Additionally, non-microbial sequence data such as those from humans was obtained from the LifeSeq Database from Incyte Pharmaceuticals, Palo Alto, Calif., as well as from public sources such as Genbank.
Where required, Incyte nucleotide sequences were translated into protein sequences in all six possible reading frames. GTC supplied predicted protein sequences with their data. In the case of other nucleotide sequences, the program CRITICA (Badger, J. and Olsen, G., Molecular Biology and Evolution 16: 512-524 (1999) was used. The sequences were stored in flat files on a UNIX computer system. Each predicted amino acid sequences used was greater than 90 amino acids.
Each predicted protein sequence was compared to every other sequence (an “all-against-all” comparison). The program FASTA (Pearson, W. R. Methods in Molecular Biology 132: 185-219 (2000)) was used for this comparison, using ktup=2 as a parameter, and all scores above the default cutoff were stored. The output was processed and stored in a PostGres 95 database. Graphical user interfaces, using web browser technology were constructed to query the database.
A Concordance Analysis was performed on the data. A total of 560 fungal conserved polynucleotides were identified (FCGs) of which 125 polynucleotides had a known function and 435 polynucleotides had an unknown or partially known function (denoted Conserved Unknown Reading Frames or CURFs). 49 CURFs were selected and tested for essentiality in S. cervisiae, and 11 of these were found essential in C. albicans (see below).
In many cases, the function of the essential polynucleotides determined herein was suggested by the results of similarity searches. Table 2 lists the putative functions of these polynucleotides encoded by SEQ ID NO: 1 through to SEQ ID NO: 11.
The putative function of the essential polypeptides was determined using computer-aided bioinformatic approaches including motif searching. The motif searching approach involved using hidden Markov models (e.g., Profile HMM, Bateman et al, Nucleic Acids Research 28: 263-266 (2000)). Global sequence similarity searches were performed using the amino acid sequences of all the conserved essential polynucleotide sequence against a non-redundant protein database using the Smith-Waterman algorithm with default parameters on a TimeLogic DeCypher system (Crystal Bay, Nev.).
The percentage sequence similarity between these subsets of CURFs and S. cervisiae is listed in Table 1. Percent similarity ranged from 61% to 87%. Additionally, these sequences were aligned with A. fumigatus, S. pombe and Homo sapiens. The percentage identity between the 11 essential polynucleotides of the invention and homologues of these species including those from S. cervisiae is listed in Table 2. Based on this analysis, the CURFs of the invention should be homologues to polynucleotides from additional fungal species.







TABLE 1







Similarity Between Candida albicans, CURFs and S. cervisiae















Species
Genbank



Seq ID
Clone
Sequence
of Closest
Accessions for


No.
ID No.
Name
Homolog
Closest Homolog
% Similarity





12
FCG5
CaYLR100wORF
Saccharomyces
Z73272, S64936
75%





cerevisiae


13
FCG6
CaYDR341cORF
Saccharomyces
S70106
79%





cerevisiae


14
FCG7
CaYLR022cORF
Saccharomyces
Z73149, S64849
73%





cerevisiae


15
FCG8
CaYOL077cORF
Saccharomyces
Z74819, S66770
84%





cerevisiae


16
FCG10
CaYNL132wORF
Saccharomyces
Z46843
87%





cerevisiae


17
FCG12
CaYGR145wORF
Saccharomyces
X85807
73%





cerevisiae


18
FCG13
CaYDR412wORF
Saccharomyces
U33007, S69697
61%





cerevisiae


19
FCG14
CaYOL010wORF
Saccharomyces
Z74752, Q08096
81%





cerevisiae


20
FCG15
CaYOR004wORF
Saccharomyces
Z74912, S61987
73%





cerevisiae


21
FCG16
CaYOR056cORF
Saccharomyces
Z70678, S66939
62%





cerevisiae


22
FCG17
CaYLR009wORF
Saccharomyces
Z73181, S64831
82%





cerevisiae









Table 2 also includes the putative function and putative cellular role based on the annotation of the CURF homologues.







TABLE 2







Fungal CURFs Annotation, Similarity and Identity





















%










Identity





% Identity
% Identity
% Identity
with


SEQ

Name in
with
with
with
human


ID #
FCG #
YPD
S. cerevisiae
A. fumigatus*
S. pombe
(d2)
Function*
Cellular Role**


















12
5
ERG27/
60
34, 15
31
22
3-keto sterol
Ergosterol




YLR100w




reductase
biosynthesis


13
6
YDR341c/
65
54, 51
25
23
Arginine tRNA
Protein synthesis




RRS1




synthetase


14
7
YLR022c
56
46, 22
46
10
Unknown
Unknown


15
8
YOL077c/
70
51, 43
7
36
Unknown/required
Protein synthesis




BRX1




for









biopolynucleotidesis









of the 60S ribosomal









subunit


16
10
YNL132w/
74
68, 65
62
12
Unknown (P-loop)
Unknown/killer




KRE33





toxin resistant


17
12
YGR145w
57
38, 31
42
8
Component of NuA3
Unknown/WD









histone
domain, G-beta









acetyltransferase
repeat


18
13
YDR412w
43
48, 9
20
16
Unknown
Unknown


19
14
YOL010w/
68
38, 41
41
22
RNA cyclase-like
RNA




RCL1





processing/modification


20
15
YOR004w
55
41, 21
46
13
Unknown
Unknown


21
16
YOR056c/
46
43, 9
45
15
Unknown/nin one-p
Unknown/associated




NOB1




binding protein
with the 26S










proteasome


22
17
YLR009w
73
46, 31
34
7
RNA-binding
Protein synthesis









protein/ribosome-









associated





*The two numbers represent percent identities derived from alignment and query sequences, respective






FIG. 1 depicts the alignments of the top hits from a nonredundant database (Bristol-Meyers Squibb, Princeton, N.J.) containing sequences included from Genbank (infra). The aligned sequences of FIG. 1 containing “NR” in their sequence name depict the top hit from this database. All of the top hits are S. cervisiae species. Additionally, FIG. 1 depicts the alignment between the top hit in the DERWENT patent database (Alexandria, Va.). Table 3 shows the Genbank Accession No. and/or patent or patent application number in which the aligned CURF homolog sequence can be found. As is evident from the alignment, the percent identity for each of the 11 essential polynucleotides of the invention is less than that from the non-redundant database hits.







TABLE 3







Sequences Aligning With CURFs of the Invention










Sequence Name
ID
Patent No.
Species





PAT_PROT\BMSPATENT_AAB94675
AAB94675
WO 200107628-A2
Saccharomyces





cerevisiae


PAT_PROT\BMSPATENT_AAW33110
AAW33110
JP 09263600-A
Yeast


PAT_PROT\BMSPATENT_AAB95680
AAB95680
EP1074617-A2
Homo sapiens


PAT_PROT\BMSPATENT_AAG46965
AAG46965
EP 1033405-A2
Arabidopsis





thaliana


PAT_PROT\BMSPATENT_AAB43803
AAB43803
WO 200055351-A1
Homo sapiens


PAT_PROT\BMSPATENT_AAB42957
AAB42957
WO 200058473-A2
Homo sapiens


PAT_PROT\BMSPATENT_AAB19089
AAB19089
WO 200058520-A1
Saccharomyces





cerevisiae


PAT_PROT\BMSPATENT_AAB93917
AAB93917
EP 1074617-A2
Homo sapiens


PAT_PROT\BMSPATENT_AAW60075
AAW60075
WO 9745535-A1
Saccharomyces





cerevisiae


PAT_PROT\BMSPATENT_AAB62453
AAB62453
US 6221597-B1
Saccharomyces





cerevisiae


PAT_PROT\BMSPATENT_AAG48012
AAG48012
EP 1033406-A2
Arabidopsis





thaliana


PAT_PROT\BMSPATENT_AAB09929
AAB09929
JP 2000116383-A
Homo sapiens









FIG. 2 depicts the alignments between the CURFs of the invention and sequences from A. fumigatus. As seen in Table 2, the percent identity between these homologues ranges from 34% to 68%.
Example 2
Strains and Growth Media Used for Identifying Essential Genes
49 CURFs were selected and tested for essentiality in S. cervisiae and then in C. albicans. Three polynucleotides whose functions were known, ERG1, RAM2 and NMT1, were also used for verification of essentiality in C. albicans. 
The strains used for this analysis included the C. albicans strains SC5314 (wild type, BMS collection) and its derivative BWP17 (ura3Δ::λimm434/ura3Δ::λimm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG) obtained from A. P. Mitchell of Columbia University. The yeast strain of S. cerevisiae used is ATCC 201390 (MATa/MATα his3Δ1/his3Δ1 leu2Δ0/leu2Δ1 lys2Δ0/LYS2 met15Δ0/MET15 ura3Δ0/ura30); Escherichia coli strain DH5α was used for plasmids propagation.
Yeast extract/peptone/dextrose (YPD), synthetic complete medium (SC), and synthetic dextrose (SD) were prepared according to the standard procedure described by Sherman, F. et al. Methods Enzymol. 184:3-21 (1991). 5-Fluoroorotic acid (FOA) plates were used to select for the Ura− revertant strains (Sherman, et al. (1991)). Spider (Liu, et al., Science 266:1723-1725 (1994)) and LBC media (Lee, et al., Sabouraudia 13:148-153 (1975)) were used to induce hyphal growth. Twenty percent bovine serum (Köhler, et al., Proc. Natl. Acad. Sci. 93:13223-13228 (1996)) and medium 199 (Gibco BRL, Gaithersburg, Md.) were used to induce germ tube formation. Uridine (25 μg/ml) was added according to Fonzi & Irwin (Fonzi, et al., Genetics 134:717-728 (1993)) when needed to grow uridine-auxotrophic C. albicans strains. Other supplements such as histidine and arginine were added to a concentration described by Sherman, F. et al. Methods Enzymol. 184:3-21 (1991). Strains were grown at 30° C., unless otherwise noted. The storage and maintenance of C. albicans, which prevented chromosomal instability, was as previously described (Perepnikhatka, et al., J. Bacteriol. 181:4041-4049).
Example 3
Evaluation of Gene Essentiality in S. cervisiae 
To evaluate polynucleotide essentiality for the homologues of the sequences encoding essential polynucleotides including SEQ ID NO: 1 through to SEQ ID NO: 11 in S. cervisiae, one copy of the essential polynucleotides of the invention were disrupted in a diploid strain background. The resulting heterozygous strains were then sporulated and the tetrads dissected to determine essentiality.
Disruption of the first allele of the polynucleotides of the invention was achieved via a PCR-based polynucleotide disruption approach where a PCR product containing the URA3 marker flanked by 40-50 bp of polynucleotide specific sequence was introduced into diploid yeast cells to replace the wild type copy of the polynucleotide via homologous recombination. Confirmation PCR was used to verify polynucleotide replacement using primers designed within 100 bp upstream or downstream of the site of crossover. A 2:2 ratio of segregation of meiotic progenies after tetrad dissection indicated that the polynucleotide of interest was essential for cell survival or synthetic medium. Gene disruption techniques are well documented in the literature for S. cervisiae and can be found at the web site (sequences-stanford.edu/group deletion project).
Example 4
PCR Based Gene Disruption in C. albicans 
Gene essentiality in C. albicans was identified by two methods: PCR-based polynucleotide disruption and promoter swapping. Gene disruption was accomplished using a PCR-based polynucleotide disruption tool (Wilson et al., J. Bacteriol. 181:1868-1874 (1999)).
The PCR-based polynucleotide disruption system used herein was purchased from Dr. A. P. Mitchell of Columbia University. This system requires the use of two markers to create homozygous disruptions in C. albicans. A triply-marked auxotroph (Ura− Arg− His−) strain BWP17 and three sets of plasmids -pGEM-URA3, pRS-ARG4ΔSpeI and pGEM-HIS1, each carrying a unique marker -URA3, ARG4 or HIS1 was used herein. A sequential disruption (from start to stop codons) of the two copies of any single polynucleotide can be achieved with any combination of the two selective markers.
The general scheme of the approach is illustrated in FIG. 3. Basically, the scheme involves (1) design a pair of PCR primers which incorporate sequences that are able to anneal to plasmids containing markers. Examples of such marker containing plasmids include pGEM-URA3, pRS-ARSΔSpeI and pGEM-HIS1 (see FIG. 3). An example of sequences which may be incorporated into the primers includes 5DR, (SEQ ID NO: 221) and 3DR (SEQ ID NO: 222). The forward primer additionally contains about 50 to about 60 bp of flanking sequences derived from the start codon region of a polynucleotide of interest or an open reading frame (ORF) of interest and this flanking sequence is attached to the 5′ end of the forward (e.g., 5DR) primer. The reverse primer additionally contains 50-60 bp of flanking sequences that were derived from the stop codon region of the polynucleotide of interest or ORF of interest attached to the 3′ end of the reverse primer (e.g., 3DR, SEQ ID NO: 222). (2) A selective marker (e.g. URA3) is then amplified from one of the plasmid templates resulting in a PCR product which includes short regions of homology to the polynucleotide or ORF of interest on both ends that allows for homologous recombination at the chromosomal locus when introduced into cells. (3) The PCR product is then used to transform the strain (in this case BWP17) and transformants are selected that grow on an appropriate selective medium (e.g., SC-Uridine). (4) Total DNA is isolated from the transformants and the presence of the PCR constructs are verified using detection primers which are common primers used for all three plasmids. The correct construct should have its chromosomal allele replaced with the PCR fragment introduced via homologous recombination. (5) Finally, once the first allele of the polynucleotide or ORF is disrupted, a second round of transformation with the PCR product amplified from a different marker (e.g., ARG4 in pRS-ARG4ΔSpeI) can be conducted to disrupt the remaining allele.
FIG. 3 depicts PCR-based polynucleotide disruption in C. albicans using YFG1 (or your favorite polynucleotide or the polynucleotide of interest). As seen in FIG. 3, a first copy of YFG1 is disrupted. Disrupting the second polynucleotide copy in a heterozygous strain (YFG1/yfg1) would give rise to a null mutant that has the remaining copy of the polynucleotide disrupted (yfg1/yfg1). In the FIG., 5′-KOP and 3′-KOP are a pair of polynucleotide disruption primers that are designed based on the common primers 5DR and 3DR (see below). Confirmation PCR primers are also indicated in the FIG and they include, in pairs, 5′ gene/3′-polynucleotide detecting primers, 5′ gene/3′ plasmid detecting primers and 5′ plasmid detecting primer and 3′ polynucleotide detecting primer.
Table 4 lists the SEQ ID Nos. for the primers which contain 5DR and 3DR along with appropriate flanking sequences (under the column “PCR based knockout primers, Mitchell's”) as well as the confirmation primers which may be used for this methodology. These knockout and confirmation primers were used for both the essential polynucleotides of the invention encoded by SEQ ID NO: 1 through to SEQ ID NO: 11 as well as the polynucleotide encoded by SEQ ID NO: 45. Three polynucleotides whose homologues are known to be essential in S. cervisiae, CaERG, CaRAM2 and CaNMT1 were also tested. CaAaH1 and CaNMT1 are homologues of S. cervisiae known to not be essential and these were also examined.







TABLE 4







PCR-Knockout and Gene Confirmation Primers


















PCR based
PCR-based







knockout
knockout




SEQ ID
Gene specific
Gene specific
primers
primers



Seq.
NO of
confirmation
confirmation
(Mitchell's)
(Mitchell's)


Gene Name
ID No.
translation
primers-5′)
primers-3′)
forward
reverse)
















CaYLR100wORF
1
12
159
160
195
196


CaYDR341cORF
2
13
161
162
197
198


CaYLR022cORF
3
14
163
164
199
200


CaYOL077cORF
4
15
165
166
201
202


CaYNL132wORF
5
16
167
168
203
204


CaYGR145wORF
6
17
171
172
207
208


CaYDR412wORF
7
18
173
174
209
210


CaYOL010wORF
8
19
175
176
211
212


CaYOR004wORF
9
20
177
178
213
214


YOR056cORF
10
21
179
180
215
216


YOR009wORF
11
22
181
182
217
218


CaYJR072c
—
45
169
170
205
206


CaERG
—
—
151
152
185
186


CaRAM2
—
—
153
154
187
188


CaPFY1
—
—
155
156
189
190


CaNMT1
—
—
157
158
191/193
192/194


CaAAH1
—
—
183
184
219
220









FIG. 4 depicts the results this polynucleotide disruption scheme using CaAAH1. The knockout primers used for this assay are listed in SEQ ID NO: 219 and SEQ ID NO: 220.
Panels A and B depict the identification of a heterozygous AAH1/aah1 construct. Lane M in both panels shows a 1 kb marker with sizes of some of the fragments indicated. Lane 1 in Panel A depicts a wild type strain that did not result in any PCR products when using a 5′ primer (e.g. SEQ ID NO: 221) which anneals to any of the plasmids used herein (pGEM-URA3, pRS-ARSΔSpeI and pGEM-HIS1) and a 3′ primer which anneals to the 3′ region of the AAHI polynucleotide (e.g., SEQ ID NOs: 184). The wild type strain, however seen in panel B, Lane 1, did produce the wild type polynucleotide fragment of 0.9 kb when using 5′ and 3′ primers which anneal to the 5′ and 3′ regions of AAH1 (e.g., SEQ ID NOs 183-184). Lane 2 (Panel A) shows the construct of the heterozygous strain that gives a 2.2 kb band via a common 5′ plasmid primer and a 3′ polynucleotide detecting primer (e.g., SEQ ID NOs: 221 and 184, respectively, for example). Panel B, lane 2 shows the expected size fragments of 0.9 kb and 2.3 kb when using the 5′ and 3′ polynucleotide primers.
Panel C and D show the identification of the homozygous aah1/aah1 null mutant. Lane M is the 1 kb marker on both panels. Lane 3 contains the heterozygous strain used for obtaining the double disruptant of AAH1. Lane 4 shows the aah1/aah1 construct which correctly results in two PCR bands of 1.6 and 2.2 kb, respectively (panel C), via a 5′ common primer and a 3′ polynucleotide specific primer (primers 5′ detect/3′ gene) and two bands of 1.7 kb and 2.3 kb bands, respectively, via the 5′ and 3′ polynucleotide specific primers (panel D).
Table 5 lists the results of this methodology for the essential polynucleotides of the invention encoded by SEQ ID NO:1 through to SEQ ID NO:11, SEQ ID NO:45. Genes with known essential function were also examined. These include the C. albicans homologues of ERG1, RAM2 and NMT1. The non-essential gene, PFY1 was also tested.







TABLE 5







Essential Genes Identified By PCR-Based Knockout


and Promoter Swapping Cassette

















Essentiality
Essentiality



SEQ



via PCR-
via MET3


ID#

FCG #
Name in YPD
based KO
promoter
Functions
















—
Known Genes
1
ERG1
Essential
Essential
Squaline








epoxidase


—

2
RAM2/YKL019w
Essential
Essential
Alpha subunit of








farnesyl








transferase


—

3
PFY1/YOR122c
Not
Not
Cell polarity






essential
essential


—

4
NMT1/YLR195c
ND*
Essential
N-myristoyl








transferase


12
Unknown
5
ERG27/YLR100w
Essential
Essential
3-keto sterol



polynucleotides




reductase


13
or CURFs
6
YDR341c/RRS1
Essential
Essential
Arginine tRNA








synthetase


14

7
YLR022c
Essential
Essential
Unknown


15

8
YOL077c/BRX1
Essential
Essential
Unknown


16

10
YNL132w/KRE33
?**
Essential
Unknown (P-








loop)


45

11
YJR072c
Essential
Not
Unknown







essential


17

12
YGR145w
?**
Essential
Component of








NuA3 histone








acetyltransferase


18

13
YDR412w
?**
Essential
Unknown


19

14
YOL010w/RCL1
Essential
Essential
RNA processing


20

15
YOR004w
Essential
Essential
Unknown


21

16
YOR056c
?**
Essential
Protein








degradation


22

17
YLR009w
?**
Essential
Protein synthesis





*Not determined.


**? Essentiality couldn't be established by this methodology.






The results as shown in Table 5 show that essentiality could not be ascertained using this methodology for CaYNL132w, CaYGR145w, CaYDR412w, CaYOR056c and CaYLR009w. The remainder of the polynucleotides encoded by SEQ ID NO: 1 through SEQ ID NO: 11 were determined to be essential. As expected, CaERG1 and CaRAM2 were found to be essential and PFY1 was found not to be essential. Essentiality for NMT1 was not determined.
Example 5
Construction of MET3 Promoter Plasmids
Two plasmids, pUMP and pAMP, were constructed which contain a MET3 promoter cassette with one plasmid harboring the URA3 polynucleotide and the other harboring ARG4 as the selective marker, respectively (see FIG. 5 for plasmid maps). The C. albicans MET3 promoter region was amplified by PCR from the total DNA of strain SC5324 using the primers MET3SPHI (SEQ ID NO:222) and MET3NCOI (SEQ ID NO:224). The primers contain the SpeI and NcoI restriction sites, respectively. To construct pUMP, the MET3 promoter PCR product was cut with restriction enzymes SphI and NcoI, gel-purified and ligated to pGEM-URA3 that was linearized by SphI and NcoI. This placed the MET3 promoter sequence adjacent to URA3 yet in opposite orientation to avoid transcription read-through (FIG. 5). Construction of pAMP involved two steps. First, the C. albicans ARG4 polynucleotide was released from pRS-ARG4ΔSpeI after digestion with SacI and KpnI (blunt-ended), and then ligated to the SacII (blunt-ended) and SacI sites of pGEM-URA3 thus replacing URA3. The resulting plasmid was named pGEM-ARG4. Second, pGEM-ARG4 was linearized with SphI and NcoI, gel-purified and ligated with the MET3 promoter PCR product treated with SphI and NcoI, yielding plasmid pAMP.
Example 6
Promoter Swapping
Similar to the PCR-based polynucleotide disruption approach, two common primer sequences that allow annealing to the plasmid template were designed based on pUMP and pAMP. The forward common primer MET3PF (Seq ID NO:225) is derived from the same sequence as primer 3DR described above while the reverse common primer MET3PR (SEQ ID NO:224) is derived from MET3 promoter sequence so that the primer will anneal to the sequence right in front of the ATG start codon. In order to replace the endogenous promoter of the polynucleotide of interest, the MET3 promoter swapping cassette with either URA3 or ARG4 as the selective marker is amplified from the plasmid pUMP or pAMP, respectively, using a pair of primers designed similarly to the ones used in the PCR-based polynucleotide disruption technique described above. The forward primer contains 50-60 bp of flanking sequences that are derived of sequences 500-1000 bp upstream of the ATG codon of the polynucleotide of interest to ensure it would anneal upstream or on the boundary of the endogenous promoter and this portion of the forward primer is attached to the 5′ end of the forward common promoter primer MET3 PF. The reverse primer has the 50-60 bp of flanking sequences which are derived from the start codon region of the polynucleotide or ORF including ATG attached to the 3′ end of reverse common primer MET3PR. The resulting PCR product contains the MET3 promoter cassette that is flanked by 50-60 bp of sequences, on either end, homologous to the upstream promoter region and to the coding region of the polynucleotide of interest, respectively.
Once introduced into the cells heterozygous for the polynucleotide of interest obtained via the regular PCR-based polynucleotide disruption approach (supra), the MET3 promoter cassette would replace the endogenous promoter of the remaining allele via homologous recombination.
FIG. 6 depicts this promoter swapping scheme. The first copy of YFG1, as seen in the FIG, is disrupted via the PCR-based polynucleotide disruption approach. ARG4 is the selective marker used in this example. The native promoter of the remaining copy of YFG1 is subsequently replaced by the MET3 promoter via homologous recombination. Primers used for confirmation PCR in pairs include 5′ promoter (i.e., a promoter-specific confirmation primer, upstream of the introduced MET3 promoter)/3′-PTURA (e.g. SEQ ID NO: 114 and SEQ ID NO: 146), 5′-PTURA/3′-promoter (i.e., a promoter-specific confirmation reverse primer, downstream of the ATG start codon (see, e.g., SEQ ID NO: 147 and SEQ ID NO: 115), 5′-gene/3′ polynucleotide (e.g., if using CaERG as YFG1, SEQ ID NO: 151 and SEQ ID NO: 152). If, alternatively, URA3 is used as the selective marker and acts to disrupt the first copy of YFG1, the primer pair 5′-PTARG and 3′-PTARG will replace 5′-PTURA and 3′-PTURA, respectively, for confirmation PCR (i.e., SEQ ID NO: 149 and SEQ ID NO: 150).
For example, FIG. 7 shows the results of confirmation PCR for C. albicans MET3P-ERG/erg1::ARG4 strains. Lanes labeled “M” contain 1 kb DNA markers with some fragment sizes indicated. Lanes 1 and 2 are transformants putatively not containing the correct constructs. Lanes 3 to 6 putatively contain the correct constructs (see below regarding methionine and cysteine construct regulation). The primer pair, SEQ ID NOs 147 and 115, respectively, are used. These primers correspond to 5′PTURA and sequences downstream of the ATG start codon of CaERG. The resulting PCR was 1.6 kb (Lanes 3-6) indicating the correct construct. Similarly, the primer pair SEQ ID NOs 114, and 146 is expected to yield a 1.2 kb single band since this primer pair corresponds to a sequence upstream of the ERG promoter and 3′-PTURA. Additionally, the FIG shows the expected single 2.4 kb band for a correct construct resulting from use of the polynucleotide specific primers SEQ ID NO: 151 and SEQ ID NO: 152.
Table 6 lists the SEQ ID NOs of the MET3 promoter swapping primers which may be used to remove the promoters associated with the essential polynucleotides encoded by SEQ ID NO: 1 through to SEQ ID NO: 11, SEQ ID NO: 45 and known essential and non-essential C. albicans homologues of S. cervisiae described herein. Additionally, this table lists the SEQ ID Nos. for PCR confirmation primers for promoter constructs. The 5′ primers listed in this table are upstream of the introduced MET3 promoter and the introduced selective marker. The 3′ primers are located downstream of the ATG start codon of the polynucleotide. SEQ ID NOs for polynucleotide specific primers which may be used for confirmation to detect MET3 promoter strain constructs in Candida albicans are listed in Table 4.







TABLE 6







MET3 Promoter Swapping Primers and Promoter Construct Confirmation Primers


















PCR
PCR





MET3
MET3
confirmation
confirmation





Promoter
Promoter
primers for
primers for




SEQ ID
Swapping
Swapping
promoter
promoter



Seq ID
NO of
Primers
Primers
constructs
constructs


Gene Name
No.
translation
(forward)
(reverse)
(5′)
(3′)
















CaYLR100wORF
1
12
88
89
121
122


CaYDR341cORF
2
13
90
91
123
124


CaYLR022cORF
3
14
92
93)
125
126


CaYOL077cORF
4
15
94
95
127
128


CaYNL132wORF
5
16
96
97
129
130


CaYGR145wORF
6
17
100
101
133
134


CaYDR412wORF
7
18
102
103
135
136


CaYOL010wORF
8
19
104
105
137
138


CaYOR004wORF
9
20
106
107
139
140


YOR056cORF
10
21
108
109
141
142


YOR009wORF
11
22
110
111
143
144


CaYJR072c
—
45
98
99
131
132


CaERG
—
—
80
81
114
115


RAM2
—
—
82
83
116
117


PFY1
—
—
84
85
118
119


CaNMT1
—
—
86
87
—
120


CaAAH1
—
—
112
113
145
146









Example 7
Further Means to Identify MET3 Promoter Constructs
MET3 promoter constructs were identified by phenotypic analysis via down-regulation of the MET3 promoter as well as by a PCR confirmation method described below. Phenotypic analysis was conducted first for two reasons. First, if the polynucleotide of interest is essential for cell growth, switching the MET3 promoter by adding methionine and cysteine in the growing culture will block the cell growth yielding no growth or inhibited growth phenotype. In this way, the correct constructs are identified without screening a large number of transformants via a PCR confirmation test. Second, the inability to inhibit cell growth via down-regulation of the MET3 promoter would be an early indication that the polynucleotide being tested might not be essential. Therefore, the final conclusion on whether a polynucleotide is essential depends on both the phenotypic analysis and PCR confirmation tests.
FIG. 8 shows examples of phenotypes containing constructs resulting in down regulation of polynucleotide expression by methionine and cysteine. This figure depicts the phenotypes resulting from the incorporation of correct and incorrect promoter swapping cassettes. 1/10 serial dilutions of cells were made and 5 μl of diluted cells were spotted from left to right on SD medium (left panel) and SD supplemented with 2.5 mM of methionine and 0.5 mM of cysteine (right panel). The known essential polynucleotides are “turned off” by the addition of the amino acids when the correct construct was introduced into the strain after 48 hours at 30° C. as evident by the lack of growth seen on the right panel. There is no difference in growth when the polynucleotide is non-essential without regard to the incorporation of the appropriate construct.
Example 8
Drug Hypersensitivity
In order to determine if essential polynucleotide products could be reduced in fungal cells resulting in drug hypersensitivity, a high throughput whole cell assay was designed. The known C. albicans essential polynucleotide corresponding ERG1 polynucleotide in S. cervisiae was tested. CaERG 1 codes for squaline epoxidase and is known to be inhibited by terbinafine. The first polynucleotide copy was disrupted as described above and the second polynucleotide copy was modified by promoter swapping with MET3 promoter, also as described above.
The optimal cell density needed to control cell growth in a 384-well plate within the log phase after 18 hours incubation at 35° C. was first determined. For this determination, 50 ml of SD+His broth (minimal media with histidine) was inoculated with colonies grown on an SD plate with histidine and shaken at 35° C. overnight. Plates which contain colonies were streaked out once a week and stored on the bench at room temperature to eliminate any shock to the cells upon culturing. Cells were then diluted to varying concentrations in SD+His medium. 35 μl of the diluted cells were dispensed into wells of a 384-well plate containing 8 μl of 10.26% DMSO. To these wells, 10 μl of SD+His broth was added for a final volume in ach well of 53 μl. The plate was sealed and incubated at 35° C. At different time intervals, the plate was withdrawn from the incubator and the cell growth was determined by reading absorbance at 590 nm on a Perkin Elmer 7000 machine. 7.5×105 cfu/ml was found to be the optimal stock concentration of cell density for inoculation of the 384 well plate within the log phase after 18 hours incubation at 35° C.
In order to determine the titratability of the MET3 promoter and the optimal methionine concentration to be used for drug sensitivity tests 50 mol of SD+His broth medium was inoculated with colonies grown on an SD+His plate and shaken at 35° C. overnight. Cells were diluted from these cultures to 757,002 cfu/ml in SD+His medium. 35 μl of the diluted cells were dispensed into wells of a 384-well plate containing 8 μl of 10.26% DMSO. 10 μl of methionine stock solution made in SD+His broth was added to the plates. 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.3125 mM, 0.1563 mM, 0.078 mM, 0.039 mM and no methionine concentrations were added to the plate. Cell growth was monitored between 16 and 24 hours by reading the optical density at 595 nm. FIG. 9, depicts the cell growth over time with the different methionine concentrations. As is evident from FIG. 9 cell growth increases with decreasing concentration of methionine. A 0.5 mM concentration of methionine reduces cell growth by approximately 50%.







TABLE 7







Sensitivity of the MET3P-ERG1 Construct to Antifungal Drugs


MICs μg/ml at 20 hrs














Terbinafine

Sordarin

Fluconazole















−Met
+Met
−Met
+Met
−Met
+Met







4 or 8
0.5
0.5
0.5
1
1










In order to assess whether cells containing the ERG1-MET promoter construct would display hypersensitivity to terbinafine upon the addition of methionine in comparison to cells for which no methionine was added, cells were grown in the 384 well-plate as described above. A final concentration of 0.05 mM methinone was added to wells within one plate of cells and a control plate, in which SD+His broth was added in lieu of methionine was added to a second plate. 8 μl of various concentrations of terbinafine in 10.26% DMSO was added to both the control and test plates. The concentrations of terbinafine added to different wells ranged from 163 μg/ml to 0.02 μg/ml. FIG. 10 shows the results of the sensitivity of MET3 promoter-CaERG1 cells to terbinafine in the absence and presence of methionine and lists the particular concentrations of terbinafine inhibitor used. After the addition of this inhibitor, both the control and experimental plates were incubated at 35° C. After 18 hours of incubation, the OD readings were taken of the plates to measure cell growth (595 nm). FIG. 10 shows that a 4-8 fold increase in sensitivity to the specific inhibitor terbinafine which is known to target the CaERG1 polynucleotide product squalene epoxidase were obtained in the presence of methionine comparing to the no methionine control. Increased sensitivity of cells containing the ERG1-MET3 was confirmed in a separate experiment using a 96-well format plate. Table 7 shows that when the promoter activity was down regulated by the presence of methionine in these experiments, sensitivity to terbinafine increased by 16-32 fold.
Furthermore, results of additional experiments conducted with the drugs sordarin and fluconazole which are not known to be inhibitors of the CaERG1 polynucleotide product confirm that sensitivity to these drugs is not altered by the presence of methionine.
These results establish that the MET3 promoter is titratable and target polynucleotide expression directed by the promoter, when down-regulated, result in cells more sensitive to specific drugs.
Example 9
High Throughput Screen
In order to test compounds on polynucleotide products efficiently and quickly, a high throughput screen was used. Using the known target ERG1 under the control of the MET3 promoter and a specific inhibitor as a control, a high throughput screen was carried out as follows. Overnight cultures were seeded from multiple colonies grown on an SD+His plate into 50 mls of SD+His broth. The culture was shaken overnight at 35° C. Plates which contain colonies were freshly streaked once per week and stored on the bench at room temperature to eliminate any shock to the cells upon culturing. Stock solution was prepared from the overnight culture. 0.350 mls of overnight culture were added to 14 mls of SD+His media at room temperature. The stock solution is adjusted to contain 1.0×107 cells. The stock solution is diluted in preparation for use to contain 757,002 cells/ml. 26,500 cells/well are plated. 35 μl of the 757,002 cells/ml solution is added to each well. A 500 ml culture is used for 37 plates. For a 500 ml culture 37.85 ml of stock solution was added to 500 mls of SD+His at room temperature. In order to prepare the plates, 35 μl of Millipore water is added to each well in columns 1-24 of the plates. 100% DMSO is added to wells in columns 21-24 using a stacking multidrop.
Preparation of QC Plates
Two plates are used as control plates. One of the control plates contains 4 ul of compounds and antibiotics in each well with known MICs in 100% DMSO. Wells also contain 162.6 μg/ml to 0 μg/ml of terbinafine titration ranging from 162.6 μg/ml to 0 μg/ml. The blank plate contains 4 μl of 100% DMSO in all wells. These are diluted as above and 8 μl stamped into daughter plates as well. SD+His media is added only to columns 23 and 24 using the stacking multipdrop. 35 μl of Candia albicans culture is added to columns 1-22 using a Multidrop on the microbial robot. 10 μl of 2.65 mM methionine which has been diluted from 250 mM in SD+His broth is added to all wells with a multidrop. Plates are placed in an incubator at 35° C. for 18 hours. Readings to assess the amount of cell culture growth are taken at OD590 using a Perkin Elmer 7000 spectrophotometer using the following additional settings: Gain 50, Integration Time 40, 3 flashes, flash delay 10, Dark #10, Top Read, X-direction.
The screen is run using 384 well plates containing 4 μl of 100% DMSO of a 1 mM stock in the last four columns. 25 μl of water is added to columns 1-24 and 8 μl of each compound is transferred to a “daughter” plate on a Cybio Cybiwell. The daughter plates used were clear tissue culture treated plates from Becton Dickinson (Bedford, Mass., catalog number 358058).
Example 10
Method of Cloning the Fungal CURFs of the Present Invention
The fungal CURFs of the present invention may be cloned into expression vectors for protein purification. Such vectors would be useful in the further elucidation of polynucleotide function, and enable the application of biochemical assays and the development of screening assays either biophysically or biochemically. All 11 CURFs may be amplified by PCR from the genome of C. albicans and cloned to the GatewayTM Expression System (Invitrogen, CA) for overexpression in E. coli. The GatewayTM Expression System allows for construction of either 6×His-tagged or GST-tagged fusion proteins to facilitate easy and high yield protein overexpression and purification.
In brief, the cloning procedure involves three steps: 1) PCR amplifying the polynucleotide of interest from C. albicans genomic DNA by a primer pair of the corresponding gene; 2) cloning of PCR products via the BP reaction to the Donor vector pDONR201 to create an entry clone; 3) sequencing the cloned polynucleotide in the entry clone via PCR primer pair SeqL-A (5′-TCGCGTTAACGCTAGCATGGATCTC-3′ (SEQ ID NO:227) and SeqL-B (5′-GTAACATCAGAGATTTTGAGACAC-3′ (SEQ ID NO:228)). Once the entry clone is made, an overexpression construct containing either 6× Histidine tag or GST-tag can be created by transferring polynucleotides from entry clones into destination vectors such as pDEST 17 via the LR reaction.
The PCR reactions are carried out using Pfu TruboTM DNA polymerase (Stragegene, La Jolla, Calif. 92037) as described in the preceding session (session 3. DNA manipulations). As for the polynucleotide cloning procedure, the manufacture protocol as described in GATEWAYTM Cloning Technology Instruction Manual may be followed essentially as described (Invitrogen, Carlsbad, Calif. 92008).
Representative primer pairs for cloning the fungal CURFs of the present invention are provided below.














SEQ











Primer

ID










Name
Primer Sequence
NO:













FCG5-fp
GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGTTCCGCG
229




TGGTAGCATGTCACTTTTAAAGGATTC


 


FCG5-rp
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAAGGTTG
230



ACGTGTATTTACTATTTG


 


FCG6-fp
GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGTTCCGCG
231



TGGTAGCATGTCAGTCGAAACAATTAG


 


FCG6-rp
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACATACG
232



ATTAACTGGAGTCAAAC


 


FCG7-fp
GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGTTCCGCG
233



TGGTAGCATGGCGGTGATTAATCAACC


 


FCG7-rp
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATTCCTT
234



TATGGCAGACATATC


 


FCG8-fp
GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGTTCCGCG
235



TGGTAGCATGTCAGCTATCTATAAGGC


 


FCG8-rp
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATTTAAA
236



TAAAGCATCATTGG


 


FCG10-fp
GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGTTCCGCG
237



TGGTAGCATGGGTAAAAAAGCAATTGATG


 


FCG10-rp
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATTTTTT
238



TGATTTCTTTGATTTC


 


FCG12-fp
GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGTTCCGCG
239



TGGTAGCATGGTTTTAAAATCAACAAC


 


FCG12-rp
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACATACC
240



TCTAAACTTATTCTTG


 


FCG13-fp
GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGTTCCGCG
241



TGGTAGCATGGCAGGATTTAAAAAGAATAG


 


FCG13-rp
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACTTCTT
242



GCCCTTTGATTTTG


 


FCG14-fp
GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGTTCCGCG
243



TGGTAGCATGTCCAGTGTTGCTTCCAAAAAG


 


FCG14-rp
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAAGCTAT
244



TTTTTTAGAAACATTG


 


FCG15-fp
GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGTTCCGCG
245



TGGTAGCATGAGACAAAAGCGTGCCAAG


 


FCG15-rp
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGTTGTT
246



GCTTCGTTCACTTGC


 


FCG16-fp
GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGTTCCGCG
247



TGGTAGCATGTCTGAAACAAAAAATATTG


 


FCG16-rp
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACTTTCT
248



TTTCTTTTTGGAAG


 


FCG17-fp
GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGTTCCGCG
249



TGGTAGCATGAGGATTTATCAATGTCA


 


FCG17-rp
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAACACGT
250



TTTTGTGTCACTTTC









Example 11
Biochemical Demonstration that CaYLR100w is a 3-Keto Sterol Reductase Involved in C-4 Sterol Demethylation
Overnight cultures were diluted into fresh SD+histidine (His) broth and grown at 30° C. until logarithmic growth was established. Cultures were harvested by centrifugation. Cells were then separated into tubes at a final density of 0.25 OD600 containing either fresh SD+His broth or SD+His broth containing 5 mM methionine (Met) and 2.5 mM cysteine (Cys). At the end of either a 0.75, 1.5, 3 or 4.5 hour incubation period, the OD600 values of each tube were measured and cultures were adjusted to a density of 0.25 OD600 units per ml (2 mls per tube). Cultures were then pulsed with 2 μCi/tube of [3H]-acetic acid for 60 minutes.
At the end of the labeling period, cells were pelleted and rinsed 1× with medium. The washed pellet was resuspended with 1 ml of 0.1 N HCl, transferred to glass screw-capped tubes, and incubated for 5 minutes at 85° C. Two mls of 90% ethanol: 15% KOH: 0.25% pyrogallol (w/v) was added and the sample allowed to saponify for 30 minutes at 85° C.
Tubes were extracted with three mls of petroleum ether with vigorous vortexing. The ether layer was removed and the residual aqueous layer was extracted a second time with 3 mls of petroleum ether. The ether layers were pooled and evaporated to dryness under an N2 stream.
Dried samples were dissolved in petroleum ether and spotted onto a 20 cm×20 cm silica gel 60 TLC plate. The plate was developed in a solvent system of benzene: ethyl acetate (99.5:0.5% v/v) until solvent front was 2 cm from the top of the TLC plate. The plate was dried completely then exposed for 1 to 3 days against X-ray film. The location of radiolabeled spots on the TLC plate was determined by matching the exposed spots on the autoradiogram. Results are shown in FIGS. 22A and B.
The affect of downregulating CaYLR100w expression on cell growth was also assessed by tracking the absorbance at OD600 for both the caerg1Δ/PMET3-CaERG1 (FIG. 23A) and fcg5Δ/PMET3-FCG5 (FIG. 23B) in the presence or absence of methionine and cysteine.
In order to quantitate the radioactivity associated with the ergosterol pathway products, the silica gel corresponding to the exposed spots was excised from the plate and mixed with 0.5 ml of water, then mixed with 5 mls of scintillation cocktail prior to scintillation counting. [14C]-cholesterol was used as a standard for the migration of ergosterol on the TLC plate. Results are shown in FIG. 24.
Example 12
Biochemical Demonstration that CaYDR341c is Involved in Whole Cell Protein Synthesis
A plate containing SD+His agar was inoculated with C. albicans mutant fcg6Δ/PMET3-FCG6 and incubated for 2 days at 30° C. An overnight culture was prepared from several colonies in SD+His broth and incubated at 30° C. on a rotary wheel for 18 hours. The overnight culture was diluted to 0.125 OD600 and incubated at 30° C. until 0.28 OD600 was achieved. A 100 μl aliquot of this culture was pipetted into the wells of a 96-well filter plate. Half of the wells then received 50 μl of SD+His broth, while the other half received 50 μl of SD+His broth containing 15 mM Met and 7.5 mM Cys solution to give a final concentration of methionine and cysteine of 5 mM and 2.5 mM, respectively. The plate was incubated at 30° C. without shaking for 3.5 hours prior to labelling.
A 4× radiolabelled amino acid labelling stock was prepared by adding 20 μl of 1 mCi/ml [3H]-leucine (42.5 Ci/mmole) or [3H]-arginine (57 Ci/mmole) to 1 ml of SD+His broth. Wells were labelled for 1 hour at 30° C. with the addition of 50 μl of the above [3H]-leucine or [3H]-arginine solution into the well (Final radioactive concentration of 5 μCi/ml or 1 μCi per well). After one hour, labelling was terminated with the addition of 100 μl of 20% trichloroacetic acid (TCA) per well (final [TCA]=6.7%). Plates were incubated at 4° C. overnight to precipitate proteins and amino acid-charged tRNA. Precipitates were collected by filtering on a vacuum manifold, washing filter wells 2× with 10% TCA, then 2× with water and finally 1× with ethanol. The plate was counted by adding 100 μl of Microscint-A scintillation fluid to the dried filter plate, then counting in a Packard Top-count scintillation counter. Results are shown in FIGS. 25A and 25B.
Example 13
Bacterial Expression of a Polypeptide
A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in herein, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5′ end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and XbaI correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.
The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, that expresses the lacI repressor and also confers kanamycin resistance (Kanr). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased polynucleotide expression.
Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 6000×g). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at 4 degree C. The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind to the Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).
Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.
The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM imidazole. Imidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4 degree C. or frozen at −80 degree C.
Other methods of purifying a polypeptide of the present invention are known in the art or disclosed elsewhere herein.
Example 14
Purification of a Polypeptide from an Inclusion Body
The following alternative method can be used to purify a polypeptide expressed in E. coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4-10 degree C.
Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10 degree C. and the cells harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer.
The cells are then lysed by passing the solution through a microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000×g for 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×g centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4 degree C. overnight to allow further GuHCl extraction.
Following high speed centrifugation (30,000×g) to remove insoluble particles, the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded diluted protein solution is kept at 4 degree C. without mixing for 12 hours prior to further purification steps.
To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0.16 um membrane filter with appropriate surface area (e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perceptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.
Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros HQ-50, Perceptive Biosystems) and weak anion (Poros CM-20, Perceptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10 column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A280 monitoring of the effluent. Fractions containing the polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.
The resultant polypeptide should exhibit greater than 95% purity after the above refolding and purification steps. No major contaminant bands should be observed from Coomassie blue stained 16% SDS-PAGE gel when 5 ug of purified protein is loaded. The purified protein can also be tested for endotoxin/LPS contamination, and typically the LPS content is less than 0.1 ng/ml according to LAL assays.
Other methods of purifying a polypeptide of the present invention are known in the art or disclosed elsewhere herein.
Example 15
Cloning and Expression of a Polypeptide in a Baculovirus Expression System
In this example, the plasmid shuttle vector pAc373 is used to insert a polynucleotide into a baculovirus to express a polypeptide. A typical baculovirus expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites, which may include, for example BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40 (“SV40”) is often used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase polynucleotide from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin polynucleotide. The inserted polynucleotides are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned polynucleotide.
Many other baculovirus vectors can be used in place of the vector above, such as pVL941 and pAcIM1, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al., Virology 170:31-39 (1989).
A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 12, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites at the 5′ end of the primers in order to clone the amplified product into the expression vector. Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon and the naturally associated leader sequence identified elsewhere herein (if applicable), is amplified using the PCR protocol described herein. If the naturally occurring signal sequence is used to produce the protein, the vector used does not need a second signal peptide. Alternatively, the vector can be modified to include a baculovirus leader sequence, using the standard methods described in Summers et al., “A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,” Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).
The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratapolynucleotide Cloning Systems, La Jolla, Calif.) cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.
Five ug of a plasmid containing the polynucleotide is co-transformed with 1.0 ug of a commercially available linearized baculovirus DNA (“BaculoGoldtm baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of BaculoGoldtm virus DNA and 5 ug of the plasmid are mixed in a sterile well of a microtiter plate containing 50 ul of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27 degrees C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27 degrees C. for four days.
After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10.) After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ul of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4 degree C.
To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection (“MOI”) of about 2. If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of 35S-methionine and 5 uCi 35S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).
Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the produced protein.
Other methods of purifying a polypeptide of the present invention are known in the art or disclosed elsewhere herein.
Example 16
Expression of the Fungal Conserved Essential Polypeptides in Mammalian Cells
The polypeptide of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome. The co-transformation with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transformed cells.
The transformed polynucleotide can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the polynucleotide of interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.
A polynucleotide of the present invention is amplified according to the protocol outlined in herein. If the naturally occurring signal sequence is used to produce the protein, the vector does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.) The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
The amplified fragment is then digested with the same restriction enzyme and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR polynucleotide is used for transformation. Five μg of an expression plasmid is cotransformed with 0.5 ug of the plasmid pSVneo using lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo polynucleotide from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 uM. Expression of the desired polynucleotide product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.
Other methods of purifying a polypeptide of the present invention are known in the art or disclosed elsewhere herein.
Example 17
Method of Enhancing the Biological Activity/Functional Characteristics of Invention Through Molecular Evolution
Although many of the most biologically active proteins known are highly effective for their specified function in an organism, they often possess characteristics that make them undesirable for transgenic, therapeutic, pharmaceutical, and/or industrial applications. Among these traits, a short physiological half-life is the most prominent problem, and is present either at the level of the protein, or the level of the proteins mRNA. The ability to extend the half-life, for example, would be particularly important for a proteins use in polynucleotide therapy, transgenic animal production, the bioprocess production and purification of the protein, and use of the protein as a chemical modulator among others. Therefore, there is a need to identify novel variants of isolated proteins possessing characteristics which enhance their application as a therapeutic for treating diseases of animal origin, in addition to the proteins applicability to common industrial and pharmaceutical applications.
Thus, one aspect of the present invention relates to the ability to enhance specific characteristics of invention through directed molecular evolution. Such an enhancement may, in a non-limiting example, benefit the inventions utility as an essential component in a kit, the inventions physical attributes such as its solubility, structure, or codon optimization, the inventions specific biological activity, including any associated enzymatic activity, the proteins enzyme kinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity, protein-DNA binding activity, antagonist/inhibitory activity (including direct or indirect interaction), agonist activity (including direct or indirect interaction), the proteins antigenicity (e.g., where it would be desirable to either increase or decrease the antigenic potential of the protein), the immunogenicity of the protein, the ability of the protein to form dimers, trimers, or multimers with either itself or other proteins, the antigenic efficacy of the invention, including its subsequent use a preventative treatment for disease or disease states, or as an effector for targeting diseased genes. Moreover, the ability to enhance specific characteristics of a protein may also be applicable to changing the characterized activity of an enzyme to an activity completely unrelated to its initially characterized activity. Other desirable enhancements of the invention would be specific to each individual protein, and would thus be well known in the art and contemplated by the present invention.
For example, an engineered CURF may be constitutively active upon binding of its cognate ligand. Alternatively, an engineered CURF may be constitutively active in the absence of ligand binding. In yet another example, an engineered CURF may be capable of being activated with less than all of the regulatory factors and/or conditions typically required for CURF activation (e.g., ligand binding, phosphorylation, conformational changes, etc.). Such CURFs would be useful in screens to identify CURF modulators, among other uses described herein.
Directed evolution is comprised of several steps. The first step is to establish a library of variants for the polynucleotide or protein of interest. The most important step is to then select for those variants that entail the activity you wish to identify. The design of the screen is essential since your screen should be selective enough to eliminate non-useful variants, but not so stringent as to eliminate all variants. The last step is then to repeat the above steps using the best variant from the previous screen. Each successive cycle, can then be tailored as necessary, such as increasing the stringency of the screen, for example.
Over the years, there have been a number of methods developed to introduce mutations into macromolecules. Some of these methods include, random mutagenesis, “error-prone” PCR, chemical mutagenesis, site-directed mutagenesis, and other methods well known in the art (for a comprehensive listing of current mutagenesis methods, see Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). Typically, such methods have been used, for example, as tools for identifying the core functional region(s) of a protein or the function of specific domains of a protein (if a multi-domain protein). However, such methods have more recently been applied to the identification of macromolecule variants with specific or enhanced characteristics.
Random mutagenesis has been the most widely recognized method to date. Typically, this has been carried out either through the use of “error-prone” PCR (as described in Moore, J., et al, Nature Biotechnology 14:458, (1996), or through the application of randomized synthetic oligonucleotides corresponding to specific regions of interest (as described by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), and Hill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approaches have limits to the level of mutagenesis that can be obtained. However, either approach enables the investigator to effectively control the rate of mutagenesis. This is particularly important considering the fact that mutations beneficial to the activity of the enzyme are fairly rare. In fact, using too high a level of mutagenesis may counter or inhibit the desired benefit of a useful mutation.
While both of the aforementioned methods are effective for creating randomized pools of macromolecule variants, a third method, termed “DNA Shuffling”, or “sexual PCR” (WPC, Stemmer, PNAS, 91:10747, (1994)) has recently been elucidated. DNA shuffling has also been referred to as “directed molecular evolution”, “exon-shuffling”, “directed enzyme evolution”, “in vitro evolution”, and “artificial evolution”. Such reference terms are known in the art and are encompassed by the invention. This new, preferred, method apparently overcomes the limitations of the previous methods in that it not only propagates positive traits, but simultaneously eliminates negative traits in the resulting progeny.
DNA shuffling accomplishes this task by combining the principal of in vitro recombination, along with the method of “error-prone” PCR. In effect, you begin with a randomly digested pool of small fragments of your gene, created by Dnase I digestion, and then introduce said random fragments into an “error-prone” PCR assembly reaction. During the PCR reaction, the randomly sized DNA fragments not only hybridize to their cognate strand, but also may hybridize to other DNA fragments corresponding to different regions of the polynucleotide of interest—regions not typically accessible via hybridization of the entire polynucleotide. Moreover, since the PCR assembly reaction utilizes “error-prone” PCR reaction conditions, random mutations are introduced during the DNA synthesis step of the PCR reaction for all of the fragments-further diversifying the potential hybridation sites during the annealing step of the reaction.
A variety of reaction conditions could be utilized to carry-out the DNA shuffling reaction. However, specific reaction conditions for DNA shuffling are provided, for example, in PNAS, 91:10747, (1994). Briefly:
Prepare the DNA substrate to be subjected to the DNA shuffling reaction. Preparation may be in the form of simply purifying the DNA from contaminating cellular material, chemicals, buffers, oligonucleotide primers, deoxynucleotides, RNAs, etc., and may entail the use of DNA purification kits as those provided by Qiagen, Inc., or by the Promega, Corp., for example.
Once the DNA substrate has been purified, it would be subjected to Dnase I digestion. About 2-4 ug of the DNA substrate(s) would be digested with 0.0015 units of Dnase I (Sigma) per ul in 100 ul of 50 mM Tris-HCL, pH 7.4/1 mM MgCl2 for 10-20 min. at room temperature. The resulting fragments of 10-50 bp could then be purified by running them through a 2% low-melting point agarose gel by electrophoresis onto DE81 ion-exchange paper (Whatman) or could be purified using Microcon concentrators (Amicon) of the appropriate molecular weight cuttoff, or could use oligonucleotide purification columns (Qiagen), in addition to other methods known in the art. If using DE81 ion-exchange paper, the 10-50 bp fragments could be eluted from said paper using 1 M NaCL, followed by ethanol precipitation.
The resulting purified fragments would then be subjected to a PCR assembly reaction by re-suspension in a PCR mixture containing: 2 mM of each dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM Tris.HCL, pH 9.0, and 0.1% Triton X-100, at a final fragment concentration of 10-30 ng/ul. No primers are added at this point. Taq DNA polymerase (Promega) would be used at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 C for 60 s; 94 C for 30 s, 50-55 C for 30 s, and 72 C for 30 s using 30-45 cycles, followed by 72 C for 5 min using an MJ Research (Cambridge, Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a 1:40 dilution of the resulting primerless product would then be introduced into a PCR mixture (using the same buffer mixture used for the assembly reaction) containing 0.8 um of each primer and subjecting this mixture to 15 cycles of PCR (using 94 C for 30 s, 50 C for 30 s, and 72 C for 30 s). The referred primers would be primers corresponding to the nucleic acid sequences of the polynucleotide(s) utilized in the shuffling reaction. Said primers could consist of modified nucleic acid base pairs using methods known in the art and referred to else where herein, or could contain additional sequences (i.e., for adding restriction sites, mutating specific base-pairs, etc.).
The resulting shuffled, assembled, and amplified product can be purified using methods well known in the art (e.g., Qiagen PCR purification kits) and then subsequently cloned using appropriate restriction enzymes.
Although a number of variations of DNA shuffling have been published to date, such variations would be obvious to the skilled artisan and are encompassed by the invention. The DNA shuffling method can also be tailored to the desired level of mutagenesis using the methods described by Zhao, et al. (Nucl Acid Res., 25(6): 1307-1308, (1997).
As described above, once the randomized pool has been created, it can then be subjected to a specific screen to identify the variant possessing the desired characteristic(s). Once the variant has been identified, DNA corresponding to the variant could then be used as the DNA substrate for initiating another round of DNA shuffling. This cycle of shuffling, selecting the optimized variant of interest, and then re-shuffling, can be repeated until the ultimate variant is obtained. Examples of model screens applied to identify variants created using DNA shuffling technology may be found in the following publications: J. C., Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al., Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat. Biotech., 15:436-438, (1997).
DNA shuffling has several advantages. First, it makes use of beneficial mutations. When combined with screening, DNA shuffling allows the discovery of the best mutational combinations and does not assume that the best combination contains all the mutations in a population. Secondly, recombination occurs simultaneously with point mutagenesis. An effect of forcing DNA polymerase to synthesize full-length genes from the small fragment DNA pool is a background mutagenesis rate. In combination with a stringent selection method, enzymatic activity has been evolved up to 16000 fold increase over the wild-type form of the enzyme. In essence, the background mutagenesis yielded the genetic variability on which recombination acted to enhance the activity.
A third feature of recombination is that it can be used to remove deleterious mutations. As discussed above, during the process of the randomization, for every one beneficial mutation, there may be at least one or more neutral or inhibitory mutations. Such mutations can be removed by including in the assembly reaction an excess of the wild-type random-size fragments, in addition to the random-size fragments of the selected mutant from the previous selection. During the next selection, some of the most active variants of the polynucleotide/polypeptide/enzyme, should have lost the inhibitory mutations.
Finally, recombination enables parallel processing. This represents a significant advantage since there are likely multiple characteristics that would make a protein more desirable (e.g. solubility, activity, etc.). Since it is increasingly difficult to screen for more than one desirable trait at a time, other methods of molecular evolution tend to be inhibitory. However, using recombination, it would be possible to combine the randomized fragments of the best representative variants for the various traits, and then select for multiple properties at once.
DNA shuffling can also be applied to the polynucleotides and polypeptides of the present invention to decrease their immunogenicity in a specified host, particularly if the polynucleotides and polypeptides provide a therapeutic use. For example, a particular variant of the present invention may be created and isolated using DNA shuffling technology. Such a variant may have all of the desired characteristics, though may be highly immunogenic in a host due to its novel intrinsic structure. Specifically, the desired characteristic may cause the polypeptide to have a non-native structure which could no longer be recognized as a “self” molecule, but rather as a “foreign”, and thus activate a host immune response directed against the novel variant. Such a limitation can be overcome, for example, by including a copy of the polynucleotide sequence for a xenobiotic ortholog of the native protein in with the polynucleotide sequence of the novel variant polynucleotide in one or more cycles of DNA shuffling. The molar ratio of the ortholog and novel variant DNAs could be varied accordingly. Ideally, the resulting hybrid variant identified would contain at least some of the coding sequence which enabled the xenobiotic protein to evade the host immune system, and additionally, the coding sequence of the original novel variant that provided the desired characteristics.
Likewise, the invention encompasses the application of DNA shuffling technology to the evolution of polynucleotides and polypeptides of the invention, wherein one or more cycles of DNA shuffling include, in addition to the polynucleotide template DNA, oligonucleotides coding for known allelic sequences, optimized codon sequences, known variant sequences, known polynucleotide polymorphism sequences, known ortholog sequences, known homolog sequences, additional homologous sequences, additional non-homologous sequences, sequences from another species, and any number and combination of the above.
In addition to the described methods above, there are a number of related methods that may also be applicable, or desirable in certain cases. Representative among these are the methods discussed in PCT applications WO 98/31700, and WO 98/32845, which are hereby incorporated by reference. Furthermore, related methods can also be applied to the polynucleotide sequences of the present invention in order to evolve invention for creating ideal variants for use in polynucleotide therapy, protein engineering, evolution of whole cells containing the variant, or in the evolution of entire enzyme pathways containing polynucleotides of the invention as described in PCT applications WO 98/13485, WO 98/13487, WO 98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech., 15:436-438, (1997), respectively.
Additional methods of applying “DNA Shuffling” technology to the polynucleotides and polypeptides of the present invention, including their proposed applications, may be found in U.S. Pat. No. 5,605,793; PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCT Application No. WO 97/35966; and PCT Application No. WO 98/42832; PCT Application No. The forgoing are hereby incorporated in their entirety herein for all purposes.
Example 18
Method of Creating N- and C-Terminal Deletion Mutants Corresponding to the Fungal Essential Conserved Polypeptides of the Present Invention
As described elsewhere herein, the present invention encompasses the creation of N- and C-terminal deletion mutants, in addition to any combination of N- and C-terminal deletions thereof, corresponding to the fungal essential conserved polypeptides of the present invention. A number of methods are available to one skilled in the art for creating such mutants. Such methods may include a combination of PCR amplification and polynucleotide cloning methodology. Although one of skill in the art of molecular biology, through the use of the teachings provided or referenced herein, and/or otherwise known in the art as standard methods, could readily create each deletion mutant of the present invention, exemplary methods are described below.
Briefly, using the isolated cDNA clone encoding the full-length fungal essential conserved polypeptides sequence (as described in herein, for example), appropriate primers of about 15-25 nucleotides derived from the desired 5′ and 3′ positions of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 may be designed to PCR amplify, and subsequently clone, the intended N- and/or C-terminal deletion mutant. Such primers could comprise, for example, an initiation and stop codon for the 5′ and 3′ primer, respectively. Such primers may also comprise restriction sites to facilitate cloning of the deletion mutant post amplification. Moreover, the primers may comprise additional sequences, such as, for example, flag-tag sequences, kozac sequences, or other sequences discussed and/or referenced herein.
Representative PCR amplification conditions are provided below, although the skilled artisan would appreciate that other conditions may be required for efficient amplification. A 100 ul PCR reaction mixture may be prepared using long of the template DNA (cDNA clone of fungal essential conserved polypeptides), 200 uM 4dNTPs, 1 uM primers, 0.25 U Taq DNA polymerase (PE), and standard Taq DNA polymerase buffer. Typical PCR cycling condition are as follows:
20-25 cycles:

    
    
        45 sec, 93 degrees
        2 min, 50 degrees
        2 min, 72 degrees
    
    


1 cycle: 10 min, 72 degrees
After the final extension step of PCR, 5U Klenow Fragment may be added and incubated for 15 min at 30 degrees.
Upon digestion of the fragment with the NotI and SalI restriction enzymes, the fragment could be cloned into an appropriate expression and/or cloning vector which has been similarly digested (e.g., pSport1, among others). The skilled artisan would appreciate that other plasmids could be equally substituted, and may be desirable in certain circumstances. The digested fragment and vector are then ligated using a DNA ligase, and then used to transform competent E. coli cells using methods provided herein and/or otherwise known in the art.
The 5′ primer sequence for amplifying any additional N-terminal deletion mutants may be determined by reference to the following formula: (S+(X*3)) to ((S+(X*3))+25), wherein ‘S’ is equal to the nucleotide position of the initiating start codon of the fungal essential conserved polypeptides polynucleotide (SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), and ‘X’ is equal to the most N-terminal amino acid of the intended N-terminal deletion mutant. The first term will provide the start 5′ nucleotide position of the 5′ primer, while the second term will provide the end 3′ nucleotide position of the 5′ primer corresponding to sense strand of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. Once the corresponding nucleotide positions of the primer are determined, the final nucleotide sequence may be created by the addition of applicable restriction site sequences to the 5′ end of the sequence, for example. As referenced herein, the addition of other sequences to the 5′ primer may be desired in certain circumstances (e.g., kozac sequences, etc.).
The 3′ primer sequence for amplifying any additional N-terminal deletion mutants may be determined by reference to the following formula: (S+(X*3)) to ((S+(X*3))−25), wherein ‘S’ is equal to the nucleotide position of the initiating start codon of the fungal essential conserved polypeptides polynucleotide (SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11), and ‘X’ is equal to the most C-terminal amino acid of the intended N-terminal deletion mutant. The first term will provide the start 5′ nucleotide position of the 3′ primer, while the second term will provide the end 3′ nucleotide position of the 3′ primer corresponding to the anti-sense strand of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. Once the corresponding nucleotide positions of the primer are determined, the final nucleotide sequence may be created by the addition of applicable restriction site sequences to the 5′ end of the sequence, for example. As referenced herein, the addition of other sequences to the 3′ primer may be desired in certain circumstances (e.g., stop codon sequences, etc.). The skilled artisan would appreciate that modifications of the above nucleotide positions may be necessary for optimizing PCR amplification.
The same general formulas provided above may be used in identifying the 5′ and 3′ primer sequences for amplifying any C-terminal deletion mutant of the present invention. Moreover, the same general formulas provided above may be used in identifying the 5′ and 3′ primer sequences for amplifying any combination of N-terminal and C-terminal deletion mutant of the present invention. The skilled artisan would appreciate that modifications of the above nucleotide positions may be necessary for optimizing PCR amplification.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., polynucleotide therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.
It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.
The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is hereby incorporated herein by reference. Further, the hard copy of the sequence listing submitted herewith and the corresponding computer readable form are both incorporated herein by reference in their entireties.













TABLE 8





Atom
Atom

Residue





No.
Type
Residue
Position
X Coord.
Y Coord.
Z Coord.





















1
N
MET
1
89.886
75.280
32.999


2
CA
MET
1
89.364
75.553
31.649


3
C
MET
1
89.998
74.642
30.605


4
O
MET
1
89.639
74.684
29.424


5
CB
MET
1
89.592
77.010
31.266


6
CG
MET
1
88.726
77.938
32.110


7
SD
MET
1
86.945
77.666
31.958


8
CE
MET
1
86.766
77.911
30.174


9
N
SER
2
90.875
73.766
31.066


10
CA
SER
2
91.477
72.765
30.173


11
C
SER
2
90.562
71.553
30.024


12
O
SER
2
90.684
70.769
29.079


13
CB
SER
2
92.812
72.318
30.759


14
OG
SER
2
93.346
71.313
29.906


15
N
LEU
3
89.584
71.463
30.903


16
CA
LEU
3
88.626
70.369
30.827


17
C
LEU
3
87.524
70.715
29.839


18
O
LEU
3
87.647
70.463
28.637


19
CB
LEU
3
88.021
70.153
32.209


20
CG
LEU
3
89.080
69.793
33.245


21
CD1
LEU
3
88.496
69.821
34.652


22
CD2
LEU
3
89.706
68.436
32.949


23
N
LEU
4
86.535
71.430
30.342


24
CA
LEU
4
85.284
71.656
29.608


25
C
LEU
4
85.357
72.691
28.489


26
O
LEU
4
86.225
73.574
28.494


27
CB
LEU
4
84.214
72.144
30.590


28
CG
LEU
4
83.396
71.037
31.260


29
CD1
LEU
4
82.789
70.108
30.217


30
CD2
LEU
4
84.168
70.240
32.307


31
N
LYS
5
84.612
72.370
27.437


32
CA
LYS
5
84.031
73.345
26.489


33
C
LYS
5
84.957
74.020
25.465


34
O
LYS
5
84.446
74.621
24.513


35
CB
LYS
5
83.421
74.435
27.376


36
CG
LYS
5
82.435
75.357
26.664


37
CD
LYS
5
81.128
74.640
26.345


38
CE
LYS
5
80.431
74.158
27.615


39
NZ
LYS
5
80.114
75.280
28.516


40
N
ASP
6
86.257
73.802
25.530


41
CA
ASP
6
87.159
74.650
24.742


42
C
ASP
6
87.397
74.157
23.312


43
O
ASP
6
86.456
73.984
22.526


44
CB
ASP
6
88.471
74.821
25.508


45
CG
ASP
6
88.321
75.848
26.641


46
OD1
ASP
6
87.198
76.283
26.862


47
OD2
ASP
6
89.349
76.418
26.983


48
N
SER
7
88.672
74.011
22.988


49
CA
SER
7
89.145
73.697
21.632


50
C
SER
7
88.514
72.438
21.025


51
O
SER
7
88.172
71.482
21.734


52
CB
SER
7
90.657
73.551
21.737


53
OG
SER
7
91.198
73.565
20.429


54
N
THR
8
88.406
72.441
19.705


55
CA
THR
8
87.672
71.390
18.976


56
C
THR
8
88.549
70.157
18.740


57
O
THR
8
89.432
70.163
17.872


58
CB
THR
8
87.225
71.958
17.632


59
OG1
THR
8
86.924
73.337
17.790


60
CG2
THR
8
85.986
71.244
17.101


61
N
VAL
9
88.240
69.095
19.468


62
CA
VAL
9
89.052
67.864
19.461


63
C
VAL
9
88.716
66.858
18.348


64
O
VAL
9
87.574
66.381
18.210


65
CB
VAL
9
88.864
67.236
20.837


66
CG1
VAL
9
87.417
67.358
21.295


67
CG2
VAL
9
89.374
65.801
20.933


68
N
ALA
10
89.752
66.537
17.581


69
CA
ALA
10
89.677
65.513
16.523


70
C
ALA
10
90.813
64.488
16.650


71
O
ALA
10
91.980
64.803
16.388


72
CB
ALA
10
89.764
66.209
15.170


73
N
VAL
11
90.453
63.258
16.982


74
CA
VAL
11
91.437
62.195
17.268


75
C
VAL
11
91.539
61.146
16.149


76
O
VAL
11
90.544
60.489
15.823


77
CB
VAL
11
90.980
61.523
18.561


78
CG1
VAL
11
91.917
60.401
18.992


79
CG2
VAL
11
90.831
62.546
19.680


80
N
ILE
12
92.742
60.942
15.628


81
CA
ILE
12
92.972
59.966
14.539


82
C
ILE
12
93.796
58.764
15.013


83
O
ILE
12
94.903
58.951
15.530


84
CB
ILE
12
93.779
60.645
13.435


85
CG1
ILE
12
93.256
62.027
13.090


86
CG2
ILE
12
93.809
59.789
12.171


87
CD1
ILE
12
94.165
62.659
12.045


88
N
THR
13
93.306
57.554
14.785


89
CA
THR
13
94.070
56.350
15.154


90
C
THR
13
95.005
55.881
14.043


91
O
THR
13
94.634
55.818
12.864


92
CB
THR
13
93.122
55.200
15.478


93
OG1
THR
13
92.574
54.687
14.267


94
CG2
THR
13
91.997
55.636
16.409


95
N
GLY
14
96.213
55.525
14.441


96
CA
GLY
14
97.161
54.894
13.521


97
C
GLY
14
97.808
55.923
12.610


98
O
GLY
14
97.975
55.688
11.407


99
N
THR
15
98.289
56.996
13.211


100
CA
THR
15
98.884
58.094
12.435


101
C
THR
15
100.352
57.874
12.097


102
O
THR
15
100.981
58.711
11.445


103
CB
THR
15
98.704
59.392
13.191


104
OG1
THR
15
99.332
59.272
14.464


105
CG2
THR
15
97.218
59.630
13.395


106
N
SER
16
100.870
56.728
12.505


107
CA
SER
16
102.194
56.294
12.065


108
C
SER
16
102.096
55.512
10.749


109
O
SER
16
103.120
55.069
10.217


110
CB
SER
16
102.836
55.441
13.154


111
OG
SER
16
102.054
54.269
13.345


112
N
SER
17
100.878
55.273
10.281


113
CA
SER
17
100.681
54.748
8.926


114
C
SER
17
100.698
55.929
7.969


115
O
SER
17
100.332
57.041
8.370


116
CB
SER
17
99.327
54.046
8.806


117
OG
SER
17
98.292
55.024
8.690


118
N
ASN
18
100.919
55.659
6.692


119
CA
ASN
18
100.924
56.744
5.699


120
C
ASN
18
99.521
57.307
5.479


121
O
ASN
18
99.384
58.526
5.310


122
CB
ASN
18
101.475
56.235
4.370


123
CG
ASN
18
102.928
55.774
4.494


124
OD1
ASN
18
103.611
56.038
5.490


125
ND2
ASN
18
103.401
55.137
3.437


126
N
LEU
19
98.507
56.495
5.739


127
CA
LEU
19
97.126
56.986
5.720


128
C
LEU
19
96.908
58.004
6.833


129
O
LEU
19
96.700
59.178
6.507


130
CB
LEU
19
96.138
55.833
5.890


131
CG
LEU
19
95.523
55.353
4.572


132
CD1
LEU
19
96.546
54.670
3.669


133
CD2
LEU
19
94.360
54.401
4.840


134
N
GLY
20
97.258
57.651
8.064


135
CA
GLY
20
97.099
58.566
9.204


136
C
GLY
20
97.902
59.854
9.058


137
O
GLY
20
97.345
60.942
9.244


138
N
PHE
21
99.119
59.729
8.556


139
CA
PHE
21
99.992
60.879
8.302


140
C
PHE
21
99.402
61.858
7.280


141
O
PHE
21
99.301
63.056
7.577


142
CB
PHE
21
101.295
60.294
7.761


143
CG
PHE
21
102.427
61.277
7.488


144
CD1
PHE
21
103.369
61.522
8.476


145
CD2
PHE
21
102.539
61.898
6.250


146
CE1
PHE
21
104.413
62.403
8.236


147
CE2
PHE
21
103.582
62.782
6.010


148
CZ
PHE
21
104.518
63.035
7.003


149
N
ASN
22
98.811
61.337
6.214


150
CA
ASN
22
98.297
62.201
5.138


151
C
ASN
22
96.938
62.783
5.513


152
O
ASN
22
96.661
63.955
5.224


153
CB
ASN
22
98.173
61.403
3.833


154
CG
ASN
22
99.487
61.347
3.041


155
OD1
ASN
22
99.576
61.892
1.933


156
ND2
ASN
22
100.427
60.547
3.514


157
N
ILE
23
96.239
62.076
6.385


158
CA
ILE
23
94.959
62.561
6.894


159
C
ILE
23
95.167
63.633
7.958


160
O
ILE
23
94.469
64.652
7.920


161
CB
ILE
23
94.207
61.366
7.468


162
CG1
ILE
23
93.907
60.367
6.356


163
CG2
ILE
23
92.920
61.817
8.143


164
CD1
ILE
23
93.420
59.026
6.884


165
N
ALA
24
96.291
63.564
8.653


166
CA
ALA
24
96.643
64.595
9.630


167
C
ALA
24
97.036
65.895
8.940


168
O
ALA
24
96.499
66.952
9.298


169
CB
ALA
24
97.812
64.090
10.466


170
N
VAL
25
97.686
65.764
7.793


171
CA
VAL
25
98.053
66.930
6.986


172
C
VAL
25
96.819
67.624
6.417


173
O
VAL
25
96.673
68.845
6.579


174
CB
VAL
25
98.928
66.448
5.832


175
CG1
VAL
25
99.155
67.543
4.799


176
CG2
VAL
25
100.256
65.886
6.323


177
N
ARG
26
95.838
66.830
6.019


178
CA
ARG
26
94.610
67.387
5.449


179
C
ARG
26
93.708
68.021
6.504


180
O
ARG
26
93.249
69.153
6.301


181
CB
ARG
26
93.860
66.260
4.752


182
CG
ARG
26
92.512
66.730
4.218


183
CD
ARG
26
91.817
65.623
3.438


184
NE
ARG
26
90.494
66.049
2.952


185
CZ
ARG
26
90.231
66.321
1.673


186
NH1
ARG
26
91.215
66.311
0.771


187
NH2
ARG
26
88.995
66.668
1.307


188
N
LEU
27
93.686
67.456
7.700


189
CA
LEU
27
92.817
68.009
8.743


190
C
LEU
27
93.395
69.284
9.342


191
O
LEU
27
92.669
70.285
9.407


192
CB
LEU
27
92.625
66.965
9.832


193
CG
LEU
27
91.974
65.706
9.273


194
CD1
LEU
27
91.890
64.621
10.332


195
CD2
LEU
27
90.598
65.989
8.687


196
N
LEU
28
94.717
69.362
9.385


197
CA
LEU
28
95.391
70.567
9.892


198
C
LEU
28
95.351
71.734
8.913


199
O
LEU
28
95.574
72.884
9.304


200
CB
LEU
28
96.855
70.239
10.134


201
CG
LEU
28
97.049
69.355
11.351


202
CD1
LEU
28
98.502
68.912
11.454


203
CD2
LEU
28
96.602
70.083
12.612


204
N
GLU
29
95.030
71.442
7.665


205
CA
GLU
29
94.918
72.481
6.651


206
C
GLU
29
93.458
72.767
6.294


207
O
GLU
29
93.198
73.589
5.406


208
CB
GLU
29
95.709
72.017
5.434


209
CG
GLU
29
97.180
71.850
5.810


210
CD
GLU
29
97.957
71.148
4.702


211
OE1
GLU
29
99.131
70.870
4.920


212
OE2
GLU
29
97.397
70.963
3.631


213
N
GLY
30
92.522
72.138
6.989


214
CA
GLY
30
91.111
72.326
6.644


215
C
GLY
30
90.197
72.693
7.814


216
O
GLY
30
89.600
73.777
7.818


217
N
LEU
31
90.095
71.796
8.781


218
CA
LEU
31
89.063
71.884
9.829


219
C
LEU
31
89.438
70.833
10.885


220
O
LEU
31
90.117
69.876
10.500


221
CB
LEU
31
87.738
71.540
9.136


222
CG
LEU
31
86.548
72.401
9.557


223
CD1
LEU
31
86.776
73.871
9.224


224
CD2
LEU
31
85.267
71.906
8.899


225
N
PRO
32
88.985
70.884
12.135


226
CA
PRO
32
87.797
71.615
12.626


227
C
PRO
32
87.936
73.130
12.788


228
O
PRO
32
89.020
73.715
12.721


229
CB
PRO
32
87.482
71.015
13.959


230
CG
PRO
32
88.559
70.020
14.344


231
CD
PRO
32
89.473
69.934
13.141


232
N
ASP
33
86.784
73.740
13.018


233
CA
ASP
33
86.674
75.186
13.246


234
C
ASP
33
86.515
75.501
14.740


235
O
ASP
33
87.335
75.023
15.533


236
CB
ASP
33
85.535
75.753
12.389


237
CG
ASP
33
84.282
74.882
12.397


238
OD1
ASP
33
84.154
74.046
11.508


239
OD2
ASP
33
83.440
75.104
13.259


240
N
ASN
34
85.527
76.319
15.098


241
CA
ASN
34
85.293
76.753
16.494


242
C
ASN
34
86.579
77.395
17.038


243
O
ASN
34
87.417
77.829
16.236


244
CB
ASN
34
84.818
75.535
17.310


245
CG
ASN
34
84.139
75.916
18.630


246
OD1
ASN
34
83.591
77.018
18.748


247
ND2
ASN
34
84.380
75.111
19.650


248
N
LYS
35
86.650
77.650
18.333


249
CA
LYS
35
87.902
78.104
18.939


250
C
LYS
35
88.971
77.002
18.899


251
O
LYS
35
89.005
76.078
19.722


252
CB
LYS
35
87.615
78.596
20.357


253
CG
LYS
35
86.655
77.669
21.095


254
CD
LYS
35
86.204
78.254
22.430


255
CE
LYS
35
85.093
77.404
23.040


256
NZ
LYS
35
84.607
77.970
24.310


257
N
GLU
36
89.744
77.069
17.825


258
CA
GLU
36
90.947
76.261
17.591


259
C
GLU
36
90.680
74.804
17.227


260
O
GLU
36
90.026
74.049
17.956


261
CB
GLU
36
91.883
76.335
18.792


262
CG
GLU
36
92.414
77.746
18.992


263
CD
GLU
36
93.409
77.769
20.148


264
OE1
GLU
36
93.519
78.811
20.780


265
OE2
GLU
36
94.114
76.784
20.303


266
N
ILE
37
91.153
74.465
16.041


267
CA
ILE
37
91.287
73.075
15.597


268
C
ILE
37
92.404
72.375
16.368


269
O
ILE
37
93.575
72.767
16.287


270
CB
ILE
37
91.651
73.146
14.112


271
CG1
ILE
37
92.372
71.907
13.588


272
CG2
ILE
37
92.459
74.409
13.822


273
CD1
ILE
37
92.838
72.121
12.152


274
N
THR
38
92.052
71.349
17.119


275
CA
THR
38
93.099
70.588
17.810


276
C
THR
38
93.104
69.125
17.408


277
O
THR
38
92.309
68.303
17.889


278
CB
THR
38
92.979
70.724
19.317


279
OG1
THR
38
91.601
70.838
19.637


280
CG2
THR
38
93.698
71.970
19.822


281
N
LEU
39
94.049
68.825
16.532


282
CA
LEU
39
94.226
67.461
16.040


283
C
LEU
39
95.084
66.641
17.001


284
O
LEU
39
96.140
67.095
17.461


285
CB
LEU
39
94.908
67.536
14.682


286
CG
LEU
39
94.739
66.235
13.913


287
CD1
LEU
39
93.260
65.961
13.678


288
CD2
LEU
39
95.482
66.288
12.588


289
N
VAL
40
94.569
65.476
17.352


290
CA
VAL
40
95.277
64.533
18.222


291
C
VAL
40
95.571
63.236
17.465


292
O
VAL
40
94.689
62.391
17.258


293
CB
VAL
40
94.407
64.237
19.439


294
CG1
VAL
40
95.137
63.320
20.412


295
CG2
VAL
40
93.987
65.522
20.144


296
N
VAL
41
96.816
63.096
17.049


297
CA
VAL
41
97.230
61.918
16.287


298
C
VAL
41
97.787
60.829
17.197


299
O
VAL
41
98.730
61.030
17.973


300
CB
VAL
41
98.240
62.352
15.236


301
CG1
VAL
41
97.554
63.244
14.209


302
CG2
VAL
41
99.460
63.037
15.839


303
N
THR
42
97.145
59.679
17.132


304
CA
THR
42
97.494
58.587
18.033


305
C
THR
42
98.205
57.431
17.337


306
O
THR
42
97.961
57.119
16.163


307
CB
THR
42
96.219
58.083
18.686


308
OG1
THR
42
95.491
57.317
17.741


309
CG2
THR
42
95.341
59.221
19.191


310
N
SER
43
99.107
56.824
18.088


311
CA
SER
43
99.837
55.626
17.642


312
C
SER
43
100.556
54.981
18.827


313
O
SER
43
100.892
55.653
19.807


314
CB
SER
43
100.827
56.013
16.543


315
OG
SER
43
101.686
54.913
16.259


316
N
ARG
44
100.757
53.676
18.746


317
CA
ARG
44
101.459
52.940
19.803


318
C
ARG
44
102.985
53.014
19.690


319
O
ARG
44
103.688
52.663
20.646


320
CB
ARG
44
101.022
51.482
19.705


321
CG
ARG
44
101.223
50.950
18.291


322
CD
ARG
44
100.784
49.499
18.153


323
NE
ARG
44
101.051
49.019
16.788


324
CZ
ARG
44
100.740
47.791
16.367


325
NH1
ARG
44
101.032
47.421
15.117


326
NH2
ARG
44
100.150
46.930
17.198


327
N
THR
45
103.492
53.564
18.599


328
CA
THR
45
104.947
53.577
18.397


329
C
THR
45
105.560
54.935
18.719


330
O
THR
45
105.853
55.701
17.795


331
CB
THR
45
105.249
53.189
16.949


332
OG1
THR
45
104.499
54.026
16.071


333
CG2
THR
45
104.845
51.748
16.664


334
N
LEU
46
105.921
55.135
19.979


335
CA
LEU
46
106.467
56.426
20.471


336
C
LEU
46
107.481
57.136
19.547


337
O
LEU
46
107.112
58.216
19.065


338
CB
LEU
46
107.042
56.227
21.877


339
CG
LEU
46
107.720
57.482
22.432


340
CD1
LEU
46
106.786
58.689
22.457


341
CD2
LEU
46
108.290
57.221
23.821


342
N
PRO
47
108.613
56.550
19.151


343
CA
PRO
47
109.556
57.327
18.334


344
C
PRO
47
109.059
57.624
16.914


345
O
PRO
47
109.341
58.715
16.405


346
CB
PRO
47
110.810
56.509
18.284


347
CG
PRO
47
110.562
55.162
18.942


348
CD
PRO
47
109.138
55.209
19.465


349
N
LYS
48
108.124
56.835
16.411


350
CA
LYS
48
107.609
57.068
15.066


351
C
LYS
48
106.443
58.052
15.095


352
O
LYS
48
106.344
58.883
14.185


353
CB
LYS
48
107.209
55.729
14.459


354
CG
LYS
48
106.732
55.880
13.020


355
CD
LYS
48
106.666
54.528
12.318


356
CE
LYS
48
105.844
53.522
13.113


357
NZ
LYS
48
105.751
52.235
12.406


358
N
VAL
49
105.808
58.186
16.252


359
CA
VAL
49
104.770
59.211
16.419


360
C
VAL
49
105.430
60.576
16.537


361
O
VAL
49
105.027
61.520
15.848


362
CB
VAL
49
103.989
58.973
17.707


363
CG1
VAL
49
102.821
59.943
17.809


364
CG2
VAL
49
103.477
57.552
17.812


365
N
LYS
50
106.592
60.583
17.168


366
CA
LYS
50
107.379
61.807
17.312


367
C
LYS
50
108.039
62.222
15.995


368
O
LYS
50
108.070
63.418
15.676


369
CB
LYS
50
108.438
61.516
18.366


370
CG
LYS
50
109.450
62.644
18.511


371
CD
LYS
50
110.526
62.273
19.524


372
CE
LYS
50
111.233
60.976
19.136


373
NZ
LYS
50
111.911
61.099
17.833


374
N
GLU
51
108.307
61.251
15.137


375
CA
GLU
51
108.840
61.561
13.810


376
C
GLU
51
107.747
62.074
12.880


377
O
GLU
51
107.973
63.092
12.215


378
CB
GLU
51
109.478
60.303
13.240


379
CG
GLU
51
110.743
59.956
14.015


380
CD
GLU
51
111.190
58.535
13.691


381
OE1
GLU
51
110.319
57.697
13.496


382
OE2
GLU
51
112.387
58.287
13.741


383
N
VAL
52
106.522
61.613
13.083


384
CA
VAL
52
105.382
62.134
12.320


385
C
VAL
52
105.040
63.559
12.752


386
O
VAL
52
104.875
64.427
11.881


387
CB
VAL
52
104.181
61.215
12.544


388
CG1
VAL
52
102.881
61.839
12.047


389
CG2
VAL
52
104.400
59.849
11.905


390
N
ILE
53
105.251
63.850
14.028


391
CA
ILE
53
105.049
65.211
14.538


392
C
ILE
53
106.077
66.171
13.950


393
O
ILE
53
105.679
67.146
13.296


394
CB
ILE
53
105.175
65.209
16.060


395
CG1
ILE
53
104.095
64.358
16.713


396
CG2
ILE
53
105.110
66.633
16.604


397
CD1
ILE
53
102.713
64.968
16.523


398
N
SER
54
107.321
65.718
13.885


399
CA
SER
54
108.407
66.559
13.372


400
C
SER
54
108.303
66.773
11.866


401
O
SER
54
108.422
67.915
11.402


402
CB
SER
54
109.731
65.875
13.688


403
OG
SER
54
109.812
65.706
15.097


404
N
ASP
55
107.812
65.763
11.168


405
CA
ASP
55
107.647
65.864
9.719


406
C
ASP
55
106.548
66.848
9.348


407
O
ASP
55
106.817
67.793
8.596


408
CB
ASP
55
107.261
64.498
9.161


409
CG
ASP
55
108.347
63.448
9.381


410
OD1
ASP
55
109.514
63.816
9.349


411
OD2
ASP
55
107.992
62.275
9.448


412
N
ILE
56
105.428
66.790
10.050


413
CA
ILE
56
104.287
67.619
9.654


414
C
ILE
56
104.346
69.052
10.192


415
O
ILE
56
103.907
69.962
9.475


416
CB
ILE
56
103.010
66.896
10.064


417
CG1
ILE
56
102.924
65.564
9.327


418
CG2
ILE
56
101.766
67.729
9.782


419
CD1
ILE
56
101.613
64.840
9.612


420
N
LYS
57
105.152
69.298
11.212


421
CA
LYS
57
105.321
70.692
11.648


422
C
LYS
57
106.362
71.433
10.799


423
O
LYS
57
106.394
72.669
10.789


424
CB
LYS
57
105.650
70.742
13.144


425
CG
LYS
57
106.926
70.021
13.578


426
CD
LYS
57
108.186
70.869
13.420


427
CE
LYS
57
109.433
70.095
13.828


428
NZ
LYS
57
110.644
70.890
13.580


429
N
LYS
58
107.116
70.684
10.008


430
CA
LYS
58
108.105
71.262
9.097


431
C
LYS
58
107.647
71.117
7.638


432
O
LYS
58
108.324
71.597
6.714


433
CB
LYS
58
109.402
70.493
9.334


434
CG
LYS
58
110.588
71.042
8.553


435
CD
LYS
58
111.831
70.188
8.762


436
CE
LYS
58
112.970
70.660
7.867


437
NZ
LYS
58
112.577
70.587
6.453


438
N
TYR
59
106.433
70.613
7.463


439
CA
TYR
59
105.938
70.141
6.161


440
C
TYR
59
105.530
71.273
5.230


441
O
TYR
59
104.337
71.522
5.042


442
CB
TYR
59
104.747
69.218
6.408


443
CG
TYR
59
104.173
68.540
5.167


444
CD1
TYR
59
104.931
67.599
4.483


445
CD2
TYR
59
102.896
68.862
4.724


446
CE1
TYR
59
104.415
66.983
3.351


447
CE2
TYR
59
102.381
68.248
3.591


448
CZ
TYR
59
103.141
67.310
2.905


449
OH
TYR
59
102.626
66.693
1.785


450
N
ILE
60
106.521
71.697
4.461


451
CA
ILE
60
106.478
72.782
3.464


452
C
ILE
60
106.645
74.138
4.157


453
O
ILE
60
105.835
75.068
4.015


454
CB
ILE
60
105.240
72.723
2.552


455
CG1
ILE
60
105.067
71.342
1.917


456
CG2
ILE
60
105.312
73.775
1.444


457
CD1
ILE
60
106.270
70.953
1.068


458
N
VAL
61
107.682
74.201
4.979


459
CA
VAL
61
108.174
75.496
5.462


460
C
VAL
61
109.508
75.795
4.789


461
O
VAL
61
109.797
76.939
4.417


462
CB
VAL
61
108.402
75.431
6.968


463
CG1
VAL
61
108.808
76.799
7.516


464
CG2
VAL
61
107.177
74.920
7.705


465
N
ALA
62
110.197
74.722
4.435


466
CA
ALA
62
111.578
74.827
3.944


467
C
ALA
62
111.745
75.150
2.458


468
O
ALA
62
112.852
75.522
2.050


469
CB
ALA
62
112.290
73.517
4.250


470
N
LYS
63
110.686
75.064
1.669


471
CA
LYS
63
110.806
75.472
0.262


472
C
LYS
63
110.642
76.978
0.219


473
O
LYS
63
109.511
77.472
0.200


474
CB
LYS
63
109.729
74.809
−0.582


475
CG
LYS
63
109.796
73.290
−0.499


476
CD
LYS
63
108.864
72.661
−1.527


477
CE
LYS
63
107.470
73.268
−1.443


478
NZ
LYS
63
106.553
72.627
−2.397


479
N
ILE
64
111.766
77.667
0.091


480
CA
ILE
64
111.847
79.094
0.431


481
C
ILE
64
111.394
79.211
1.885


482
O
ILE
64
110.192
79.325
2.156


483
CB
ILE
64
110.992
79.943
−0.511


484
CG1
ILE
64
111.431
79.747
−1.958


485
CG2
ILE
64
111.070
81.420
−0.135


486
CD1
ILE
64
110.575
80.568
−2.917


487
N
PRO
65
112.375
79.336
2.773


488
CA
PRO
65
112.347
78.723
4.128


489
C
PRO
65
111.428
79.349
5.192


490
O
PRO
65
111.592
79.071
6.386


491
CB
PRO
65
113.764
78.757
4.612


492
CG
PRO
65
114.649
79.398
3.561


493
CD
PRO
65
113.746
79.677
2.376


494
N
THR
66
110.506
80.195
4.774


495
CA
THR
66
109.546
80.827
5.674


496
C
THR
66
108.125
80.458
5.226


497
O
THR
66
107.143
81.105
5.612


498
CB
THR
66
109.787
82.336
5.626


499
OG1
THR
66
108.960
82.976
6.590


500
CG2
THR
66
109.499
82.932
4.250


501
N
LYS
67
108.039
79.413
4.414


502
CA
LYS
67
106.775
78.990
3.791


503
C
LYS
67
105.694
78.621
4.808


504
O
LYS
67
105.974
78.192
5.934


505
CB
LYS
67
107.058
77.831
2.843


506
CG
LYS
67
106.530
78.124
1.444


507
CD
LYS
67
107.093
79.440
0.917


508
CE
LYS
67
106.606
79.744
−0.492


509
NZ
LYS
67
107.174
81.013
−0.976


510
N
VAL
68
104.465
78.660
4.317


511
CA
VAL
68
103.250
78.705
5.148


512
C
VAL
68
102.816
77.397
5.830


513
O
VAL
68
102.077
77.465
6.823


514
CB
VAL
68
102.153
79.205
4.195


515
CG1
VAL
68
102.157
78.433
2.875


516
CG2
VAL
68
100.753
79.218
4.812


517
N
ASN
69
103.414
76.269
5.495


518
CA
ASN
69
102.895
75.015
6.042


519
C
ASN
69
103.658
74.516
7.271


520
O
ASN
69
104.317
73.468
7.260


521
CB
ASN
69
102.884
73.967
4.950


522
CG
ASN
69
101.739
74.138
3.959


523
OD1
ASN
69
101.368
75.248
3.556


524
ND2
ASN
69
101.184
73.000
3.582


525
N
LYS
70
103.403
75.197
8.375


526
CA
LYS
70
103.989
74.856
9.681


527
C
LYS
70
102.951
74.225
10.617


528
O
LYS
70
102.887
74.550
11.810


529
CB
LYS
70
104.525
76.150
10.281


530
CG
LYS
70
103.444
77.226
10.317


531
CD
LYS
70
103.984
78.548
10.848


532
CE
LYS
70
105.005
79.158
9.893


533
NZ
LYS
70
104.381
79.501
8.603


534
N
VAL
71
102.182
73.290
10.083


535
CA
VAL
71
101.002
72.775
10.796


536
C
VAL
71
101.333
71.931
12.031


537
O
VAL
71
101.953
70.863
11.979


538
CB
VAL
71
100.129
72.018
9.798


539
CG1
VAL
71
99.320
72.990
8.946


540
CG2
VAL
71
100.952
71.090
8.913


541
N
GLU
72
100.898
72.466
13.161


542
CA
GLU
72
101.145
71.849
14.469


543
C
GLU
72
99.968
70.998
14.937


544
O
GLU
72
98.804
71.296
14.646


545
CB
GLU
72
101.428
72.958
15.478


546
CG
GLU
72
102.670
73.770
15.105


547
CD
GLU
72
103.947
72.947
15.276


548
OE1
GLU
72
103.841
71.838
15.787


549
OE2
GLU
72
105.014
73.511
15.078


550
N
PHE
73
100.296
69.975
15.708


551
CA
PHE
73
99.303
68.980
16.145


552
C
PHE
73
99.807
68.146
17.325


553
O
PHE
73
101.008
67.889
17.471


554
CB
PHE
73
98.899
68.076
14.973


555
CG
PHE
73
99.943
67.173
14.292


556
CD1
PHE
73
101.267
67.563
14.117


557
CD2
PHE
73
99.524
65.950
13.792


558
CE1
PHE
73
102.170
66.715
13.489


559
CE2
PHE
73
100.422
65.102
13.165


560
CZ
PHE
73
101.746
65.478
13.019


561
N
ASP
74
98.866
67.729
18.154


562
CA
ASP
74
99.170
66.985
19.384


563
C
ASP
74
99.292
65.492
19.105


564
O
ASP
74
98.605
64.964
18.223


565
CB
ASP
74
98.024
67.205
20.369


566
CG
ASP
74
97.831
68.692
20.654


567
OD1
ASP
74
96.690
69.133
20.648


568
OD2
ASP
74
98.835
69.375
20.809


569
N
TYR
75
100.182
64.829
19.822


570
CA
TYR
75
100.303
63.371
19.688


571
C
TYR
75
99.831
62.653
20.950


572
O
TYR
75
99.883
63.204
22.056


573
CB
TYR
75
101.742
62.977
19.356


574
CG
TYR
75
102.800
63.152
20.448


575
CD1
TYR
75
103.479
64.357
20.584


576
CD2
TYR
75
103.107
62.084
21.284


577
CE1
TYR
75
104.445
64.503
21.572


578
CE2
TYR
75
104.072
62.228
22.272


579
CZ
TYR
75
104.736
63.439
22.414


580
OH
TYR
75
105.688
63.585
23.399


581
N
LEU
76
99.317
61.451
20.755


582
CA
LEU
76
98.848
60.625
21.873


583
C
LEU
76
99.269
59.166
21.674


584
O
LEU
76
98.985
58.550
20.639


585
CB
LEU
76
97.326
60.783
21.943


586
CG
LEU
76
96.674
60.217
23.206


587
CD1
LEU
76
95.406
60.986
23.554


588
CD2
LEU
76
96.379
58.726
23.103


589
N
LEU
77
99.940
58.619
22.671


590
CA
LEU
77
100.364
57.217
22.597


591
C
LEU
77
99.232
56.275
22.987


592
O
LEU
77
98.761
56.283
24.129


593
CB
LEU
77
101.531
56.997
23.546


594
CG
LEU
77
102.737
57.836
23.151


595
CD1
LEU
77
103.848
57.673
24.181


596
CD2
LEU
77
103.223
57.464
21.754


597
N
VAL
78
98.832
55.439
22.046


598
CA
VAL
78
97.774
54.467
22.336


599
C
VAL
78
98.002
53.153
21.593


600
O
VAL
78
98.177
53.088
20.369


601
CB
VAL
78
96.398
55.058
22.021


602
CG1
VAL
78
96.198
55.289
20.532


603
CG2
VAL
78
95.276
54.175
22.557


604
N
ASP
79
98.063
52.100
22.383


605
CA
ASP
79
98.191
50.758
21.816


606
C
ASP
79
96.799
50.248
21.465


607
O
ASP
79
95.998
49.953
22.360


608
CB
ASP
79
98.900
49.834
22.801


609
CG
ASP
79
99.139
48.476
22.146


610
OD1
ASP
79
98.187
47.701
22.131


611
OD2
ASP
79
100.131
48.355
21.449


612
N
PHE
80
96.643
49.881
20.202


613
CA
PHE
80
95.324
49.584
19.620


614
C
PHE
80
94.715
48.234
20.035


615
O
PHE
80
93.532
47.992
19.778


616
CB
PHE
80
95.526
49.607
18.108


617
CG
PHE
80
94.263
49.770
17.271


618
CD1
PHE
80
93.423
50.853
17.492


619
CD2
PHE
80
93.966
48.852
16.273


620
CE1
PHE
80
92.279
51.014
16.722


621
CE2
PHE
80
92.822
49.013
15.504


622
CZ
PHE
80
91.980
50.094
15.727


623
N
THR
81
95.486
47.385
20.695


624
CA
THR
81
94.955
46.110
21.190


625
C
THR
81
94.863
46.114
22.715


626
O
THR
81
94.475
45.110
23.325


627
CB
THR
81
95.899
44.988
20.777


628
OG1
THR
81
97.084
45.101
21.554


629
CG2
THR
81
96.274
45.072
19.304


630
N
ASP
82
95.280
47.208
23.326


631
CA
ASP
82
95.387
47.252
24.782


632
C
ASP
82
94.319
48.167
25.371


633
O
ASP
82
94.459
49.396
25.339


634
CB
ASP
82
96.791
47.759
25.107


635
CG
ASP
82
97.175
47.561
26.572


636
OD1
ASP
82
96.366
47.903
27.426


637
OD2
ASP
82
98.337
47.272
26.805


638
N
MET
83
93.448
47.566
26.167


639
CA
MET
83
92.316
48.296
26.752


640
C
MET
83
92.726
49.307
27.825


641
O
MET
83
92.173
50.411
27.831


642
CB
MET
83
91.374
47.277
27.378


643
CG
MET
83
90.910
46.246
26.357


644
SD
MET
83
89.797
44.977
27.001


645
CE
MET
83
88.450
46.045
27.560


646
N
VAL
84
93.859
49.084
28.473


647
CA
VAL
84
94.337
50.025
29.492


648
C
VAL
84
95.044
51.217
28.848


649
O
VAL
84
94.928
52.345
29.341


650
CB
VAL
84
95.302
49.282
30.411


651
CG1
VAL
84
95.840
50.193
31.510


652
CG2
VAL
84
94.632
48.055
31.018


653
N
SER
85
95.475
51.026
27.612


654
CA
SER
85
96.097
52.112
26.861


655
C
SER
85
95.021
52.939
26.162


656
O
SER
85
95.185
54.152
25.986


657
CB
SER
85
97.047
51.500
25.844


658
OG
SER
85
97.690
52.560
25.158


659
N
ILE
86
93.855
52.335
25.996


660
CA
ILE
86
92.686
53.061
25.496


661
C
ILE
86
92.027
53.848
26.630


662
O
ILE
86
91.561
54.970
26.401


663
CB
ILE
86
91.714
52.045
24.900


664
CG1
ILE
86
92.355
51.328
23.715


665
CG2
ILE
86
90.407
52.710
24.478


666
CD1
ILE
86
91.426
50.274
23.123


667
N
LEU
87
92.252
53.406
27.860


668
CA
LEU
87
91.808
54.169
29.034


669
C
LEU
87
92.754
55.335
29.311


670
O
LEU
87
92.306
56.406
29.742


671
CB
LEU
87
91.803
53.253
30.253


672
CG
LEU
87
90.837
52.084
30.101


673
CD1
LEU
87
91.009
51.080
31.235


674
CD2
LEU
87
89.393
52.562
30.018


675
N
SER
88
93.989
55.200
28.856


676
CA
SER
88
94.959
56.292
28.964


677
C
SER
88
94.628
57.378
27.952


678
O
SER
88
94.492
58.548
28.337


679
CB
SER
88
96.353
55.745
28.682


680
OG
SER
88
96.615
54.705
29.614


681
N
ALA
89
94.223
56.954
26.764


682
CA
ALA
89
93.771
57.894
25.733


683
C
ALA
89
92.504
58.622
26.171


684
O
ALA
89
92.483
59.861
26.166


685
CB
ALA
89
93.475
57.106
24.462


686
N
TYR
90
91.609
57.875
26.799


687
CA
TYR
90
90.368
58.421
27.354


688
C
TYR
90
90.638
59.532
28.366


689
O
TYR
90
90.331
60.694
28.065


690
CB
TYR
90
89.631
57.270
28.034


691
CG
TYR
90
88.319
57.646
28.712


692
CD1
TYR
90
88.261
57.799
30.092


693
CD2
TYR
90
87.176
57.812
27.945


694
CE1
TYR
90
87.061
58.145
30.703


695
CE2
TYR
90
85.977
58.155
28.554


696
CZ
TYR
90
85.924
58.325
29.928


697
OH
TYR
90
84.744
58.722
30.519


698
N
TYR
91
91.493
59.248
29.338


699
CA
TYR
91
91.772
60.218
30.402


700
C
TYR
91
92.566
61.434
29.924


701
O
TYR
91
92.260
62.555
30.351


702
CB
TYR
91
92.552
59.510
31.506


703
CG
TYR
91
93.028
60.440
32.620


704
CD1
TYR
91
92.110
61.002
33.499


705
CD2
TYR
91
94.382
60.732
32.748


706
CE1
TYR
91
92.543
61.854
34.505


707
CE2
TYR
91
94.817
61.585
33.754


708
CZ
TYR
91
93.895
62.144
34.629


709
OH
TYR
91
94.323
63.007
35.613


710
N
GLU
92
93.378
61.267
28.893


711
CA
GLU
92
94.135
62.405
28.364


712
C
GLU
92
93.228
63.356
27.592


713
O
GLU
92
93.244
64.563
27.866


714
CB
GLU
92
95.223
61.870
27.443


715
CG
GLU
92
96.227
61.025
28.218


716
CD
GLU
92
97.142
60.272
27.257


717
OE1
GLU
92
97.630
60.896
26.324


718
OE2
GLU
92
97.363
59.089
27.486


719
N
LEU
93
92.242
62.799
26.911


720
CA
LEU
93
91.314
63.633
26.151


721
C
LEU
93
90.253
64.247
27.057


722
O
LEU
93
89.965
65.443
26.910


723
CB
LEU
93
90.688
62.769
25.068


724
CG
LEU
93
91.769
62.330
24.086


725
CD1
LEU
93
91.321
61.171
23.206


726
CD2
LEU
93
92.263
63.505
23.249


727
N
ASN
94
89.971
63.583
28.167


728
CA
ASN
94
89.051
64.142
29.164


729
C
ASN
94
89.678
65.279
29.965


730
O
ASN
94
88.978
66.243
30.297


731
CB
ASN
94
88.639
63.044
30.139


732
CG
ASN
94
87.589
62.134
29.516


733
OD1
ASN
94
87.788
60.922
29.374


734
ND2
ASN
94
86.453
62.731
29.205


735
N
LYS
95
90.996
65.284
30.062


736
CA
LYS
95
91.680
66.325
30.824


737
C
LYS
95
92.072
67.528
29.960


738
O
LYS
95
92.358
68.604
30.502


739
CB
LYS
95
92.924
65.676
31.426


740
CG
LYS
95
93.585
66.538
32.494


741
CD
LYS
95
94.823
65.847
33.051


742
CE
LYS
95
95.487
66.680
34.141


743
NZ
LYS
95
96.711
66.025
34.628


744
N
ARG
96
92.049
67.361
28.646


745
CA
ARG
96
92.482
68.449
27.759


746
C
ARG
96
91.367
69.046
26.895


747
O
ARG
96
91.602
70.072
26.244


748
CB
ARG
96
93.578
67.914
26.843


749
CG
ARG
96
94.782
67.410
27.632


750
CD
ARG
96
95.437
68.522
28.443


751
NE
ARG
96
96.531
67.983
29.264


752
CZ
ARG
96
96.708
68.308
30.546


753
NH1
ARG
96
95.879
69.171
31.135


754
NH2
ARG
96
97.720
67.776
31.235


755
N
TYR
97
90.240
68.357
26.781


756
CA
TYR
97
89.116
68.857
25.965


757
C
TYR
97
87.775
68.472
26.576


758
O
TYR
97
86.725
69.004
26.180


759
CB
TYR
97
89.170
68.216
24.580


760
CG
TYR
97
90.472
68.420
23.816


761
CD1
TYR
97
90.771
69.666
23.285


762
CD2
TYR
97
91.357
67.360
23.660


763
CE1
TYR
97
91.965
69.857
22.603


764
CE2
TYR
97
92.550
67.550
22.975


765
CZ
TYR
97
92.850
68.799
22.451


766
OH
TYR
97
94.041
68.994
21.789


767
N
LYS
98
87.827
67.341
27.266


768
CA
LYS
98
86.706
66.693
27.980


769
C
LYS
98
85.762
65.900
27.063


770
O
LYS
98
85.142
64.922
27.505


771
CB
LYS
98
85.925
67.738
28.769


772
CG
LYS
98
84.921
67.091
29.711


773
CD
LYS
98
85.623
66.210
30.735


774
CE
LYS
98
84.611
65.475
31.603


775
NZ
LYS
98
83.716
66.428
32.276


776
N
HIS
99
85.690
66.279
25.800


777
CA
HIS
99
84.854
65.566
24.836


778
C
HIS
99
85.698
65.139
23.648


779
O
HIS
99
86.914
65.363
23.617


780
CB
HIS
99
83.758
66.507
24.341


781
CG
HIS
99
83.036
67.263
25.439


782
ND1
HIS
99
82.392
66.738
26.500


783
CD2
HIS
99
82.926
68.631
25.541


784
CE1
HIS
99
81.886
67.736
27.251


785
NE2
HIS
99
82.217
68.907
26.658


786
N
ILE
100
85.065
64.400
22.755


787
CA
ILE
100
85.660
64.060
21.459


788
C
ILE
100
84.602
64.278
20.388


789
O
ILE
100
83.589
63.571
20.338


790
CB
ILE
100
86.162
62.617
21.448


791
CG1
ILE
100
87.331
62.445
22.411


792
CG2
ILE
100
86.579
62.189
20.044


793
CD1
ILE
100
87.928
61.050
22.320


794
N
ASP
101
84.819
65.279
19.551


795
CA
ASP
101
83.794
65.618
18.565


796
C
ASP
101
84.046
64.852
17.280


797
O
ASP
101
83.106
64.441
16.591


798
CB
ASP
101
83.844
67.115
18.282


799
CG
ASP
101
83.682
67.902
19.579


800
OD1
ASP
101
84.566
68.706
19.850


801
OD2
ASP
101
82.841
67.512
20.380


802
N
TYR
102
85.311
64.575
17.024


803
CA
TYR
102
85.665
63.801
15.837


804
C
TYR
102
86.571
62.624
16.191


805
O
TYR
102
87.640
62.809
16.789


806
CB
TYR
102
86.364
64.739
14.859


807
CG
TYR
102
85.493
65.865
14.309


808
CD1
TYR
102
85.950
67.176
14.338


809
CD2
TYR
102
84.240
65.578
13.783


810
CE1
TYR
102
85.155
68.200
13.835


811
CE2
TYR
102
83.444
66.599
13.284


812
CZ
TYR
102
83.905
67.907
13.308


813
OH
TYR
102
83.139
68.909
12.753


814
N
LEU
103
86.135
61.427
15.834


815
CA
LEU
103
86.965
60.227
16.040


816
C
LEU
103
87.226
59.521
14.704


817
O
LEU
103
86.334
59.402
13.858


818
CB
LEU
103
86.270
59.285
17.019


819
CG
LEU
103
87.140
58.077
17.356


820
CD1
LEU
103
88.428
58.505
18.049


821
CD2
LEU
103
86.384
57.067
18.205


822
N
PHE
104
88.476
59.159
14.479


823
CA
PHE
104
88.873
58.537
13.220


824
C
PHE
104
89.514
57.163
13.423


825
O
PHE
104
90.667
57.066
13.860


826
CB
PHE
104
89.899
59.481
12.617


827
CG
PHE
104
90.113
59.326
11.126


828
CD1
PHE
104
89.744
60.364
10.284


829
CD2
PHE
104
90.680
58.171
10.608


830
CE1
PHE
104
89.930
60.240
8.917


831
CE2
PHE
104
90.864
58.045
9.242


832
CZ
PHE
104
90.487
59.079
8.400


833
N
ILE
105
88.822
56.121
13.001


834
CA
ILE
105
89.388
54.771
13.084


835
C
ILE
105
89.999
54.370
11.741


836
O
ILE
105
89.305
54.100
10.751


837
CB
ILE
105
88.308
53.787
13.506


838
CG1
ILE
105
87.659
54.228
14.813


839
CG2
ILE
105
88.912
52.398
13.684


840
CD1
ILE
105
88.619
54.104
15.992


841
N
ASN
106
91.320
54.363
11.750


842
CA
ASN
106
92.129
54.080
10.559


843
C
ASN
106
93.088
52.913
10.780


844
O
ASN
106
93.343
52.137
9.850


845
CB
ASN
106
92.907
55.354
10.231


846
CG
ASN
106
94.166
55.089
9.406


847
OD1
ASN
106
94.114
54.689
8.237


848
ND2
ASN
106
95.295
55.346
10.042


849
N
ALA
107
93.516
52.730
12.020


850
CA
ALA
107
94.500
51.675
12.320


851
C
ALA
107
93.962
50.266
12.052


852
O
ALA
107
92.876
49.912
12.526


853
CB
ALA
107
94.916
51.798
13.780


854
N
ALA
108
94.724
49.491
11.291


855
CA
ALA
108
94.349
48.102
10.964


856
C
ALA
108
95.503
47.316
10.326


857
O
ALA
108
96.252
47.850
9.500


858
CB
ALA
108
93.172
48.128
9.995


859
N
GLN
109
95.624
46.053
10.706


860
CA
GLN
109
96.628
45.145
10.116


861
C
GLN
109
96.222
44.652
8.728


862
O
GLN
109
95.029
44.589
8.399


863
CB
GLN
109
96.761
43.908
10.991


864
CG
GLN
109
97.253
44.203
12.399


865
CD
GLN
109
97.059
42.934
13.219


866
OE1
GLN
109
98.009
42.346
13.748


867
NE2
GLN
109
95.817
42.491
13.245


868
N
GLY
110
97.217
44.202
7.979


869
CA
GLY
110
96.990
43.640
6.641


870
C
GLY
110
97.864
42.411
6.376


871
O
GLY
110
99.064
42.534
6.105


872
N
VAL
111
97.253
41.238
6.448


873
CA
VAL
111
97.985
39.985
6.200


874
C
VAL
111
98.128
39.713
4.705


875
O
VAL
111
97.141
39.513
3.988


876
CB
VAL
111
97.252
38.830
6.876


877
CG1
VAL
111
97.869
37.482
6.516


878
CG2
VAL
111
97.243
39.018
8.387


879
N
TYR
112
99.369
39.750
4.251


880
CA
TYR
112
99.674
39.524
2.837


881
C
TYR
112
99.713
38.037
2.523


882
O
TYR
112
100.270
37.240
3.292


883
CB
TYR
112
101.014
40.179
2.532


884
CG
TYR
112
101.031
41.635
2.976


885
CD1
TYR
112
101.982
42.075
3.889


886
CD2
TYR
112
100.086
42.517
2.468


887
CE1
TYR
112
101.968
43.397
4.315


888
CE2
TYR
112
100.071
43.836
2.894


889
CZ
TYR
112
101.005
44.268
3.824


890
OH
TYR
112
100.888
45.523
4.366


891
N
GLY
113
99.153
37.709
1.369


892
CA
GLY
113
99.019
36.328
0.890


893
C
GLY
113
100.308
35.531
1.000


894
O
GLY
113
101.399
36.036
0.696


895
N
GLY
114
100.163
34.354
1.587


896
CA
GLY
114
101.280
33.429
1.804


897
C
GLY
114
102.421
34.110
2.544


898
O
GLY
114
103.470
34.360
1.939


899
N
ILE
115
102.143
34.501
3.781


900
CA
ILE
115
103.098
35.183
4.681


901
C
ILE
115
104.011
36.173
3.954


902
O
ILE
115
105.104
35.822
3.501


903
CB
ILE
115
103.909
34.118
5.425


904
CG1
ILE
115
102.987
33.262
6.283


905
CG2
ILE
115
104.972
34.748
6.320


906
CD1
ILE
115
102.364
34.104
7.391


907
N
ASP
116
103.524
37.397
3.813


908
CA
ASP
116
104.239
38.492
3.121


909
C
ASP
116
104.863
38.079
1.784


910
O
ASP
116
106.075
38.222
1.580


911
CB
ASP
116
105.277
39.173
4.034


912
CG
ASP
116
106.383
38.264
4.582


913
OD1
ASP
116
106.189
37.770
5.683


914
OD2
ASP
116
107.475
38.303
4.026


915
N
TRP
117
104.001
37.634
0.877


916
CA
TRP
117
104.351
37.325
−0.523


917
C
TRP
117
105.541
36.372
−0.640


918
O
TRP
117
106.483
36.673
−1.383


919
CB
TRP
117
104.734
38.604
−1.280


920
CG
TRP
117
104.127
39.915
−0.806


921
CD1
TRP
117
102.835
40.362
−0.976


922
CD2
TRP
117
104.831
40.955
−0.092


923
NE1
TRP
117
102.722
41.579
−0.384


924
CE2
TRP
117
103.887
41.963
0.171


925
CE3
TRP
117
106.137
41.088
0.353


926
CZ2
TRP
117
104.261
43.079
0.903


927
CZ3
TRP
117
106.506
42.214
1.077


928
CH2
TRP
117
105.569
43.205
1.354


929
N
THR
118
105.515
35.252
0.062


930
CA
THR
118
106.665
34.343
0.000


931
C
THR
118
106.358
33.046
−0.736


932
O
THR
118
105.476
32.967
−1.599


933
CB
THR
118
107.203
34.048
1.398


934
OG1
THR
118
106.163
33.515
2.207


935
CG2
THR
118
107.738
35.310
2.067


936
N
GLY
119
107.196
32.063
−0.460


937
CA
GLY
119
107.144
30.780
−1.166


938
C
GLY
119
108.479
30.562
−1.872


939
O
GLY
119
109.538
30.707
−1.248


940
N
ALA
120
108.425
30.485
−3.194


941
CA
ALA
120
109.630
30.293
−4.031


942
C
ALA
120
110.400
31.591
−4.332


943
O
ALA
120
111.066
31.703
−5.367


944
CB
ALA
120
109.210
29.634
−5.341


945
N
VAL
121
110.307
32.549
−3.423


946
CA
VAL
121
110.858
33.889
−3.627


947
C
VAL
121
112.146
34.116
−2.828


948
O
VAL
121
112.917
35.036
−3.127


949
CB
VAL
121
109.781
34.863
−3.142


950
CG1
VAL
121
110.149
36.320
−3.401


951
CG2
VAL
121
108.431
34.548
−3.777


952
N
LEU
122
112.410
33.249
−1.864


953
CA
LEU
122
113.513
33.515
−0.928


954
C
LEU
122
114.776
32.694
−1.181


955
O
LEU
122
114.738
31.615
−1.781


956
CB
LEU
122
113.018
33.260
0.491


957
CG
LEU
122
111.914
34.237
0.885


958
CD1
LEU
122
111.360
33.897
2.264


959
CD2
LEU
122
112.416
35.678
0.848


960
N
GLU
123
115.867
33.224
−0.642


961
CA
GLU
123
117.206
32.597
−0.641


962
C
GLU
123
117.803
32.442
−2.035


963
O
GLU
123
117.599
31.417
−2.694


964
CB
GLU
123
117.160
31.225
0.030


965
CG
GLU
123
116.674
31.300
1.473


966
CD
GLU
123
116.833
29.935
2.136


967
OE1
GLU
123
117.828
29.285
1.843


968
OE2
GLU
123
116.064
29.654
3.044


969
N
VAL
124
118.603
33.418
−2.434


970
CA
VAL
124
119.271
33.347
−3.740


971
C
VAL
124
120.788
33.231
−3.575


972
O
VAL
124
121.396
32.234
−3.980


973
CB
VAL
124
118.931
34.603
−4.543


974
CG1
VAL
124
119.558
34.552
−5.934


975
CG2
VAL
124
117.422
34.795
−4.657


976
N
LEU
125
121.373
34.221
−2.923


977
CA
LEU
125
122.829
34.262
−2.756


978
C
LEU
125
123.240
33.876
−1.340


979
O
LEU
125
122.760
34.458
−0.360


980
CB
LEU
125
123.289
35.681
−3.104


981
CG
LEU
125
124.793
35.906
−2.950


982
CD1
LEU
125
125.353
36.685
−4.134


983
CD2
LEU
125
125.130
36.608
−1.636


984
N
GLN
126
124.064
32.845
−1.244


985
CA
GLN
126
124.646
32.474
0.050


986
C
GLN
126
126.170
32.619
0.022


987
O
GLN
126
126.857
32.103
−0.866


988
CB
GLN
126
124.218
31.057
0.424


989
CG
GLN
126
122.700
30.982
0.583


990
CD
GLN
126
122.275
29.634
1.157


991
OE1
GLN
126
123.068
28.947
1.809


992
NE2
GLN
126
121.021
29.285
0.925


993
N
SER
127
126.657
33.421
0.952


994
CA
SER
127
128.090
33.727
1.086


995
C
SER
127
128.510
33.368
2.536


996
O
SER
127
127.922
32.387
3.004


997
CB
SER
127
128.241
35.174
0.622


998
OG
SER
127
127.964
35.197
−0.772


999
N
PRO
128
129.433
34.005
3.265


1000
CA
PRO
128
130.268
35.173
2.925


1001
C
PRO
128
131.546
34.818
2.168


1002
O
PRO
128
131.794
33.666
1.802


1003
CB
PRO
128
130.626
35.786
4.239


1004
CG
PRO
128
130.407
34.745
5.321


1005
CD
PRO
128
129.727
33.575
4.636


1006
N
ILE
129
132.325
35.858
1.913


1007
CA
ILE
129
133.635
35.708
1.272


1008
C
ILE
129
134.722
35.503
2.335


1009
O
ILE
129
135.735
34.838
2.088


1010
CB
ILE
129
133.914
36.987
0.481


1011
CG1
ILE
129
132.841
37.230
−0.577


1012
CG2
ILE
129
135.292
36.959
−0.171


1013
CD1
ILE
129
132.829
36.127
−1.631


1014
N
GLU
130
134.456
35.998
3.533


1015
CA
GLU
130
135.382
35.806
4.655


1016
C
GLU
130
134.627
35.523
5.948


1017
O
GLU
130
133.510
34.997
5.911


1018
CB
GLU
130
136.287
37.023
4.819


1019
CG
GLU
130
137.741
36.639
4.554


1020
CD
GLU
130
138.668
37.778
4.963


1021
OE1
GLU
130
138.249
38.547
5.819


1022
OE2
GLU
130
139.796
37.805
4.495


1023
N
ALA
131
135.181
35.981
7.062


1024
CA
ALA
131
134.684
35.605
8.400


1025
C
ALA
131
133.506
36.430
8.931


1026
O
ALA
131
133.565
36.960
10.046


1027
CB
ALA
131
135.845
35.703
9.381


1028
N
VAL
132
132.416
36.455
8.184


1029
CA
VAL
132
131.208
37.148
8.636


1030
C
VAL
132
130.323
36.142
9.366


1031
O
VAL
132
129.433
35.515
8.779


1032
CB
VAL
132
130.485
37.746
7.436


1033
CG1
VAL
132
129.263
38.549
7.877


1034
CG2
VAL
132
131.428
38.626
6.622


1035
N
THR
133
130.579
36.020
10.657


1036
CA
THR
133
129.957
34.971
11.470


1037
C
THR
133
128.501
35.267
11.817


1038
O
THR
133
128.191
36.158
12.614


1039
CB
THR
133
130.781
34.820
12.741


1040
OG1
THR
133
132.120
34.532
12.357


1041
CG2
THR
133
130.274
33.673
13.607


1042
N
ASN
134
127.624
34.556
11.127


1043
CA
ASN
134
126.181
34.597
11.384


1044
C
ASN
134
125.602
33.185
11.322


1045
O
ASN
134
126.244
32.276
10.783


1046
CB
ASN
134
125.511
35.495
10.343


1047
CG
ASN
134
125.508
36.948
10.815


1048
OD1
ASN
134
124.775
37.305
11.745


1049
ND2
ASN
134
126.308
37.771
10.160


1050
N
PRO
135
124.473
32.978
11.984


1051
CA
PRO
135
123.744
31.713
11.845


1052
C
PRO
135
123.371
31.454
10.388


1053
O
PRO
135
123.020
32.375
9.643


1054
CB
PRO
135
122.531
31.852
12.712


1055
CG
PRO
135
122.526
33.232
13.351


1056
CD
PRO
135
123.775
33.936
12.848


1057
N
THR
136
123.497
30.201
9.985


1058
CA
THR
136
123.233
29.825
8.591


1059
C
THR
136
121.806
29.313
8.364


1060
O
THR
136
121.360
29.183
7.218


1061
CB
THR
136
124.263
28.766
8.194


1062
OG1
THR
136
124.006
28.350
6.859


1063
CG2
THR
136
124.215
27.535
9.095


1064
N
TYR
137
121.068
29.097
9.440


1065
CA
TYR
137
119.705
28.581
9.294


1066
C
TYR
137
118.672
29.698
9.354


1067
O
TYR
137
118.418
30.291
10.407


1068
CB
TYR
137
119.451
27.531
10.367


1069
CG
TYR
137
120.279
26.266
10.155


1070
CD1
TYR
137
120.361
25.703
8.887


1071
CD2
TYR
137
120.943
25.676
11.223


1072
CE1
TYR
137
121.117
24.557
8.683


1073
CE2
TYR
137
121.700
24.529
11.020


1074
CZ
TYR
137
121.786
23.974
9.750


1075
OH
TYR
137
122.548
22.845
9.544


1076
N
LYS
138
117.987
29.861
8.234


1077
CA
LYS
138
116.993
30.930
8.052


1078
C
LYS
138
115.561
30.528
8.437


1079
O
LYS
138
114.607
31.232
8.086


1080
CB
LYS
138
117.032
31.325
6.579


1081
CG
LYS
138
118.452
31.681
6.144


1082
CD
LYS
138
118.517
32.029
4.661


1083
CE
LYS
138
119.944
32.331
4.215


1084
NZ
LYS
138
119.991
32.660
2.780


1085
N
LEU
139
115.411
29.427
9.158


1086
CA
LEU
139
114.075
28.886
9.436


1087
C
LEU
139
113.741
28.914
10.928


1088
O
LEU
139
114.477
28.348
11.746


1089
CB
LEU
139
114.059
27.445
8.930


1090
CG
LEU
139
112.689
26.788
9.065


1091
CD1
LEU
139
111.639
27.539
8.253


1092
CD2
LEU
139
112.749
25.326
8.637


1093
N
GLN
140
112.657
29.597
11.270


1094
CA
GLN
140
112.178
29.585
12.661


1095
C
GLN
140
110.672
29.884
12.753


1096
O
GLN
140
109.853
29.039
12.380


1097
CB
GLN
140
113.046
30.541
13.490


1098
CG
GLN
140
112.970
30.307
15.005


1099
CD
GLN
140
113.205
28.846
15.406


1100
OE1
GLN
140
112.398
28.276
16.150


1101
NE2
GLN
140
114.278
28.252
14.908


1102
N
LYS
141
110.319
31.087
13.182


1103
CA
LYS
141
108.913
31.433
13.464


1104
C
LYS
141
108.192
32.136
12.313


1105
O
LYS
141
107.020
32.512
12.445


1106
CB
LYS
141
108.903
32.346
14.683


1107
CG
LYS
141
109.574
31.672
15.872


1108
CD
LYS
141
109.681
32.615
17.064


1109
CE
LYS
141
110.370
31.933
18.240


1110
NZ
LYS
141
110.485
32.846
19.388


1111
N
VAL
142
108.890
32.344
11.212


1112
CA
VAL
142
108.296
33.059
10.080


1113
C
VAL
142
107.459
32.120
9.215


1114
O
VAL
142
107.995
31.358
8.403


1115
CB
VAL
142
109.426
33.667
9.256


1116
CG1
VAL
142
108.877
34.502
8.103


1117
CG2
VAL
142
110.339
34.514
10.136


1118
N
GLY
143
106.153
32.175
9.412


1119
CA
GLY
143
105.236
31.373
8.598


1120
C
GLY
143
104.068
30.824
9.411


1121
O
GLY
143
103.837
29.609
9.436


1122
N
VAL
144
103.327
31.720
10.043


1123
CA
VAL
144
102.159
31.306
10.829


1124
C
VAL
144
101.029
30.820
9.924


1125
O
VAL
144
100.886
31.246
8.773


1126
CB
VAL
144
101.681
32.466
11.699


1127
CG1
VAL
144
102.745
32.854
12.719


1128
CG2
VAL
144
101.267
33.676
10.868


1129
N
GLU
145
100.264
29.883
10.451


1130
CA
GLU
145
99.141
29.297
9.710


1131
C
GLU
145
97.992
30.291
9.595


1132
O
GLU
145
97.917
31.265
10.356


1133
CB
GLU
145
98.650
28.021
10.402


1134
CG
GLU
145
99.470
26.767
10.073


1135
CD
GLU
145
100.812
26.696
10.808


1136
OE1
GLU
145
101.667
25.956
10.348


1137
OE2
GLU
145
100.919
27.317
11.857


1138
N
SER
146
97.012
29.933
8.778


1139
CA
SER
146
95.858
30.813
8.527


1140
C
SER
146
94.937
30.950
9.738


1141
O
SER
146
94.386
32.036
9.956


1142
CB
SER
146
95.065
30.260
7.349


1143
OG
SER
146
95.906
30.299
6.204


1144
N
GLY
147
95.003
29.983
10.640


1145
CA
GLY
147
94.302
30.077
11.925


1146
C
GLY
147
94.853
31.227
12.767


1147
O
GLY
147
94.080
32.078
13.222


1148
N
ASP
148
96.169
31.391
12.748


1149
CA
ASP
148
96.821
32.438
13.540


1150
C
ASP
148
96.673
33.799
12.871


1151
O
ASP
148
96.492
34.805
13.569


1152
CB
ASP
148
98.309
32.126
13.642


1153
CG
ASP
148
98.532
30.686
14.092


1154
OD1
ASP
148
98.090
30.351
15.181


1155
OD2
ASP
148
99.049
29.919
13.290


1156
N
LYS
149
96.527
33.790
11.553


1157
CA
LYS
149
96.292
35.032
10.816


1158
C
LYS
149
94.915
35.583
11.159


1159
O
LYS
149
94.823
36.705
11.674


1160
CB
LYS
149
96.343
34.746
9.319


1161
CG
LYS
149
97.673
34.146
8.877


1162
CD
LYS
149
97.655
33.862
7.377


1163
CE
LYS
149
98.940
33.192
6.910


1164
NZ
LYS
149
98.936
32.992
5.452


1165
N
LEU
150
93.924
34.704
11.165


1166
CA
LEU
150
92.555
35.111
11.500


1167
C
LEU
150
92.422
35.461
12.978


1168
O
LEU
150
91.803
36.481
13.301


1169
CB
LEU
150
91.613
33.960
11.164


1170
CG
LEU
150
91.589
33.676
9.667


1171
CD1
LEU
150
90.907
32.345
9.368


1172
CD2
LEU
150
90.932
34.817
8.897


1173
N
GLY
151
93.175
34.771
13.819


1174
CA
GLY
151
93.242
35.081
15.251


1175
C
GLY
151
93.686
36.516
15.509


1176
O
GLY
151
92.863
37.335
15.944


1177
N
LEU
152
94.860
36.864
15.005


1178
CA
LEU
152
95.446
38.187
15.258


1179
C
LEU
152
94.704
39.318
14.553


1180
O
LEU
152
94.611
40.432
15.091


1181
CB
LEU
152
96.882
38.163
14.744


1182
CG
LEU
152
97.726
37.117
15.463


1183
CD1
LEU
152
99.067
36.920
14.766


1184
CD2
LEU
152
97.923
37.478
16.931


1185
N
VAL
153
94.032
39.007
13.459


1186
CA
VAL
153
93.282
40.044
12.758


1187
C
VAL
153
91.876
40.235
13.329


1188
O
VAL
153
91.407
41.376
13.379


1189
CB
VAL
153
93.262
39.698
11.278


1190
CG1
VAL
153
92.472
40.723
10.487


1191
CG2
VAL
153
94.687
39.644
10.746


1192
N
PHE
154
91.340
39.234
14.005


1193
CA
PHE
154
90.082
39.456
14.724


1194
C
PHE
154
90.368
40.250
15.991


1195
O
PHE
154
89.675
41.233
16.283


1196
CB
PHE
154
89.444
38.124
15.116


1197
CG
PHE
154
88.814
37.313
13.985


1198
CD1
PHE
154
88.230
37.950
12.898


1199
CD2
PHE
154
88.800
35.927
14.063


1200
CE1
PHE
154
87.650
37.200
11.883


1201
CE2
PHE
154
88.222
35.177
13.047


1202
CZ
PHE
154
87.648
35.814
11.955


1203
N
GLN
155
91.532
39.999
16.562


1204
CA
GLN
155
91.941
40.711
17.767


1205
C
GLN
155
92.182
42.192
17.499


1206
O
GLN
155
91.328
43.007
17.871


1207
CB
GLN
155
93.186
40.026
18.307


1208
CG
GLN
155
92.813
38.616
18.750


1209
CD
GLN
155
94.050
37.767
19.013


1210
OE1
GLN
155
95.155
38.083
18.557


1211
NE2
GLN
155
93.824
36.642
19.668


1212
N
ALA
156
93.154
42.520
16.667


1213
CA
ALA
156
93.480
43.940
16.499


1214
C
ALA
156
92.656
44.692
15.453


1215
O
ALA
156
92.578
45.921
15.535


1216
CB
ALA
156
94.960
44.073
16.173


1217
N
ASN
157
91.970
43.999
14.560


1218
CA
ASN
157
91.169
44.717
13.561


1219
C
ASN
157
89.689
44.750
13.933


1220
O
ASN
157
88.934
45.540
13.355


1221
CB
ASN
157
91.289
44.061
12.186


1222
CG
ASN
157
92.667
44.166
11.534


1223
OD1
ASN
157
93.724
43.984
12.155


1224
ND2
ASN
157
92.628
44.425
10.240


1225
N
VAL
158
89.271
43.909
14.866


1226
CA
VAL
158
87.862
43.921
15.278


1227
C
VAL
158
87.698
44.319
16.741


1228
O
VAL
158
87.053
45.337
17.024


1229
CB
VAL
158
87.251
42.546
15.026


1230
CG1
VAL
158
85.886
42.392
15.687


1231
CG2
VAL
158
87.167
42.244
13.536


1232
N
PHE
159
88.408
43.650
17.635


1233
CA
PHE
159
88.228
43.926
19.066


1234
C
PHE
159
88.930
45.213
19.463


1235
O
PHE
159
88.358
46.017
20.211


1236
CB
PHE
159
88.753
42.754
19.886


1237
CG
PHE
159
87.922
41.484
19.732


1238
CD1
PHE
159
88.465
40.359
19.126


1239
CD2
PHE
159
86.617
41.456
20.206


1240
CE1
PHE
159
87.700
39.208
18.985


1241
CE2
PHE
159
85.852
40.305
20.067


1242
CZ
PHE
159
86.393
39.182
19.455


1243
N
GLY
160
90.048
45.477
18.807


1244
CA
GLY
160
90.735
46.778
18.887


1245
C
GLY
160
89.776
47.963
18.728


1246
O
GLY
160
89.409
48.594
19.732


1247
N
PRO
161
89.275
48.189
17.518


1248
CA
PRO
161
88.346
49.304
17.295


1249
C
PRO
161
86.987
49.165
17.992


1250
O
PRO
161
86.403
50.201
18.324


1251
CB
PRO
161
88.154
49.368
15.813


1252
CG
PRO
161
88.873
48.205
15.157


1253
CD
PRO
161
89.584
47.470
16.277


1254
N
TYR
162
86.580
47.967
18.388


1255
CA
TYR
162
85.333
47.821
19.144


1256
C
TYR
162
85.465
48.410
20.545


1257
O
TYR
162
84.643
49.258
20.916


1258
CB
TYR
162
84.988
46.339
19.239


1259
CG
TYR
162
83.761
46.019
20.087


1260
CD1
TYR
162
83.916
45.400
21.322


1261
CD2
TYR
162
82.490
46.335
19.623


1262
CE1
TYR
162
82.802
45.107
22.097


1263
CE2
TYR
162
81.375
46.041
20.397


1264
CZ
TYR
162
81.534
45.427
21.631


1265
OH
TYR
162
80.425
45.063
22.364


1266
N
TYR
163
86.605
48.199
21.184


1267
CA
TYR
163
86.805
48.755
22.527


1268
C
TYR
163
87.115
50.247
22.463


1269
O
TYR
163
86.582
51.017
23.273


1270
CB
TYR
163
87.967
48.032
23.203


1271
CG
TYR
163
87.750
46.538
23.432


1272
CD1
TYR
163
86.523
46.068
23.884


1273
CD2
TYR
163
88.795
45.650
23.209


1274
CE1
TYR
163
86.331
44.707
24.086


1275
CE2
TYR
163
88.604
44.289
23.408


1276
CZ
TYR
163
87.372
43.822
23.843


1277
OH
TYR
163
87.175
42.470
24.015


1278
N
PHE
164
87.707
50.665
21.356


1279
CA
PHE
164
88.039
52.079
21.174


1280
C
PHE
164
86.781
52.909
20.916


1281
O
PHE
164
86.543
53.888
21.637


1282
CB
PHE
164
88.984
52.185
19.984


1283
CG
PHE
164
90.138
53.162
20.182


1284
CD1
PHE
164
90.010
54.231
21.058


1285
CD2
PHE
164
91.329
52.966
19.497


1286
CE1
PHE
164
91.068
55.112
21.238


1287
CE2
PHE
164
92.388
53.847
19.676


1288
CZ
PHE
164
92.256
54.921
20.546


1289
N
ILE
165
85.866
52.365
20.129


1290
CA
ILE
165
84.614
53.071
19.845


1291
C
ILE
165
83.641
52.982
21.012


1292
O
ILE
165
82.985
53.984
21.303


1293
CB
ILE
165
83.963
52.471
18.600


1294
CG1
ILE
165
84.799
52.733
17.357


1295
CG2
ILE
165
82.562
53.041
18.394


1296
CD1
ILE
165
84.753
54.207
16.985


1297
N
HIS
166
83.765
51.955
21.835


1298
CA
HIS
166
82.857
51.815
22.974


1299
C
HIS
166
83.221
52.795
24.088


1300
O
HIS
166
82.328
53.453
24.640


1301
CB
HIS
166
82.980
50.387
23.493


1302
CG
HIS
166
81.771
49.891
24.257


1303
ND1
HIS
166
80.787
49.115
23.764


1304
CD2
HIS
166
81.465
50.136
25.575


1305
CE1
HIS
166
79.880
48.873
24.732


1306
NE2
HIS
166
80.299
49.506
25.851


1307
N
ARG
167
84.507
53.084
24.210


1308
CA
ARG
167
84.971
53.992
25.261


1309
C
ARG
167
84.878
55.460
24.839


1310
O
ARG
167
84.712
56.348
25.681


1311
CB
ARG
167
86.424
53.640
25.563


1312
CG
ARG
167
86.886
54.282
26.863


1313
CD
ARG
167
86.013
53.799
28.014


1314
NE
ARG
167
86.405
54.413
29.289


1315
CZ
ARG
167
85.711
54.232
30.414


1316
NH1
ARG
167
84.590
53.508
30.395


1317
NH2
ARG
167
86.123
54.796
31.551


1318
N
ILE
168
84.860
55.705
23.541


1319
CA
ILE
168
84.735
57.082
23.055


1320
C
ILE
168
83.307
57.424
22.618


1321
O
ILE
168
82.966
58.605
22.449


1322
CB
ILE
168
85.755
57.263
21.938


1323
CG1
ILE
168
87.153
57.059
22.513


1324
CG2
ILE
168
85.650
58.634
21.282


1325
CD1
ILE
168
88.235
57.328
21.477


1326
N
LYS
169
82.426
56.444
22.719


1327
CA
LYS
169
81.034
56.624
22.294


1328
C
LYS
169
80.272
57.556
23.227


1329
O
LYS
169
79.560
58.444
22.747


1330
CB
LYS
169
80.390
55.245
22.292


1331
CG
LYS
169
78.945
55.253
21.819


1332
CD
LYS
169
78.429
53.823
21.728


1333
CE
LYS
169
78.649
53.087
23.045


1334
NZ
LYS
169
78.195
51.691
22.954


1335
N
HIS
170
80.675
57.563
24.487


1336
CA
HIS
170
80.076
58.469
25.470


1337
C
HIS
170
80.838
59.794
25.616


1338
O
HIS
170
80.559
60.568
26.538


1339
CB
HIS
170
79.918
57.741
26.805


1340
CG
HIS
170
81.108
56.937
27.306


1341
ND1
HIS
170
81.297
55.610
27.163


1342
CD2
HIS
170
82.174
57.419
28.027


1343
CE1
HIS
170
82.459
55.259
27.755


1344
NE2
HIS
170
82.998
56.377
28.289


1345
N
LEU
171
81.806
60.033
24.741


1346
CA
LEU
171
82.522
61.314
24.718


1347
C
LEU
171
82.047
62.176
23.553


1348
O
LEU
171
82.382
63.368
23.475


1349
CB
LEU
171
84.014
61.063
24.538


1350
CG
LEU
171
84.605
60.252
25.680


1351
CD1
LEU
171
86.092
60.025
25.442


1352
CD2
LEU
171
84.376
60.953
27.013


1353
N
LEU
172
81.326
61.547
22.639


1354
CA
LEU
172
80.775
62.247
21.476


1355
C
LEU
172
79.636
63.171
21.882


1356
O
LEU
172
78.772
62.802
22.684


1357
CB
LEU
172
80.228
61.205
20.507


1358
CG
LEU
172
81.304
60.241
20.023


1359
CD1
LEU
172
80.675
59.037
19.334


1360
CD2
LEU
172
82.306
60.935
19.106


1361
N
GLU
173
79.675
64.388
21.374


1362
CA
GLU
173
78.561
65.312
21.605


1363
C
GLU
173
77.976
65.809
20.292


1364
O
GLU
173
77.831
65.051
19.325


1365
CB
GLU
173
79.007
66.507
22.437


1366
CG
GLU
173
79.342
66.125
23.873


1367
CD
GLU
173
79.341
67.394
24.715


1368
OE1
GLU
173
78.989
67.301
25.882


1369
OE2
GLU
173
79.476
68.452
24.114


1370
N
ASN
174
77.576
67.070
20.320


1371
CA
ASN
174
77.025
67.752
19.148


1372
C
ASN
174
78.048
67.777
18.020


1373
O
ASN
174
79.189
68.223
18.201


1374
CB
ASN
174
76.684
69.185
19.550


1375
CG
ASN
174
75.774
69.201
20.777


1376
OD1
ASN
174
74.666
68.653
20.753


1377
ND2
ASN
174
76.239
69.856
21.830


1378
N
GLY
175
77.650
67.229
16.887


1379
CA
GLY
175
78.541
67.155
15.729


1380
C
GLY
175
79.602
66.083
15.944


1381
O
GLY
175
80.798
66.334
15.740


1382
N
GLY
176
79.153
64.912
16.363


1383
CA
GLY
176
80.054
63.781
16.614


1384
C
GLY
176
80.238
62.940
15.356


1385
O
GLY
176
79.480
61.994
15.105


1386
N
LYS
177
81.228
63.298
14.559


1387
CA
LYS
177
81.463
62.583
13.300


1388
C
LYS
177
82.648
61.628
13.410


1389
O
LYS
177
83.696
61.947
13.989


1390
CB
LYS
177
81.652
63.602
12.186


1391
CG
LYS
177
80.408
64.480
12.108


1392
CD
LYS
177
80.515
65.570
11.053


1393
CE
LYS
177
79.265
66.444
11.072


1394
NZ
LYS
177
78.055
65.649
10.809


1395
N
ILE
178
82.415
60.411
12.947


1396
CA
ILE
178
83.423
59.353
13.062


1397
C
ILE
178
83.671
58.625
11.743


1398
O
ILE
178
82.798
57.942
11.187


1399
CB
ILE
178
83.012
58.358
14.149


1400
CG1
ILE
178
82.964
59.032
15.515


1401
CG2
ILE
178
83.959
57.162
14.201


1402
CD1
ILE
178
82.843
58.006
16.634


1403
N
VAL
179
84.899
58.764
11.274


1404
CA
VAL
179
85.345
58.040
10.083


1405
C
VAL
179
85.796
56.644
10.496


1406
O
VAL
179
86.435
56.455
11.537


1407
CB
VAL
179
86.470
58.822
9.421


1408
CG1
VAL
179
87.012
58.121
8.182


1409
CG2
VAL
179
85.964
60.201
9.042


1410
N
TRP
180
85.398
55.675
9.698


1411
CA
TRP
180
85.625
54.267
10.005


1412
C
TRP
180
86.069
53.546
8.736


1413
O
TRP
180
85.235
53.151
7.913


1414
CB
TRP
180
84.260
53.822
10.498


1415
CG
TRP
180
84.014
52.374
10.836


1416
CD1
TRP
180
83.262
51.498
10.098


1417
CD2
TRP
180
84.469
51.654
12.000


1418
NE1
TRP
180
83.218
50.319
10.756


1419
CE2
TRP
180
83.913
50.371
11.903


1420
CE3
TRP
180
85.239
52.002
13.105


1421
CZ2
TRP
180
84.128
49.446
12.919


1422
CZ3
TRP
180
85.450
51.072
14.107


1423
CH2
TRP
180
84.899
49.797
14.019


1424
N
VAL
181
87.377
53.478
8.542


1425
CA
VAL
181
87.943
53.048
7.254


1426
C
VAL
181
87.961
51.531
7.053


1427
O
VAL
181
88.773
50.807
7.642


1428
CB
VAL
181
89.361
53.602
7.162


1429
CG1
VAL
181
90.030
53.201
5.851


1430
CG2
VAL
181
89.358
55.119
7.315


1431
N
SER
182
87.101
51.088
6.152


1432
CA
SER
182
87.000
49.675
5.788


1433
C
SER
182
87.687
49.448
4.443


1434
O
SER
182
87.797
50.364
3.620


1435
CB
SER
182
85.518
49.298
5.701


1436
OG
SER
182
85.405
47.908
5.408


1437
N
SER
183
88.237
48.260
4.273


1438
CA
SER
183
88.824
47.888
2.984


1439
C
SER
183
87.748
47.806
1.914


1440
O
SER
183
86.657
47.283
2.168


1441
CB
SER
183
89.483
46.519
3.119


1442
OG
SER
183
89.820
46.048
1.817


1443
N
LEU
184
88.128
48.128
0.687


1444
CA
LEU
184
87.212
47.997
−0.451


1445
C
LEU
184
86.898
46.550
−0.829


1446
O
LEU
184
85.847
46.305
−1.431


1447
CB
LEU
184
87.825
48.678
−1.666


1448
CG
LEU
184
87.178
50.032
−1.881


1449
CD1
LEU
184
87.533
50.584
−3.255


1450
CD2
LEU
184
85.672
49.871
−1.768


1451
N
MET
185
87.638
45.597
−0.282


1452
CA
MET
185
87.379
44.196
−0.585


1453
C
MET
185
86.312
43.603
0.331


1454
O
MET
185
85.817
42.510
0.033


1455
CB
MET
185
88.687
43.436
−0.433


1456
CG
MET
185
89.754
44.020
−1.351


1457
SD
MET
185
89.356
43.993
−3.115


1458
CE
MET
185
90.766
44.936
−3.740


1459
N
SER
186
85.812
44.385
1.279


1460
CA
SER
186
84.787
43.877
2.193


1461
C
SER
186
83.421
43.774
1.512


1462
O
SER
186
82.702
42.793
1.744


1463
CB
SER
186
84.696
44.797
3.407


1464
OG
SER
186
84.237
46.082
3.018


1465
N
SER
187
83.202
44.603
0.503


1466
CA
SER
187
81.959
44.515
−0.275


1467
C
SER
187
81.890
43.250
−1.152


1468
O
SER
187
80.938
42.489
−0.948


1469
CB
SER
187
81.762
45.777
−1.106


1470
OG
SER
187
80.575
45.600
−1.868


1471
N
PRO
188
82.848
42.949
−2.032


1472
CA
PRO
188
82.808
41.644
−2.710


1473
C
PRO
188
83.049
40.436
−1.798


1474
O
PRO
188
82.498
39.364
−2.083


1475
CB
PRO
188
83.859
41.719
−3.775


1476
CG
PRO
188
84.658
43.003
−3.618


1477
CD
PRO
188
84.009
43.749
−2.469


1478
N
LYS
189
83.629
40.634
−0.624


1479
CA
LYS
189
83.831
39.518
0.307


1480
C
LYS
189
82.533
39.083
0.970


1481
O
LYS
189
82.337
37.874
1.162


1482
CB
LYS
189
84.825
39.935
1.382


1483
CG
LYS
189
85.227
38.739
2.230


1484
CD
LYS
189
85.987
37.707
1.410


1485
CE
LYS
189
86.566
36.627
2.311


1486
NZ
LYS
189
85.514
36.045
3.153


1487
N
TYR
190
81.564
39.982
0.959


1488
CA
TYR
190
80.232
39.710
1.497


1489
C
TYR
190
79.424
38.756
0.603


1490
O
TYR
190
78.496
38.086
1.076


1491
CB
TYR
190
79.524
41.060
1.569


1492
CG
TYR
190
78.159
41.047
2.244


1493
CD1
TYR
190
78.002
40.453
3.491


1494
CD2
TYR
190
77.076
41.650
1.616


1495
CE1
TYR
190
76.762
40.454
4.110


1496
CE2
TYR
190
75.833
41.651
2.237


1497
CZ
TYR
190
75.681
41.053
3.482


1498
OH
TYR
190
74.458
41.092
4.115


1499
N
LEU
191
79.826
38.595
−0.646


1500
CA
LEU
191
79.155
37.597
−1.492


1501
C
LEU
191
80.069
36.393
−1.740


1502
O
LEU
191
79.599
35.253
−1.831


1503
CB
LEU
191
78.745
38.198
−2.834


1504
CG
LEU
191
77.399
38.916
−2.836


1505
CD1
LEU
191
77.435
40.286
−2.171


1506
CD2
LEU
191
76.886
39.071
−4.257


1507
N
SER
192
81.366
36.617
−1.599


1508
CA
SER
192
82.350
35.564
−1.887


1509
C
SER
192
82.570
34.590
−0.734


1510
O
SER
192
83.208
33.553
−0.947


1511
CB
SER
192
83.670
36.212
−2.270


1512
OG
SER
192
84.138
36.847
−1.097


1513
N
PHE
193
82.035
34.864
0.447


1514
CA
PHE
193
82.091
33.834
1.505


1515
C
PHE
193
80.862
32.908
1.451


1516
O
PHE
193
80.047
32.878
2.381


1517
CB
PHE
193
82.327
34.430
2.908


1518
CG
PHE
193
81.521
35.649
3.394


1519
CD1
PHE
193
82.173
36.621
4.138


1520
CD2
PHE
193
80.166
35.769
3.137


1521
CE1
PHE
193
81.472
37.731
4.594


1522
CE2
PHE
193
79.466
36.868
3.593


1523
CZ
PHE
193
80.115
37.857
4.318


1524
N
ASN
194
80.842
32.082
0.412


1525
CA
ASN
194
79.754
31.139
0.105


1526
C
ASN
194
78.400
31.831
−0.013


1527
O
ASN
194
77.590
31.762
0.918


1528
CB
ASN
194
79.711
30.053
1.179


1529
CG
ASN
194
80.908
29.120
1.052


1530
OD1
ASN
194
81.461
28.930
−0.039


1531
ND2
ASN
194
81.333
28.626
2.199


1532
N
ASP
195
78.152
32.521
−1.119


1533
CA
ASP
195
76.844
33.165
−1.253


1534
C
ASP
195
76.365
33.268
−2.698


1535
O
ASP
195
76.749
34.189
−3.426


1536
CB
ASP
195
76.870
34.558
−0.606


1537
CG
ASP
195
75.532
35.256
−0.832


1538
OD1
ASP
195
74.525
34.711
−0.378


1539
OD2
ASP
195
75.502
36.186
−1.629


1540
N
LEU
196
75.614
32.248
−3.096


1541
CA
LEU
196
74.792
32.207
−4.327


1542
C
LEU
196
75.504
31.851
−5.641


1543
O
LEU
196
74.825
31.324
−6.527


1544
CB
LEU
196
74.110
33.568
−4.479


1545
CG
LEU
196
73.035
33.588
−5.557


1546
CD1
LEU
196
71.872
32.691
−5.168


1547
CD2
LEU
196
72.569
35.013
−5.823


1548
N
GLN
197
76.826
31.876
−5.716


1549
CA
GLN
197
77.495
31.625
−7.019


1550
C
GLN
197
77.768
30.150
−7.354


1551
O
GLN
197
78.691
29.864
−8.126


1552
CB
GLN
197
78.811
32.406
−7.133


1553
CG
GLN
197
78.643
33.909
−7.355


1554
CD
GLN
197
78.522
34.634
−6.024


1555
OE1
GLN
197
79.272
34.344
−5.086


1556
NE2
GLN
197
77.552
35.529
−5.946


1557
N
LEU
198
76.952
29.234
−6.859


1558
CA
LEU
198
77.192
27.816
−7.158


1559
C
LEU
198
76.173
27.295
−8.173


1560
O
LEU
198
74.988
27.077
−7.871


1561
CB
LEU
198
77.169
26.991
−5.874


1562
CG
LEU
198
77.491
25.522
−6.139


1563
CD1
LEU
198
78.871
25.367
−6.772


1564
CD2
LEU
198
77.401
24.714
−4.851


1565
N
LEU
199
76.687
27.132
−9.386


1566
CA
LEU
199
75.923
26.680
−10.557


1567
C
LEU
199
74.788
27.663
−10.858


1568
O
LEU
199
73.640
27.255
−11.077


1569
CB
LEU
199
75.391
25.268
−10.287


1570
CG
LEU
199
74.863
24.571
−11.540


1571
CD1
LEU
199
75.959
24.424
−12.590


1572
CD2
LEU
199
74.263
23.214
−11.192


1573
N
ARG
200
75.144
28.944
−10.864


1574
CA
ARG
200
74.251
30.111
−11.085


1575
C
ARG
200
73.103
30.345
−10.076


1576
O
ARG
200
72.886
31.490
−9.664


1577
CB
ARG
200
73.672
30.009
−12.496


1578
CG
ARG
200
72.707
31.152
−12.791


1579
CD
ARG
200
72.182
31.099
−14.218


1580
NE
ARG
200
73.280
31.273
−15.181


1581
CZ
ARG
200
73.484
32.404
−15.862


1582
NH1
ARG
200
72.648
33.435
−15.715


1583
NH2
ARG
200
74.511
32.495
−16.710


1584
N
SER
201
72.374
29.308
−9.701


1585
CA
SER
201
71.212
29.455
−8.816


1586
C
SER
201
71.098
28.438
−7.661


1587
O
SER
201
70.934
28.914
−6.531


1588
CB
SER
201
69.947
29.426
−9.672


1589
OG
SER
201
69.999
30.533
−10.563


1590
N
PRO
202
71.146
27.116
−7.869


1591
CA
PRO
202
70.623
26.213
−6.826


1592
C
PRO
202
71.471
26.122
−5.556


1593
O
PRO
202
70.867
25.990
−4.482


1594
CB
PRO
202
70.526
24.859
−7.459


1595
CG
PRO
202
71.087
24.914
−8.866


1596
CD
PRO
202
71.423
26.371
−9.112


1597
N
ALA
203
72.757
26.445
−5.669


1598
CA
ALA
203
73.756
26.416
−4.577


1599
C
ALA
203
73.258
25.892
−3.234


1600
O
ALA
203
73.040
24.691
−3.042


1601
CB
ALA
203
74.220
27.853
−4.373


1602
N
SER
204
73.156
26.813
−2.294


1603
CA
SER
204
72.504
26.540
−1.015


1604
C
SER
204
71.573
27.704
−0.717


1605
O
SER
204
71.849
28.526
0.175


1606
CB
SER
204
73.554
26.388
0.077


1607
OG
SER
204
74.382
25.288
−0.274


1608
N
TYR
205
70.525
27.785
−1.525


1609
CA
TYR
205
69.559
28.894
−1.489


1610
C
TYR
205
68.953
29.061
−0.102


1611
O
TYR
205
68.339
28.136
0.441


1612
CB
TYR
205
68.458
28.598
−2.504


1613
CG
TYR
205
67.440
29.720
−2.692


1614
CD1
TYR
205
67.682
30.712
−3.633


1615
CD2
TYR
205
66.266
29.737
−1.947


1616
CE1
TYR
205
66.764
31.739
−3.811


1617
CE2
TYR
205
65.348
30.765
−2.122


1618
CZ
TYR
205
65.603
31.764
−3.050


1619
OH
TYR
205
64.730
32.823
−3.167


1620
N
GLU
206
69.213
30.231
0.468


1621
CA
GLU
206
68.824
30.597
1.839


1622
C
GLU
206
69.219
29.526
2.870


1623
O
GLU
206
68.413
29.086
3.697


1624
CB
GLU
206
67.323
30.866
1.848


1625
CG
GLU
206
66.884
31.671
3.064


1626
CD
GLU
206
65.396
31.985
2.939


1627
OE1
GLU
206
64.753
31.357
2.109


1628
OE2
GLU
206
64.961
32.944
3.561


1629
N
GLY
207
70.443
29.048
2.724


1630
CA
GLY
207
71.072
28.133
3.678


1631
C
GLY
207
72.475
28.647
3.961


1632
O
GLY
207
72.764
29.107
5.068


1633
N
SER
208
73.259
28.802
2.907


1634
CA
SER
208
74.578
29.414
3.091


1635
C
SER
208
74.427
30.934
3.052


1636
O
SER
208
75.151
31.666
3.737


1637
CB
SER
208
75.533
28.943
2.001


1638
OG
SER
208
76.814
29.464
2.318


1639
N
LYS
209
73.286
31.340
2.515


1640
CA
LYS
209
72.870
32.742
2.472


1641
C
LYS
209
72.260
33.237
3.795


1642
O
LYS
209
72.067
34.451
3.931


1643
CB
LYS
209
71.838
32.872
1.353


1644
CG
LYS
209
71.494
34.319
1.018


1645
CD
LYS
209
70.330
34.378
0.036


1646
CE
LYS
209
70.631
33.584
−1.229


1647
NZ
LYS
209
69.469
33.582
−2.130


1648
N
ARG
210
72.153
32.395
4.818


1649
CA
ARG
210
71.602
32.868
6.101


1650
C
ARG
210
72.531
33.875
6.775


1651
O
ARG
210
72.033
34.886
7.288


1652
CB
ARG
210
71.398
31.696
7.053


1653
CG
ARG
210
70.382
30.688
6.534


1654
CD
ARG
210
70.200
29.553
7.538


1655
NE
ARG
210
71.507
29.000
7.932


1656
CZ
ARG
210
71.842
27.716
7.779


1657
NH1
ARG
210
73.076
27.306
8.082


1658
NH2
ARG
210
70.960
26.856
7.265


1659
N
LEU
211
73.817
33.777
6.473


1660
CA
LEU
211
74.800
34.753
6.955


1661
C
LEU
211
74.493
36.137
6.394


1662
O
LEU
211
74.174
37.054
7.162


1663
CB
LEU
211
76.169
34.330
6.435


1664
CG
LEU
211
76.523
32.901
6.830


1665
CD1
LEU
211
77.734
32.408
6.046


1666
CD2
LEU
211
76.753
32.779
8.333


1667
N
VAL
212
74.245
36.164
5.094


1668
CA
VAL
212
73.998
37.412
4.373


1669
C
VAL
212
72.598
37.966
4.641


1670
O
VAL
212
72.450
39.182
4.787


1671
CB
VAL
212
74.172
37.091
2.892


1672
CG1
VAL
212
73.837
38.277
1.999


1673
CG2
VAL
212
75.586
36.592
2.615


1674
N
ASP
213
71.671
37.091
4.998


1675
CA
ASP
213
70.319
37.523
5.365


1676
C
ASP
213
70.313
38.219
6.724


1677
O
ASP
213
69.821
39.352
6.823


1678
CB
ASP
213
69.410
36.298
5.439


1679
CG
ASP
213
69.299
35.585
4.092


1680
OD1
ASP
213
69.300
36.271
3.078


1681
OD2
ASP
213
69.132
34.369
4.097


1682
N
LEU
214
71.097
37.695
7.655


1683
CA
LEU
214
71.171
38.292
8.995


1684
C
LEU
214
71.982
39.578
8.966


1685
O
LEU
214
71.561
40.584
9.548


1686
CB
LEU
214
71.849
37.300
9.930


1687
CG
LEU
214
71.030
36.025
10.089


1688
CD1
LEU
214
71.819
34.956
10.837


1689
CD2
LEU
214
69.698
36.306
10.777


1690
N
MET
215
72.932
39.616
8.046


1691
CA
MET
215
73.763
40.800
7.823


1692
C
MET
215
73.139
41.839
6.877


1693
O
MET
215
73.820
42.791
6.473


1694
CB
MET
215
75.116
40.319
7.315


1695
CG
MET
215
75.831
39.534
8.412


1696
SD
MET
215
77.574
39.143
8.127


1697
CE
MET
215
77.415
38.056
6.694


1698
N
HIS
216
71.904
41.608
6.460


1699
CA
HIS
216
71.130
42.615
5.735


1700
C
HIS
216
70.019
43.195
6.603


1701
O
HIS
216
69.474
44.260
6.286


1702
CB
HIS
216
70.480
41.971
4.517


1703
CG
HIS
216
71.307
42.019
3.252


1704
ND1
HIS
216
72.173
42.987
2.896


1705
CD2
HIS
216
71.305
41.091
2.238


1706
CE1
HIS
216
72.704
42.689
1.695


1707
NE2
HIS
216
72.169
41.515
1.289


1708
N
PHE
217
69.689
42.508
7.684


1709
CA
PHE
217
68.572
42.958
8.523


1710
C
PHE
217
69.006
43.966
9.578


1711
O
PHE
217
69.870
43.681
10.419


1712
CB
PHE
217
67.921
41.752
9.193


1713
CG
PHE
217
67.195
40.813
8.232


1714
CD1
PHE
217
66.551
41.322
7.110


1715
CD2
PHE
217
67.163
39.449
8.489


1716
CE1
PHE
217
65.895
40.465
6.236


1717
CE2
PHE
217
66.506
38.592
7.615


1718
CZ
PHE
217
65.875
39.099
6.487


1719
N
GLY
218
68.232
45.038
9.669


1720
CA
GLY
218
68.487
46.119
10.639


1721
C
GLY
218
67.887
45.856
12.025


1722
O
GLY
218
67.155
46.681
12.582


1723
N
THR
219
68.213
44.689
12.554


1724
CA
THR
219
67.816
44.247
13.889


1725
C
THR
219
69.005
43.523
14.506


1726
O
THR
219
68.964
43.095
15.667


1727
CB
THR
219
66.651
43.266
13.785


1728
OG1
THR
219
67.061
42.201
12.937


1729
CG2
THR
219
65.392
43.888
13.188


1730
N
TYR
220
70.034
43.361
13.687


1731
CA
TYR
220
71.239
42.624
14.075


1732
C
TYR
220
72.107
43.449
15.026


1733
O
TYR
220
72.784
44.398
14.617


1734
CB
TYR
220
71.995
42.317
12.785


1735
CG
TYR
220
73.052
41.223
12.885


1736
CD1
TYR
220
72.864
40.157
13.754


1737
CD2
TYR
220
74.182
41.272
12.079


1738
CE1
TYR
220
73.825
39.160
13.847


1739
CE2
TYR
220
75.145
40.275
12.171


1740
CZ
TYR
220
74.968
39.226
13.063


1741
OH
TYR
220
75.985
38.316
13.267


1742
N
ASN
221
72.053
43.086
16.297


1743
CA
ASN
221
72.810
43.804
17.327


1744
C
ASN
221
74.225
43.258
17.459


1745
O
ASN
221
74.492
42.088
17.148


1746
CB
ASN
221
72.074
43.742
18.665


1747
CG
ASN
221
71.884
42.313
19.168


1748
OD1
ASN
221
72.857
41.595
19.445


1749
ND2
ASN
221
70.627
41.935
19.321


1750
N
LYS
222
75.060
44.032
18.133


1751
CA
LYS
222
76.487
43.709
18.209


1752
C
LYS
222
76.839
42.535
19.125


1753
O
LYS
222
77.866
41.906
18.858


1754
CB
LYS
222
77.274
44.951
18.617


1755
CG
LYS
222
76.834
45.537
19.955


1756
CD
LYS
222
77.721
46.720
20.334


1757
CE
LYS
222
77.311
47.351
21.661


1758
NZ
LYS
222
75.959
47.926
21.579


1759
N
LEU
223
75.908
42.040
19.927


1760
CA
LEU
223
76.218
40.876
20.759


1761
C
LEU
223
76.147
39.604
19.918


1762
O
LEU
223
77.085
38.796
19.957


1763
CB
LEU
223
75.210
40.799
21.898


1764
CG
LEU
223
75.532
39.650
22.846


1765
CD1
LEU
223
76.920
39.821
23.457


1766
CD2
LEU
223
74.475
39.528
23.938


1767
N
GLU
224
75.269
39.631
18.926


1768
CA
GLU
224
75.137
38.505
17.996


1769
C
GLU
224
76.247
38.536
16.952


1770
O
GLU
224
76.724
37.483
16.513


1771
CB
GLU
224
73.802
38.645
17.282


1772
CG
GLU
224
72.634
38.631
18.254


1773
CD
GLU
224
71.377
39.122
17.545


1774
OE1
GLU
224
71.512
40.007
16.708


1775
OE2
GLU
224
70.302
38.795
18.028


1776
N
ARG
225
76.796
39.721
16.750


1777
CA
ARG
225
77.914
39.898
15.830


1778
C
ARG
225
79.232
39.489
16.483


1779
O
ARG
225
80.085
38.874
15.827


1780
CB
ARG
225
77.962
41.375
15.478


1781
CG
ARG
225
76.647
41.858
14.889


1782
CD
ARG
225
76.756
43.329
14.518


1783
NE
ARG
225
77.985
43.518
13.741


1784
CZ
ARG
225
78.004
43.568
12.411


1785
NH1
ARG
225
79.153
43.344
11.772


1786
NH2
ARG
225
76.854
43.548
11.734


1787
N
GLU
226
79.295
39.623
17.798


1788
CA
GLU
226
80.478
39.189
18.544


1789
C
GLU
226
80.490
37.673
18.686


1790
O
GLU
226
81.549
37.057
18.510


1791
CB
GLU
226
80.461
39.851
19.918


1792
CG
GLU
226
80.652
41.359
19.797


1793
CD
GLU
226
80.397
42.048
21.134


1794
OE1
GLU
226
79.274
42.489
21.350


1795
OE2
GLU
226
81.350
42.197
21.886


1796
N
HIS
227
79.303
37.084
18.707


1797
CA
HIS
227
79.205
35.625
18.678


1798
C
HIS
227
79.597
35.110
17.302


1799
O
HIS
227
80.460
34.230
17.227


1800
CB
HIS
227
77.779
35.192
18.986


1801
CG
HIS
227
77.300
35.542
20.381


1802
ND1
HIS
227
76.027
35.769
20.744


1803
CD2
HIS
227
78.072
35.676
21.511


1804
CE1
HIS
227
75.982
36.052
22.063


1805
NE2
HIS
227
77.247
35.995
22.536


1806
N
GLY
228
79.186
35.830
16.268


1807
CA
GLY
228
79.563
35.530
14.879


1808
C
GLY
228
81.068
35.360
14.686


1809
O
GLY
228
81.523
34.246
14.389


1810
N
ILE
229
81.845
36.378
15.027


1811
CA
ILE
229
83.301
36.276
14.832


1812
C
ILE
229
84.003
35.359
15.837


1813
O
ILE
229
84.956
34.671
15.443


1814
CB
ILE
229
83.921
37.670
14.836


1815
CG1
ILE
229
83.876
38.259
13.435


1816
CG2
ILE
229
85.350
37.680
15.369


1817
CD1
ILE
229
84.859
39.414
13.321


1818
N
LYS
230
83.374
35.105
16.972


1819
CA
LYS
230
83.920
34.135
17.923


1820
C
LYS
230
83.745
32.705
17.409


1821
O
LYS
230
84.711
31.932
17.419


1822
CB
LYS
230
83.172
34.313
19.239


1823
CG
LYS
230
83.496
33.218
20.248


1824
CD
LYS
230
82.671
33.391
21.518


1825
CE
LYS
230
81.174
33.390
21.221


1826
NZ
LYS
230
80.739
32.115
20.627


1827
N
GLN
231
82.658
32.476
16.690


1828
CA
GLN
231
82.391
31.166
16.098


1829
C
GLN
231
83.211
30.952
14.832


1830
O
GLN
231
83.768
29.862
14.657


1831
CB
GLN
231
80.902
31.102
15.796


1832
CG
GLN
231
80.123
31.182
17.102


1833
CD
GLN
231
78.675
31.604
16.871


1834
OE1
GLN
231
78.356
32.340
15.928


1835
NE2
GLN
231
77.827
31.218
17.808


1836
N
TYR
232
83.567
32.047
14.181


1837
CA
TYR
232
84.442
31.969
13.007


1838
C
TYR
232
85.881
31.654
13.407


1839
O
TYR
232
86.558
30.876
12.724


1840
CB
TYR
232
84.422
33.311
12.281


1841
CG
TYR
232
83.105
33.692
11.610


1842
CD1
TYR
232
82.199
32.712
11.223


1843
CD2
TYR
232
82.820
35.032
11.376


1844
CE1
TYR
232
81.006
33.072
10.612


1845
CE2
TYR
232
81.629
35.393
10.762


1846
CZ
TYR
232
80.725
34.411
10.380


1847
OH
TYR
232
79.562
34.762
9.734


1848
N
LEU
233
86.260
32.074
14.604


1849
CA
LEU
233
87.607
31.800
15.103


1850
C
LEU
233
87.710
30.424
15.770


1851
O
LEU
233
88.798
29.835
15.793


1852
CB
LEU
233
87.948
32.901
16.104


1853
CG
LEU
233
89.389
32.820
16.595


1854
CD1
LEU
233
90.366
32.793
15.425


1855
CD2
LEU
233
89.706
33.975
17.539


1856
N
VAL
234
86.585
29.850
16.164


1857
CA
VAL
234
86.624
28.526
16.794


1858
C
VAL
234
86.290
27.387
15.827


1859
O
VAL
234
86.002
26.272
16.282


1860
CB
VAL
234
85.706
28.475
18.014


1861
CG1
VAL
234
86.123
29.504
19.059


1862
CG2
VAL
234
84.237
28.638
17.653


1863
N
HIS
235
86.271
27.649
14.529


1864
CA
HIS
235
86.045
26.541
13.582


1865
C
HIS
235
87.222
26.300
12.631


1866
O
HIS
235
87.044
26.490
11.422


1867
CB
HIS
235
84.819
26.844
12.726


1868
CG
HIS
235
83.495
26.919
13.452


1869
ND1
HIS
235
83.164
26.322
14.617


1870
CD2
HIS
235
82.384
27.617
13.037


1871
CE1
HIS
235
81.896
26.634
14.935


1872
NE2
HIS
235
81.412
27.437
13.957


1873
N
PRO
236
88.305
25.693
13.104


1874
CA
PRO
236
89.449
25.411
12.223


1875
C
PRO
236
89.098
24.320
11.214


1876
O
PRO
236
88.663
23.229
11.594


1877
CB
PRO
236
90.548
24.964
13.137


1878
CG
PRO
236
89.984
24.763
14.535


1879
CD
PRO
236
88.524
25.175
14.459


1880
N
GLY
237
89.137
24.679
9.940


1881
CA
GLY
237
88.736
23.764
8.857


1882
C
GLY
237
87.241
23.870
8.527


1883
O
GLY
237
86.854
24.196
7.396


1884
N
ILE
238
86.438
23.790
9.579


1885
CA
ILE
238
84.973
23.751
9.507


1886
C
ILE
238
84.338
25.084
9.096


1887
O
ILE
238
83.236
25.091
8.529


1888
CB
ILE
238
84.516
23.395
10.918


1889
CG1
ILE
238
85.313
22.211
11.449


1890
CG2
ILE
238
83.030
23.074
10.955


1891
CD1
ILE
238
84.911
21.872
12.880


1892
N
PHE
239
85.124
26.148
9.126


1893
CA
PHE
239
84.631
27.460
8.706


1894
C
PHE
239
84.376
27.535
7.200


1895
O
PHE
239
83.351
28.119
6.830


1896
CB
PHE
239
85.634
28.539
9.129


1897
CG
PHE
239
87.004
28.550
8.436


1898
CD1
PHE
239
88.089
27.897
9.005


1899
CD2
PHE
239
87.174
29.255
7.251


1900
CE1
PHE
239
89.326
27.913
8.374


1901
CE2
PHE
239
88.409
29.271
6.618


1902
CZ
PHE
239
89.485
28.597
7.177


1903
N
THR
240
85.009
26.664
6.425


1904
CA
THR
240
84.827
26.696
4.972


1905
C
THR
240
83.582
25.917
4.548


1906
O
THR
240
82.966
26.232
3.521


1907
CB
THR
240
86.065
26.071
4.337


1908
OG1
THR
240
87.206
26.763
4.829


1909
CG2
THR
240
86.059
26.184
2.817


1910
N
SER
241
83.105
25.058
5.436


1911
CA
SER
241
81.923
24.257
5.122


1912
C
SER
241
80.641
24.890
5.657


1913
O
SER
241
79.547
24.414
5.332


1914
CB
SER
241
82.081
22.866
5.727


1915
OG
SER
241
82.003
22.976
7.143


1916
N
PHE
242
80.765
25.933
6.463


1917
CA
PHE
242
79.563
26.567
7.018


1918
C
PHE
242
79.475
28.065
6.750


1919
O
PHE
242
78.371
28.599
6.586


1920
CB
PHE
242
79.535
26.342
8.528


1921
CG
PHE
242
79.175
24.921
8.953


1922
CD1
PHE
242
79.857
24.314
9.999


1923
CD2
PHE
242
78.161
24.233
8.298


1924
CE1
PHE
242
79.526
23.023
10.389


1925
CE2
PHE
242
77.830
22.941
8.687


1926
CZ
PHE
242
78.513
22.335
9.733


1927
N
SER
243
80.607
28.744
6.726


1928
CA
SER
243
80.572
30.205
6.671


1929
C
SER
243
81.651
30.835
5.799


1930
O
SER
243
81.703
30.637
4.580


1931
CB
SER
243
80.724
30.730
8.092


1932
OG
SER
243
79.587
30.326
8.843


1933
N
PHE
244
82.452
31.658
6.455


1934
CA
PHE
244
83.414
32.540
5.784


1935
C
PHE
244
84.594
31.804
5.165


1936
O
PHE
244
84.956
30.681
5.539


1937
CB
PHE
244
83.919
33.587
6.774


1938
CG
PHE
244
85.093
33.185
7.668


1939
CD1
PHE
244
84.927
32.283
8.710


1940
CD2
PHE
244
86.342
33.745
7.431


1941
CE1
PHE
244
86.009
31.945
9.512


1942
CE2
PHE
244
87.423
33.407
8.232


1943
CZ
PHE
244
87.255
32.507
9.275


1944
N
PHE
245
85.207
32.503
4.227


1945
CA
PHE
245
86.346
31.972
3.490


1946
C
PHE
245
87.680
32.415
4.094


1947
O
PHE
245
88.452
31.573
4.562


1948
CB
PHE
245
86.215
32.478
2.057


1949
CG
PHE
245
87.230
31.918
1.067


1950
CD1
PHE
245
87.685
32.720
0.028


1951
CD2
PHE
245
87.692
30.616
1.197


1952
CE1
PHE
245
88.610
32.217
−0.879


1953
CE2
PHE
245
88.616
30.114
0.292


1954
CZ
PHE
245
89.075
30.913
−0.746


1955
N
GLN
246
87.973
33.707
4.036


1956
CA
GLN
246
89.284
34.193
4.498


1957
C
GLN
246
89.263
35.523
5.245


1958
O
GLN
246
88.238
36.004
5.745


1959
CB
GLN
246
90.229
34.355
3.312


1960
CG
GLN
246
90.688
33.019
2.744


1961
CD
GLN
246
91.703
33.255
1.635


1962
OE1
GLN
246
91.994
34.401
1.279


1963
NE2
GLN
246
92.239
32.168
1.111


1964
N
TYR
247
90.392
36.193
5.090


1965
CA
TYR
247
90.773
37.387
5.857


1966
C
TYR
247
90.000
38.672
5.541


1967
O
TYR
247
89.823
39.498
6.444


1968
CB
TYR
247
92.258
37.592
5.556


1969
CG
TYR
247
92.770
39.015
5.743


1970
CD1
TYR
247
92.999
39.512
7.019


1971
CD2
TYR
247
93.012
39.812
4.630


1972
CE1
TYR
247
93.439
40.818
7.183


1973
CE2
TYR
247
93.452
41.118
4.793


1974
CZ
TYR
247
93.654
41.619
6.071


1975
OH
TYR
247
93.976
42.943
6.243


1976
N
LEU
248
89.324
38.733
4.405


1977
CA
LEU
248
88.587
39.958
4.064


1978
C
LEU
248
87.218
40.017
4.750


1979
O
LEU
248
86.642
41.107
4.879


1980
CB
LEU
248
88.456
40.054
2.550


1981
CG
LEU
248
89.824
40.153
1.883


1982
CD1
LEU
248
89.720
39.989
0.371


1983
CD2
LEU
248
90.519
41.461
2.247


1984
N
ASN
249
86.881
38.936
5.445


1985
CA
ASN
249
85.672
38.877
6.267


1986
C
ASN
249
85.835
39.703
7.541


1987
O
ASN
249
84.858
40.306
8.006


1988
CB
ASN
249
85.451
37.415
6.640


1989
CG
ASN
249
84.334
37.282
7.662


1990
OD1
ASN
249
83.184
37.640
7.389


1991
ND2
ASN
249
84.686
36.770
8.829


1992
N
VAL
250
87.079
39.986
7.901


1993
CA
VAL
250
87.338
40.826
9.066


1994
C
VAL
250
87.015
42.285
8.763


1995
O
VAL
250
86.360
42.935
9.583


1996
CB
VAL
250
88.812
40.709
9.419


1997
CG1
VAL
250
89.128
41.534
10.658


1998
CG2
VAL
250
89.210
39.253
9.622


1999
N
PHE
251
87.200
42.687
7.515


2000
CA
PHE
251
86.853
44.053
7.119


2001
C
PHE
251
85.391
44.167
6.718


2002
O
PHE
251
84.824
45.266
6.773


2003
CB
PHE
251
87.742
44.503
5.970


2004
CG
PHE
251
89.097
45.032
6.420


2005
CD1
PHE
251
89.159
46.205
7.162


2006
CD2
PHE
251
90.263
44.353
6.094


2007
CE1
PHE
251
90.388
46.705
7.570


2008
CE2
PHE
251
91.492
44.854
6.503


2009
CZ
PHE
251
91.555
46.031
7.238


2010
N
THR
252
84.758
43.028
6.496


2011
CA
THR
252
83.324
43.011
6.233


2012
C
THR
252
82.586
43.215
7.548


2013
O
THR
252
81.798
44.164
7.657


2014
CB
THR
252
82.959
41.670
5.604


2015
OG1
THR
252
83.699
41.535
4.396


2016
CG2
THR
252
81.477
41.583
5.261


2017
N
TYR
253
83.087
42.570
8.591


2018
CA
TYR
253
82.520
42.754
9.929


2019
C
TYR
253
82.862
44.129
10.480


2020
O
TYR
253
81.992
44.790
11.059


2021
CB
TYR
253
83.111
41.706
10.860


2022
CG
TYR
253
82.587
41.784
12.293


2023
CD1
TYR
253
81.518
40.983
12.667


2024
CD2
TYR
253
83.176
42.634
13.223


2025
CE1
TYR
253
81.032
41.041
13.964


2026
CE2
TYR
253
82.686
42.696
14.520


2027
CZ
TYR
253
81.615
41.897
14.888


2028
OH
TYR
253
81.148
41.933
16.183


2029
N
TYR
254
84.073
44.584
10.194


2030
CA
TYR
254
84.502
45.929
10.575


2031
C
TYR
254
83.511
46.941
10.025


2032
O
TYR
254
82.754
47.513
10.821


2033
CB
TYR
254
85.900
46.161
10.003


2034
CG
TYR
254
86.542
47.526
10.263


2035
CD1
TYR
254
87.469
47.683
11.286


2036
CD2
TYR
254
86.218
48.603
9.450


2037
CE1
TYR
254
88.064
48.921
11.500


2038
CE2
TYR
254
86.805
49.840
9.669


2039
CZ
TYR
254
87.733
49.997
10.686


2040
OH
TYR
254
88.422
51.187
10.788


2041
N
GLY
255
83.315
46.918
8.714


2042
CA
GLY
255
82.385
47.830
8.038


2043
C
GLY
255
80.999
47.849
8.674


2044
O
GLY
255
80.624
48.860
9.285


2045
N
MET
256
80.400
46.675
8.787


2046
CA
MET
256
79.018
46.562
9.264


2047
C
MET
256
78.809
46.758
10.766


2048
O
MET
256
77.655
46.897
11.183


2049
CB
MET
256
78.544
45.157
8.978


2050
CG
MET
256
78.791
44.696
7.561


2051
SD
MET
256
78.303
42.976
7.363


2052
CE
MET
256
76.689
43.184
8.143


2053
N
LEU
257
79.857
46.920
11.555


2054
CA
LEU
257
79.669
47.050
13.004


2055
C
LEU
257
79.019
48.386
13.371


2056
O
LEU
257
78.154
48.417
14.259


2057
CB
LEU
257
81.045
46.936
13.651


2058
CG
LEU
257
80.989
46.917
15.175


2059
CD1
LEU
257
80.121
45.771
15.684


2060
CD2
LEU
257
82.394
46.821
15.761


2061
N
PHE
258
79.148
49.359
12.482


2062
CA
PHE
258
78.553
50.676
12.724


2063
C
PHE
258
77.065
50.789
12.394


2064
O
PHE
258
76.463
51.766
12.867


2065
CB
PHE
258
79.320
51.729
11.937


2066
CG
PHE
258
80.392
52.446
12.749


2067
CD1
PHE
258
80.933
51.850
13.881


2068
CD2
PHE
258
80.813
53.710
12.364


2069
CE1
PHE
258
81.903
52.511
14.618


2070
CE2
PHE
258
81.786
54.372
13.099


2071
CZ
PHE
258
82.332
53.772
14.226


2072
N
LEU
259
76.443
49.752
11.842


2073
CA
LEU
259
75.019
49.870
11.493


2074
C
LEU
259
74.121
49.804
12.723


2075
O
LEU
259
73.080
50.467
12.753


2076
CB
LEU
259
74.610
48.860
10.408


2077
CG
LEU
259
74.818
47.367
10.685


2078
CD1
LEU
259
73.706
46.752
11.526


2079
CD2
LEU
259
74.892
46.606
9.366


2080
N
PHE
260
74.601
49.199
13.794


2081
CA
PHE
260
73.874
49.317
15.052


2082
C
PHE
260
74.591
50.315
15.940


2083
O
PHE
260
73.976
51.317
16.335


2084
CB
PHE
260
73.803
47.972
15.752


2085
CG
PHE
260
73.151
48.047
17.128


2086
CD1
PHE
260
73.680
47.319
18.186


2087
CD2
PHE
260
72.043
48.861
17.341


2088
CE1
PHE
260
73.094
47.390
19.445


2089
CE2
PHE
260
71.458
48.932
18.599


2090
CZ
PHE
260
71.985
48.195
19.647


2091
N
TYR
261
75.914
50.195
15.934


2092
CA
TYR
261
76.794
50.921
16.862


2093
C
TYR
261
76.510
52.413
16.915


2094
O
TYR
261
75.931
52.895
17.893


2095
CB
TYR
261
78.229
50.713
16.406


2096
CG
TYR
261
79.209
50.424
17.531


2097
CD1
TYR
261
78.863
50.692
18.849


2098
CD2
TYR
261
80.449
49.873
17.233


2099
CE1
TYR
261
79.759
50.415
19.872


2100
CE2
TYR
261
81.346
49.595
18.254


2101
CZ
TYR
261
80.998
49.866
19.571


2102
OH
TYR
261
81.877
49.557
20.580


2103
N
LEU
262
76.831
53.133
15.856


2104
CA
LEU
262
76.569
54.569
15.922


2105
C
LEU
262
75.295
54.927
15.180


2106
O
LEU
262
74.576
55.833
15.615


2107
CB
LEU
262
77.733
55.362
15.346


2108
CG
LEU
262
79.046
55.114
16.082


2109
CD1
LEU
262
80.087
56.101
15.582


2110
CD2
LEU
262
78.905
55.257
17.594


2111
N
ALA
263
74.907
54.092
14.231


2112
CA
ALA
263
73.730
54.410
13.424


2113
C
ALA
263
72.444
54.386
14.236


2114
O
ALA
263
71.895
55.453
14.537


2115
CB
ALA
263
73.634
53.436
12.263


2116
N
ARG
264
72.064
53.223
14.732


2117
CA
ARG
264
70.785
53.160
15.435


2118
C
ARG
264
70.912
53.543
16.903


2119
O
ARG
264
70.030
54.241
17.421


2120
CB
ARG
264
70.211
51.754
15.327


2121
CG
ARG
264
70.160
51.295
13.876


2122
CD
ARG
264
69.243
50.091
13.688


2123
NE
ARG
264
69.527
49.004
14.641


2124
CZ
ARG
264
70.107
47.852
14.297


2125
NH1
ARG
264
70.673
47.726
13.097


2126
NH2
ARG
264
70.249
46.881
15.202


2127
N
PHE
265
72.077
53.315
17.487


2128
CA
PHE
265
72.233
53.583
18.918


2129
C
PHE
265
72.576
55.043
19.226


2130
O
PHE
265
72.332
55.490
20.352


2131
CB
PHE
265
73.307
52.644
19.461


2132
CG
PHE
265
73.513
52.710
20.970


2133
CD1
PHE
265
72.609
52.082
21.817


2134
CD2
PHE
265
74.598
53.398
21.499


2135
CE1
PHE
265
72.788
52.144
23.193


2136
CE2
PHE
265
74.776
53.461
22.875


2137
CZ
PHE
265
73.871
52.834
23.722


2138
N
LEU
266
73.047
55.795
18.242


2139
CA
LEU
266
73.282
57.228
18.463


2140
C
LEU
266
72.407
58.117
17.582


2141
O
LEU
266
72.689
59.314
17.445


2142
CB
LEU
266
74.752
57.582
18.287


2143
CG
LEU
266
75.563
57.153
19.505


2144
CD1
LEU
266
77.004
57.632
19.387


2145
CD2
LEU
266
74.942
57.704
20.784


2146
N
GLY
267
71.406
57.524
16.946


2147
CA
GLY
267
70.385
58.309
16.238


2148
C
GLY
267
69.679
59.199
17.255


2149
O
GLY
267
69.496
60.404
17.046


2150
N
SER
268
69.184
58.560
18.300


2151
CA
SER
268
68.784
59.293
19.502


2152
C
SER
268
69.974
59.270
20.462


2153
O
SER
268
70.671
58.253
20.513


2154
CB
SER
268
67.572
58.617
20.138


2155
OG
SER
268
67.949
57.306
20.543


2156
N
PRO
269
70.258
60.363
21.156


2157
CA
PRO
269
69.610
61.665
20.953


2158
C
PRO
269
70.041
62.338
19.650


2159
O
PRO
269
71.106
62.034
19.100


2160
CB
PRO
269
70.020
62.490
22.131


2161
CG
PRO
269
71.136
61.770
22.871


2162
CD
PRO
269
71.322
60.442
22.158


2163
N
TYR
270
69.341
63.421
19.352


2164
CA
TYR
270
69.386
64.107
18.045


2165
C
TYR
270
70.530
65.128
17.903


2166
O
TYR
270
70.346
66.183
17.284


2167
CB
TYR
270
68.058
64.852
17.883


2168
CG
TYR
270
66.840
64.192
18.537


2169
CD1
TYR
270
66.302
64.749
19.695


2170
CD2
TYR
270
66.249
63.065
17.973


2171
CE1
TYR
270
65.204
64.164
20.305


2172
CE2
TYR
270
65.149
62.479
18.584


2173
CZ
TYR
270
64.630
63.025
19.753


2174
OH
TYR
270
63.559
62.425
20.379


2175
N
HIS
271
71.720
64.774
18.364


2176
CA
HIS
271
72.851
65.714
18.429


2177
C
HIS
271
73.739
65.760
17.183


2178
O
HIS
271
74.823
66.357
17.243


2179
CB
HIS
271
73.692
65.374
19.653


2180
CG
HIS
271
72.991
65.721
20.953


2181
ND1
HIS
271
72.236
66.813
21.170


2182
CD2
HIS
271
73.024
65.008
22.126


2183
CE1
HIS
271
71.773
66.783
22.438


2184
NE2
HIS
271
72.263
65.671
23.028


2185
N
ASN
272
73.257
65.227
16.070


2186
CA
ASN
272
74.031
65.168
14.819


2187
C
ASN
272
75.300
64.365
15.024


2188
O
ASN
272
76.355
64.900
15.388


2189
CB
ASN
272
74.345
66.564
14.278


2190
CG
ASN
272
73.238
67.056
13.345


2191
OD1
ASN
272
73.417
67.063
12.117


2192
ND2
ASN
272
72.157
67.549
13.922


2193
N
ILE
273
75.130
63.060
14.938


2194
CA
ILE
273
76.234
62.121
15.143


2195
C
ILE
273
76.285
61.171
13.952


2196
O
ILE
273
75.308
60.472
13.665


2197
CB
ILE
273
75.995
61.350
16.443


2198
CG1
ILE
273
75.929
62.296
17.642


2199
CG2
ILE
273
77.080
60.301
16.667


2200
CD1
ILE
273
75.682
61.542
18.944


2201
N
SER
274
77.414
61.147
13.266


2202
CA
SER
274
77.503
60.335
12.050


2203
C
SER
274
78.778
59.505
11.946


2204
O
SER
274
79.866
60.003
11.627


2205
CB
SER
274
77.344
61.233
10.830


2206
OG
SER
274
75.986
61.665
10.803


2207
N
GLY
275
78.592
58.218
12.185


2208
CA
GLY
275
79.626
57.204
11.968


2209
C
GLY
275
79.423
56.520
10.615


2210
O
GLY
275
78.342
55.999
10.298


2211
N
TYR
276
80.473
56.577
9.816


2212
CA
TYR
276
80.446
56.057
8.442


2213
C
TYR
276
81.116
54.703
8.267


2214
O
TYR
276
81.462
54.017
9.234


2215
CB
TYR
276
81.215
57.018
7.550


2216
CG
TYR
276
80.423
58.219
7.074


2217
CD1
TYR
276
80.647
59.475
7.620


2218
CD2
TYR
276
79.467
58.044
6.085


2219
CE1
TYR
276
79.937
60.569
7.145


2220
CE2
TYR
276
78.758
59.135
5.612


2221
CZ
TYR
276
79.006
60.397
6.130


2222
OH
TYR
276
78.551
61.494
5.439


2223
N
ILE
277
81.099
54.268
7.016


2224
CA
ILE
277
81.955
53.168
6.549


2225
C
ILE
277
82.756
53.676
5.347


2226
O
ILE
277
82.301
53.580
4.202


2227
CB
ILE
277
81.102
51.981
6.119


2228
CG1
ILE
277
80.113
51.563
7.197


2229
CG2
ILE
277
81.988
50.794
5.759


2230
CD1
ILE
277
79.317
50.345
6.726


2231
N
ALA
278
83.945
54.191
5.611


2232
CA
ALA
278
84.744
54.875
4.579


2233
C
ALA
278
85.571
53.914
3.737


2234
O
ALA
278
86.340
53.117
4.283


2235
CB
ALA
278
85.696
55.835
5.276


2236
N
ALA
279
85.449
54.022
2.425


2237
CA
ALA
279
86.256
53.184
1.532


2238
C
ALA
279
87.174
54.020
0.639


2239
O
ALA
279
86.821
54.374
−0.493


2240
CB
ALA
279
85.313
52.337
0.697


2241
N
ASN
280
88.427
54.094
1.062


2242
CA
ASN
280
89.453
54.944
0.419


2243
C
ASN
280
90.168
54.352
−0.802


2244
O
ASN
280
91.287
54.798
−1.082


2245
CB
ASN
280
90.535
55.272
1.448


2246
CG
ASN
280
90.068
56.260
2.513


2247
OD1
ASN
280
89.280
57.179
2.248


2248
ND2
ASN
280
90.599
56.064
3.706


2249
N
ALA
281
89.607
53.337
−1.448


2250
CA
ALA
281
90.249
52.691
−2.613


2251
C
ALA
281
91.579
51.998
−2.274


2252
O
ALA
281
92.052
52.110
−1.137


2253
CB
ALA
281
90.422
53.736
−3.719


2254
N
PRO
282
92.029
51.096
−3.138


2255
CA
PRO
282
93.356
50.483
−2.967


2256
C
PRO
282
94.496
51.504
−3.076


2257
O
PRO
282
94.923
51.901
−4.168


2258
CB
PRO
282
93.451
49.436
−4.034


2259
CG
PRO
282
92.210
49.496
−4.910


2260
CD
PRO
282
91.342
50.604
−4.341


2261
N
VAL
283
94.998
51.871
−1.908


2262
CA
VAL
283
96.073
52.859
−1.763


2263
C
VAL
283
97.452
52.226
−1.954


2264
O
VAL
283
97.716
51.131
−1.450


2265
CB
VAL
283
95.947
53.410
−0.343


2266
CG1
VAL
283
96.767
54.681
−0.123


2267
CG2
VAL
283
94.489
53.697
−0.010


2268
N
ALA
284
98.370
53.019
−2.486


2269
CA
ALA
284
99.788
52.647
−2.683


2270
C
ALA
284
100.648
52.639
−1.410


2271
O
ALA
284
101.868
52.442
−1.490


2272
CB
ALA
284
100.390
53.649
−3.668


2273
N
ALA
285
100.014
52.773
−0.254


2274
CA
ALA
285
100.715
52.813
1.031


2275
C
ALA
285
101.303
51.448
1.370


2276
O
ALA
285
101.231
50.507
0.570


2277
CB
ALA
285
99.728
53.214
2.122


2278
N
ALA
286
101.706
51.286
2.620


2279
CA
ALA
286
102.365
50.039
3.035


2280
C
ALA
286
101.423
48.836
3.105


2281
O
ALA
286
101.880
47.697
2.987


2282
CB
ALA
286
103.000
50.264
4.400


2283
N
LEU
287
100.123
49.090
3.146


2284
CA
LEU
287
99.130
48.011
3.138


2285
C
LEU
287
98.574
47.705
1.743


2286
O
LEU
287
97.891
46.687
1.587


2287
CB
LEU
287
97.970
48.417
4.047


2288
CG
LEU
287
98.389
48.579
5.503


2289
CD1
LEU
287
97.272
49.206
6.327


2290
CD2
LEU
287
98.820
47.248
6.112


2291
N
GLY
288
98.933
48.488
0.736


2292
CA
GLY
288
98.311
48.321
−0.585


2293
C
GLY
288
99.341
48.157
−1.696


2294
O
GLY
288
99.003
47.793
−2.831


2295
N
GLN
289
100.578
48.483
−1.356


2296
CA
GLN
289
101.764
48.238
−2.193


2297
C
GLN
289
101.870
49.229
−3.343


2298
O
GLN
289
100.866
49.755
−3.843


2299
CB
GLN
289
101.804
46.808
−2.743


2300
CG
GLN
289
101.684
45.732
−1.666


2301
CD
GLN
289
102.648
45.990
−0.515


2302
OE1
GLN
289
103.814
46.348
−0.716


2303
NE2
GLN
289
102.121
45.833
0.687


2304
N
THR
290
103.067
49.249
−3.911


2305
CA
THR
290
103.429
50.169
−5.005


2306
C
THR
290
102.858
49.798
−6.383


2307
O
THR
290
103.271
50.380
−7.392


2308
CB
THR
290
104.948
50.200
−5.117


2309
OG1
THR
290
105.383
48.930
−5.586


2310
CG2
THR
290
105.607
50.480
−3.770


2311
N
LYS
291
102.009
48.783
−6.438


2312
CA
LYS
291
101.281
48.451
−7.664


2313
C
LYS
291
99.888
49.073
−7.666


2314
O
LYS
291
99.104
48.827
−8.593


2315
CB
LYS
291
101.181
46.938
−7.805


2316
CG
LYS
291
102.474
46.365
−8.371


2317
CD
LYS
291
102.733
46.948
−9.756


2318
CE
LYS
291
104.023
46.420
−10.369


2319
NZ
LYS
291
104.240
47.015
−11.696


2320
N
GLN
292
99.537
49.746
−6.581


2321
CA
GLN
292
98.276
50.481
−6.554


2322
C
GLN
292
98.465
51.943
−6.905


2323
O
GLN
292
99.397
52.611
−6.448


2324
CB
GLN
292
97.590
50.319
−5.209


2325
CG
GLN
292
96.979
48.928
−5.126


2326
CD
GLN
292
96.143
48.677
−6.381


2327
OE1
GLN
292
95.377
49.543
−6.829


2328
NE2
GLN
292
96.334
47.502
−6.957


2329
N
ASN
293
97.504
52.446
−7.658


2330
CA
ASN
293
97.624
53.791
−8.226


2331
C
ASN
293
97.025
54.891
−7.351


2332
O
ASN
293
97.257
56.077
−7.618


2333
CB
ASN
293
96.946
53.787
−9.593


2334
CG
ASN
293
97.620
52.765
−10.508


2335
OD1
ASN
293
98.847
52.605
−10.488


2336
ND2
ASN
293
96.808
52.098
−11.309


2337
N
CYS
294
96.321
54.528
−6.291


2338
CA
CYS
294
95.753
55.566
−5.426


2339
C
CYS
294
96.752
56.002
−4.362


2340
O
CYS
294
96.959
55.311
−3.361


2341
CB
CYS
294
94.487
55.032
−4.772


2342
SG
CYS
294
93.209
54.504
−5.934


2343
N
LYS
295
97.341
57.166
−4.569


2344
CA
LYS
295
98.307
57.715
−3.609


2345
C
LYS
295
97.686
57.944
−2.236


2346
O
LYS
295
96.462
58.023
−2.080


2347
CB
LYS
295
98.849
59.033
−4.145


2348
CG
LYS
295
99.619
58.825
−5.443


2349
CD
LYS
295
100.230
60.132
−5.936


2350
CE
LYS
295
101.046
59.919
−7.206


2351
NZ
LYS
295
101.657
61.179
−7.656


2352
N
THR
296
98.542
58.169
−1.256


2353
CA
THR
296
98.064
58.370
0.120


2354
C
THR
296
97.400
59.738
0.317


2355
O
THR
296
96.470
59.848
1.125


2356
CB
THR
296
99.243
58.212
1.068


2357
OG1
THR
296
100.209
59.203
0.746


2358
CG2
THR
296
99.896
56.844
0.913


2359
N
ALA
297
97.651
60.646
−0.614


2360
CA
ALA
297
96.948
61.929
−0.638


2361
C
ALA
297
95.546
61.825
−1.253


2362
O
ALA
297
94.722
62.716
−1.032


2363
CB
ALA
297
97.785
62.926
−1.430


2364
N
SER
298
95.225
60.709
−1.892


2365
CA
SER
298
93.850
60.521
−2.360


2366
C
SER
298
93.104
59.661
−1.345


2367
O
SER
298
91.895
59.839
−1.146


2368
CB
SER
298
93.824
59.876
−3.746


2369
OG
SER
298
94.249
58.524
−3.645


2370
N
ALA
299
93.878
58.960
−0.528


2371
CA
ALA
299
93.321
58.153
0.564


2372
C
ALA
299
93.006
58.981
1.806


2373
O
ALA
299
92.325
58.500
2.720


2374
CB
ALA
299
94.335
57.085
0.939


2375
N
CYS
300
93.465
60.220
1.831


2376
CA
CYS
300
93.036
61.121
2.893


2377
C
CYS
300
91.824
61.937
2.462


2378
O
CYS
300
91.097
62.429
3.332


2379
CB
CYS
300
94.179
62.059
3.269


2380
SG
CYS
300
94.677
63.295
2.051


2381
N
THR
301
91.457
61.846
1.194


2382
CA
THR
301
90.441
62.746
0.652


2383
C
THR
301
89.032
62.366
1.087


2384
O
THR
301
88.361
63.191
1.721


2385
CB
THR
301
90.541
62.707
−0.867


2386
OG1
THR
301
91.882
63.003
−1.232


2387
CG2
THR
301
89.629
63.733
−1.524


2388
N
ARG
302
88.717
61.082
1.026


2389
CA
ARG
302
87.358
60.640
1.349


2390
C
ARG
302
87.072
60.743
2.834


2391
O
ARG
302
86.095
61.384
3.251


2392
CB
ARG
302
87.218
59.186
0.927


2393
CG
ARG
302
87.382
59.061
−0.578


2394
CD
ARG
302
86.345
58.101
−1.155


2395
NE
ARG
302
84.997
58.246
−0.553


2396
CZ
ARG
302
84.173
59.304
−0.588


2397
NH1
ARG
302
82.917
59.161
−0.164


2398
NH2
ARG
302
84.538
60.449
−1.172


2399
N
SER
303
88.089
60.399
3.598


2400
CA
SER
303
87.967
60.421
5.044


2401
C
SER
303
88.052
61.827
5.628


2402
O
SER
303
87.298
62.125
6.561


2403
CB
SER
303
89.065
59.529
5.576


2404
OG
SER
303
88.747
58.210
5.161


2405
N
GLY
304
88.718
62.728
4.926


2406
CA
GLY
304
88.735
64.137
5.317


2407
C
GLY
304
87.357
64.753
5.115


2408
O
GLY
304
86.777
65.268
6.081


2409
N
LYS
305
86.769
64.504
3.952


2410
CA
LYS
305
85.422
65.009
3.631


2411
C
LYS
305
84.399
64.629
4.692


2412
O
LYS
305
83.895
65.510
5.401


2413
CB
LYS
305
84.951
64.381
2.325


2414
CG
LYS
305
85.780
64.803
1.122


2415
CD
LYS
305
85.329
64.041
−0.117


2416
CE
LYS
305
86.115
64.458
−1.352


2417
NZ
LYS
305
85.696
63.676
−2.527


2418
N
GLU
306
84.336
63.341
4.992


2419
CA
GLU
306
83.333
62.829
5.939


2420
C
GLU
306
83.675
63.042
7.424


2421
O
GLU
306
82.846
62.736
8.288


2422
CB
GLU
306
83.138
61.347
5.655


2423
CG
GLU
306
84.431
60.575
5.850


2424
CD
GLU
306
84.250
59.113
5.469


2425
OE1
GLU
306
83.958
58.325
6.359


2426
OE2
GLU
306
84.566
58.784
4.333


2427
N
TYR
307
84.829
63.622
7.710


2428
CA
TYR
307
85.221
63.924
9.085


2429
C
TYR
307
84.960
65.394
9.379


2430
O
TYR
307
84.723
65.785
10.527


2431
CB
TYR
307
86.724
63.695
9.163


2432
CG
TYR
307
87.305
63.397
10.538


2433
CD1
TYR
307
88.483
64.014
10.936


2434
CD2
TYR
307
86.670
62.500
11.384


2435
CE1
TYR
307
89.041
63.716
12.172


2436
CE2
TYR
307
87.228
62.198
12.615


2437
CZ
TYR
307
88.417
62.800
13.005


2438
OH
TYR
307
89.014
62.437
14.192


2439
N
LEU
308
84.943
66.184
8.318


2440
CA
LEU
308
84.841
67.641
8.449


2441
C
LEU
308
83.489
68.196
8.009


2442
O
LEU
308
83.380
69.400
7.748


2443
CB
LEU
308
85.942
68.282
7.611


2444
CG
LEU
308
87.331
67.791
8.010


2445
CD1
LEU
308
88.400
68.442
7.137


2446
CD2
LEU
308
87.614
68.037
9.490


2447
N
LEU
309
82.498
67.331
7.876


2448
CA
LEU
309
81.161
67.767
7.447


2449
C
LEU
309
80.556
68.794
8.402


2450
O
LEU
309
80.835
68.798
9.606


2451
CB
LEU
309
80.244
66.554
7.385


2452
CG
LEU
309
80.705
65.550
6.339


2453
CD1
LEU
309
79.917
64.254
6.456


2454
CD2
LEU
309
80.595
66.128
4.932


2455
N
GLU
310
79.783
69.700
7.831


2456
CA
GLU
310
79.096
70.712
8.637


2457
C
GLU
310
77.948
70.089
9.426


2458
O
GLU
310
77.307
69.127
8.987


2459
CB
GLU
310
78.587
71.830
7.731


2460
CG
GLU
310
77.682
71.301
6.624


2461
CD
GLU
310
77.201
72.444
5.734


2462
OE1
GLU
310
76.290
72.207
4.956


2463
OE2
GLU
310
77.875
73.463
5.728


2464
N
GLU
311
77.747
70.614
10.621


2465
CA
GLU
311
76.687
70.124
11.510


2466
C
GLU
311
75.309
70.610
11.067


2467
O
GLU
311
75.187
71.406
10.128


2468
CB
GLU
311
76.962
70.618
12.924


2469
CG
GLU
311
78.330
70.180
13.436


2470
CD
GLU
311
78.507
70.679
14.867


2471
OE1
GLU
311
79.358
71.534
15.059


2472
OE2
GLU
311
77.627
70.378
15.665


2473
N
GLU
312
74.296
70.033
11.698


2474
CA
GLU
312
72.881
70.388
11.488


2475
C
GLU
312
72.375
69.942
10.127


2476
O
GLU
312
71.749
70.721
9.399


2477
CB
GLU
312
72.653
71.887
11.670


2478
CG
GLU
312
72.698
72.296
13.138


2479
CD
GLU
312
71.502
71.704
13.885


2480
OE1
GLU
312
71.648
70.612
14.421


2481
OE2
GLU
312
70.475
72.362
13.928


2482
N
ILE
313
72.630
68.684
9.811


2483
CA
ILE
313
72.109
68.101
8.576


2484
C
ILE
313
71.467
66.745
8.849


2485
O
ILE
313
70.313
66.512
8.471


2486
CB
ILE
313
73.239
67.898
7.570


2487
CG1
ILE
313
74.007
69.181
7.292


2488
CG2
ILE
313
72.686
67.349
6.260


2489
CD1
ILE
313
75.069
68.941
6.228


2490
N
ASP
314
72.179
65.924
9.612


2491
CA
ASP
314
71.862
64.494
9.799


2492
C
ASP
314
71.257
63.816
8.570


2493
O
ASP
314
70.206
63.171
8.642


2494
CB
ASP
314
70.955
64.294
11.007


2495
CG
ASP
314
71.811
64.056
12.240


2496
OD1
ASP
314
72.988
63.745
12.079


2497
OD2
ASP
314
71.320
64.280
13.346


2498
N
SER
315
72.010
63.847
7.484


2499
CA
SER
315
71.586
63.202
6.239


2500
C
SER
315
72.815
62.628
5.556


2501
O
SER
315
72.718
61.871
4.581


2502
CB
SER
315
70.904
64.216
5.324


2503
OG
SER
315
70.453
63.519
4.169


2504
N
THR
316
73.938
62.828
6.228


2505
CA
THR
316
75.264
62.499
5.690


2506
C
THR
316
75.475
61.001
5.437


2507
O
THR
316
75.612
60.194
6.365


2508
CB
THR
316
76.290
63.035
6.686


2509
OG1
THR
316
76.130
62.373
7.936


2510
CG2
THR
316
76.114
64.531
6.923


2511
N
GLY
317
75.522
60.661
4.157


2512
CA
GLY
317
75.695
59.269
3.715


2513
C
GLY
317
74.533
58.385
4.161


2514
O
GLY
317
74.754
57.317
4.742


2515
N
LEU
318
73.324
58.844
3.855


2516
CA
LEU
318
72.066
58.265
4.368


2517
C
LEU
318
72.152
58.139
5.879


2518
O
LEU
318
71.622
57.187
6.463


2519
CB
LEU
318
71.769
56.915
3.733


2520
CG
LEU
318
71.482
57.046
2.245


2521
CD1
LEU
318
71.227
55.676
1.627


2522
CD2
LEU
318
70.292
57.969
2.002


2523
N
ASP
319
72.456
59.296
6.444


2524
CA
ASP
319
72.950
59.472
7.818


2525
C
ASP
319
73.419
58.182
8.467


2526
O
ASP
319
72.598
57.424
9.001


2527
CB
ASP
319
71.892
60.115
8.694


2528
CG
ASP
319
72.618
60.903
9.775


2529
OD1
ASP
319
73.511
61.641
9.362


2530
OD2
ASP
319
72.010
61.093
10.821


2531
N
ASP
320
74.732
57.984
8.462


2532
CA
ASP
320
75.368
56.828
9.130


2533
C
ASP
320
75.137
55.561
8.322


2534
O
ASP
320
74.477
55.607
7.278


2535
CB
ASP
320
74.821
56.583
10.547


2536
CG
ASP
320
75.088
57.720
11.531


2537
OD1
ASP
320
74.733
58.848
11.219


2538
OD2
ASP
320
75.845
57.463
12.460


2539
N
VAL
321
75.667
54.457
8.843


2540
CA
VAL
321
75.512
53.045
8.379


2541
C
VAL
321
75.907
52.669
6.935


2542
O
VAL
321
76.529
51.616
6.758


2543
CB
VAL
321
74.106
52.479
8.709


2544
CG1
VAL
321
72.905
53.404
8.529


2545
CG2
VAL
321
73.822
51.168
7.985


2546
N
VAL
322
75.650
53.492
5.935


2547
CA
VAL
322
75.916
53.073
4.559


2548
C
VAL
322
77.374
53.343
4.157


2549
O
VAL
322
78.097
54.114
4.806


2550
CB
VAL
322
74.899
53.790
3.675


2551
CG1
VAL
322
74.628
53.025
2.382


2552
CG2
VAL
322
73.583
53.918
4.432


2553
N
LEU
323
77.829
52.552
3.199


2554
CA
LEU
323
79.193
52.624
2.665


2555
C
LEU
323
79.429
53.943
1.927


2556
O
LEU
323
78.571
54.425
1.180


2557
CB
LEU
323
79.320
51.438
1.707


2558
CG
LEU
323
80.681
51.292
1.031


2559
CD1
LEU
323
81.796
51.097
2.052


2560
CD2
LEU
323
80.654
50.122
0.055


2561
N
TYR
324
80.565
54.558
2.199


2562
CA
TYR
324
80.940
55.792
1.508


2563
C
TYR
324
82.011
55.501
0.460


2564
O
TYR
324
83.164
55.199
0.796


2565
CB
TYR
324
81.451
56.789
2.539


2566
CG
TYR
324
80.749
58.140
2.462


2567
CD1
TYR
324
81.368
59.278
2.962


2568
CD2
TYR
324
79.489
58.228
1.883


2569
CE1
TYR
324
80.722
60.505
2.893


2570
CE2
TYR
324
78.842
59.454
1.814


2571
CZ
TYR
324
79.457
60.588
2.326


2572
OH
TYR
324
78.753
61.770
2.397


2573
N
LEU
325
81.636
55.672
−0.797


2574
CA
LEU
325
82.524
55.308
−1.909


2575
C
LEU
325
82.448
56.280
−3.082


2576
O
LEU
325
81.390
56.440
−3.700


2577
CB
LEU
325
82.121
53.927
−2.408


2578
CG
LEU
325
83.239
52.910
−2.224


2579
CD1
LEU
325
82.767
51.523
−2.630


2580
CD2
LEU
325
84.488
53.299
−3.006


2581
N
ASP
326
83.612
56.796
−3.456


2582
CA
ASP
326
83.790
57.716
−4.603


2583
C
ASP
326
82.664
58.737
−4.727


2584
O
ASP
326
81.823
58.651
−5.628


2585
CB
ASP
326
83.886
56.898
−5.888


2586
CG
ASP
326
85.135
56.018
−5.870


2587
OD1
ASP
326
85.052
54.900
−6.358


2588
OD2
ASP
326
86.127
56.451
−5.300


2589
N
THR
327
82.640
59.637
−3.752


2590
CA
THR
327
81.634
60.702
−3.506


2591
C
THR
327
80.155
60.268
−3.563


2592
O
THR
327
79.261
61.109
−3.716


2593
CB
THR
327
81.909
61.893
−4.434


2594
OG1
THR
327
81.250
63.041
−3.916


2595
CG2
THR
327
81.468
61.691
−5.881


2596
N
LEU
328
79.895
58.993
−3.330


2597
CA
LEU
328
78.527
58.485
−3.303


2598
C
LEU
328
78.291
57.640
−2.062


2599
O
LEU
328
79.223
57.243
−1.348


2600
CB
LEU
328
78.297
57.615
−4.535


2601
CG
LEU
328
78.363
58.407
−5.836


2602
CD1
LEU
328
78.377
57.477
−7.044


2603
CD2
LEU
328
77.215
59.408
−5.932


2604
N
THR
329
77.018
57.428
−1.791


2605
CA
THR
329
76.607
56.530
−0.715


2606
C
THR
329
76.186
55.198
−1.332


2607
O
THR
329
75.117
55.082
−1.942


2608
CB
THR
329
75.454
57.185
0.029


2609
OG1
THR
329
75.916
58.434
0.522


2610
CG2
THR
329
75.012
56.347
1.215


2611
N
LYS
330
77.048
54.209
−1.184


2612
CA
LYS
330
76.880
52.937
−1.888


2613
C
LYS
330
76.178
51.895
−1.016


2614
O
LYS
330
76.496
51.719
0.168


2615
CB
LYS
330
78.269
52.470
−2.311


2616
CG
LYS
330
78.224
51.479
−3.467


2617
CD
LYS
330
79.612
51.282
−4.060


2618
CE
LYS
330
79.597
50.409
−5.308


2619
NZ
LYS
330
80.942
50.318
−5.896


2620
N
GLU
331
75.240
51.193
−1.633


2621
CA
GLU
331
74.444
50.176
−0.937


2622
C
GLU
331
75.322
49.045
−0.418


2623
O
GLU
331
76.289
48.633
−1.076


2624
CB
GLU
331
73.427
49.605
−1.917


2625
CG
GLU
331
72.551
50.701
−2.510


2626
CD
GLU
331
71.575
50.097
−3.515


2627
OE1
GLU
331
71.094
49.005
−3.253


2628
OE2
GLU
331
71.287
50.767
−4.497


2629
N
TRP
332
74.950
48.506
0.731


2630
CA
TRP
332
75.735
47.422
1.334


2631
C
TRP
332
75.344
46.055
0.767


2632
O
TRP
332
74.542
45.305
1.337


2633
CB
TRP
332
75.583
47.442
2.850


2634
CG
TRP
332
76.287
46.265
3.489


2635
CD1
TRP
332
75.704
45.289
4.264


2636
CD2
TRP
332
77.690
45.933
3.391


2637
NE1
TRP
332
76.652
44.378
4.588


2638
CE2
TRP
332
77.850
44.712
4.068


2639
CE3
TRP
332
78.770
46.527
2.755


2640
CZ2
TRP
332
79.088
44.087
4.072


2641
CZ3
TRP
332
80.011
45.903
2.780


2642
CH2
TRP
332
80.168
44.685
3.434


2643
N
ASP
333
75.885
45.812
−0.417


2644
CA
ASP
333
75.711
44.584
−1.203


2645
C
ASP
333
76.243
44.870
−2.600


2646
O
ASP
333
76.329
43.985
−3.461


2647
CB
ASP
333
74.233
44.182
−1.313


2648
CG
ASP
333
73.403
45.224
−2.066


2649
OD1
ASP
333
73.106
46.258
−1.479


2650
OD2
ASP
333
73.194
45.024
−3.255


2651
N
GLU
334
76.597
46.127
−2.807


2652
CA
GLU
334
76.971
46.590
−4.139


2653
C
GLU
334
78.480
46.511
−4.324


2654
O
GLU
334
79.238
47.327
−3.790


2655
CB
GLU
334
76.471
48.023
−4.262


2656
CG
GLU
334
76.494
48.545
−5.691


2657
CD
GLU
334
75.898
49.949
−5.704


2658
OE1
GLU
334
76.107
50.655
−6.680


2659
OE2
GLU
334
75.232
50.291
−4.732


2660
N
LYS
335
78.891
45.496
−5.063


2661
CA
LYS
335
80.309
45.265
−5.349


2662
C
LYS
335
80.896
46.355
−6.241


2663
O
LYS
335
80.176
47.208
−6.774


2664
CB
LYS
335
80.445
43.893
−5.994


2665
CG
LYS
335
79.953
42.817
−5.030


2666
CD
LYS
335
80.009
41.428
−5.652


2667
CE
LYS
335
79.101
41.331
−6.872


2668
NZ
LYS
335
79.138
39.977
−7.448


2669
N
LEU
336
82.217
46.370
−6.304


2670
CA
LEU
336
82.950
47.417
−7.028


2671
C
LEU
336
82.690
47.415
−8.527


2672
O
LEU
336
83.072
46.492
−9.256


2673
CB
LEU
336
84.437
47.226
−6.775


2674
CG
LEU
336
84.791
47.591
−5.343


2675
CD1
LEU
336
86.243
47.247
−5.035


2676
CD2
LEU
336
84.515
49.070
−5.101


2677
N
LYS
337
82.131
48.521
−8.987


2678
CA
LYS
337
81.864
48.689
−10.417


2679
C
LYS
337
83.084
49.305
−11.096


2680
O
LYS
337
83.377
49.036
−12.266


2681
CB
LYS
337
80.676
49.634
−10.578


2682
CG
LYS
337
79.490
49.245
−9.698


2683
CD
LYS
337
78.896
47.887
−10.064


2684
CE
LYS
337
77.756
47.526
−9.120


2685
NZ
LYS
337
77.223
46.186
−9.414


2686
N
ASP
338
83.850
50.038
−10.306


2687
CA
ASP
338
85.076
50.673
−10.800


2688
C
ASP
338
86.268
49.748
−10.594


2689
O
ASP
338
86.562
49.346
−9.462


2690
CB
ASP
338
85.298
51.976
−10.034


2691
CG
ASP
338
84.126
52.934
−10.241


2692
OD1
ASP
338
84.141
53.642
−11.238


2693
OD2
ASP
338
83.199
52.884
−9.441


2694
N
GLN
339
86.891
49.360
−11.693


2695
CA
GLN
339
88.068
48.483
−11.629


2696
C
GLN
339
89.346
49.220
−12.028


2697
O
GLN
339
89.376
49.962
−13.018


2698
CB
GLN
339
87.829
47.284
−12.541


2699
CG
GLN
339
86.642
46.452
−12.060


2700
CD
GLN
339
86.925
45.899
−10.665


2701
OE1
GLN
339
88.066
45.537
−10.350


2702
NE2
GLN
339
85.894
45.850
−9.841


2703
N
ILE
340
90.389
49.014
−11.243


2704
CA
ILE
340
91.662
49.714
−11.472


2705
C
ILE
340
92.647
48.899
−12.311


2706
O
ILE
340
93.139
47.844
−11.886


2707
CB
ILE
340
92.273
50.054
−10.114


2708
CG1
ILE
340
91.373
51.023
−9.360


2709
CG2
ILE
340
93.674
50.639
−10.253


2710
CD1
ILE
340
92.027
51.489
−8.065


2711
N
VAL
341
92.930
49.424
−13.494


2712
CA
VAL
341
93.926
48.833
−14.399


2713
C
VAL
341
95.316
48.888
−13.764


2714
O
VAL
341
95.774
49.945
−13.313


2715
CB
VAL
341
93.907
49.629
−15.703


2716
CG1
VAL
341
94.909
49.086
−16.718


2717
CG2
VAL
341
92.504
49.647
−16.302


2718
N
ASN
342
95.914
47.718
−13.626


2719
CA
ASN
342
97.220
47.595
−12.975


2720
C
ASN
342
98.121
46.636
−13.748


2721
O
ASN
342
97.679
45.963
−14.687


2722
CB
ASN
342
97.017
47.114
−11.535


2723
CG
ASN
342
96.425
45.703
−11.460


2724
OD1
ASN
342
97.149
44.710
−11.602


2725
ND2
ASN
342
95.124
45.623
−11.231


2726
N
THR
343
99.390
46.614
−13.383


2727
CA
THR
343
100.343
45.745
−14.082


2728
C
THR
343
100.775
44.570
−13.211


2729
O
THR
343
101.618
44.707
−12.315


2730
CB
THR
343
101.554
46.575
−14.488


2731
OG1
THR
343
101.091
47.621
−15.331


2732
CG2
THR
343
102.574
45.751
−15.270


2733
N
ARG
344
100.234
43.406
−13.529


2734
CA
ARG
344
100.586
42.179
−12.806


2735
C
ARG
344
101.875
41.555
−13.334


2736
O
ARG
344
101.855
40.748
−14.270


2737
CB
ARG
344
99.448
41.183
−12.973


2738
CG
ARG
344
98.147
41.744
−12.420


2739
CD
ARG
344
96.984
40.791
−12.665


2740
NE
ARG
344
95.727
41.343
−12.132


2741
CZ
ARG
344
94.825
41.983
−12.881


2742
NH1
ARG
344
93.687
42.409
−12.331


2743
NH2
ARG
344
95.041
42.159
−14.187


2744
N
GLN
345
102.984
41.930
−12.722


2745
CA
GLN
345
104.277
41.337
−13.076


2746
C
GLN
345
104.350
39.882
−12.624


2747
O
GLN
345
103.873
39.536
−11.539


2748
CB
GLN
345
105.390
42.129
−12.403


2749
CG
GLN
345
105.437
43.564
−12.905


2750
CD
GLN
345
106.624
44.279
−12.271


2751
OE1
GLN
345
107.116
45.283
−12.797


2752
NE2
GLN
345
107.088
43.735
−11.159


2753
N
PRO
346
104.930
39.049
−13.473


2754
CA
PRO
346
105.153
37.641
−13.137


2755
C
PRO
346
106.089
37.489
−11.933


2756
O
PRO
346
105.587
37.191
−10.862


2757
CB
PRO
346
105.779
37.033
−14.353


2758
CG
PRO
346
105.987
38.115
−15.404


2759
CD
PRO
346
105.451
39.401
−14.797


2760
OXT
PRO
346
107.280
37.680
−12.116






















TABLE 9





Atom
Atom

Residue





No.
Type
Residue
Position
X Coord.
Y Coord.
Z Coord.





















1
N
MET
1
57.469
−9.726
32.676


2
CA
MET
1
56.765
−10.527
31.662


3
C
MET
1
55.259
−10.345
31.785


4
O
MET
1
54.577
−9.998
30.809


5
CB
MET
1
57.113
−12.004
31.816


6
CG
MET
1
56.425
−12.851
30.751


7
SD
MET
1
56.830
−12.425
29.042


8
CE
MET
1
58.617
−12.699
29.097


9
N
SER
2
54.797
−10.346
33.026


10
CA
SER
2
53.358
−10.282
33.302


11
C
SER
2
52.774
−8.887
33.101


12
O
SER
2
51.590
−8.778
32.766


13
CB
SER
2
53.113
−10.724
34.738


14
OG
SER
2
51.713
−10.650
34.975


15
N
VAL
3
53.636
−7.887
33.002


16
CA
VAL
3
53.173
−6.527
32.728


17
C
VAL
3
52.744
−6.415
31.272


18
O
VAL
3
51.620
−5.985
30.988


19
CB
VAL
3
54.337
−5.572
32.965


20
CG1
VAL
3
53.896
−4.123
32.794


21
CG2
VAL
3
54.960
−5.789
34.339


22
N
GLU
4
53.471
−7.133
30.433


23
CA
GLU
4
53.218
−7.113
28.996


24
C
GLU
4
52.064
−8.048
28.674


25
O
GLU
4
51.153
−7.656
27.934


26
CB
GLU
4
54.470
−7.573
28.247


27
CG
GLU
4
55.692
−6.693
28.516


28
CD
GLU
4
56.667
−7.360
29.489


29
OE1
GLU
4
57.680
−7.866
29.029


30
OE2
GLU
4
56.398
−7.322
30.686


31
N
THR
5
51.937
−9.084
29.486


32
CA
THR
5
50.857
−10.055
29.304


33
C
THR
5
49.505
−9.450
29.672


34
O
THR
5
48.580
−9.485
28.849


35
CB
THR
5
51.138
−11.253
30.203


36
OG1
THR
5
52.409
−11.786
29.849


37
CG2
THR
5
50.093
−12.349
30.026


38
N
ILE
6
49.483
−8.665
30.737


39
CA
ILE
6
48.233
−8.036
31.157


40
C
ILE
6
47.901
−6.809
30.308


41
O
ILE
6
46.732
−6.651
29.937


42
CB
ILE
6
48.343
−7.688
32.639


43
CG1
ILE
6
48.481
−8.965
33.462


44
CG2
ILE
6
47.133
−6.895
33.118


45
CD1
ILE
6
48.488
−8.667
34.957


46
N
SER
7
48.911
−6.175
29.734


47
CA
SER
7
48.644
−5.069
28.805


48
C
SER
7
48.044
−5.591
27.504


49
O
SER
7
46.955
−5.145
27.120


50
CB
SER
7
49.946
−4.339
28.495


51
OG
SER
7
50.434
−3.769
29.701


52
N
ASP
8
48.557
−6.723
27.047


53
CA
ASP
8
48.072
−7.341
25.810


54
C
ASP
8
46.652
−7.868
25.972


55
O
ASP
8
45.780
−7.522
25.163


56
CB
ASP
8
48.980
−8.516
25.451


57
CG
ASP
8
50.414
−8.067
25.173


58
OD1
ASP
8
50.575
−6.968
24.658


59
OD2
ASP
8
51.300
−8.902
25.309


60
N
SER
9
46.371
−8.453
27.125


61
CA
SER
9
45.040
−9.018
27.367


62
C
SER
9
43.982
−7.956
27.667


63
O
SER
9
42.836
−8.125
27.238


64
CB
SER
9
45.127
−10.003
28.531


65
OG
SER
9
45.568
−9.304
29.688


66
N
LEU
10
44.385
−6.792
28.151


67
CA
LEU
10
43.403
−5.731
28.397


68
C
LEU
10
43.168
−4.878
27.152


69
O
LEU
10
42.097
−4.271
27.022


70
CB
LEU
10
43.815
−4.858
29.582


71
CG
LEU
10
43.280
−5.389
30.917


72
CD1
LEU
10
41.826
−5.811
30.774


73
CD2
LEU
10
44.074
−6.561
31.476


74
N
LYS
11
44.048
−5.001
26.171


75
CA
LYS
11
43.811
−4.382
24.861


76
C
LYS
11
43.020
−5.310
23.938


77
O
LYS
11
42.591
−4.899
22.852


78
CB
LYS
11
45.149
−4.052
24.215


79
CG
LYS
11
45.910
−3.023
25.038


80
CD
LYS
11
47.275
−2.720
24.435


81
CE
LYS
11
48.039
−1.714
25.288


82
NZ
LYS
11
49.360
−1.425
24.709


83
N
GLN
12
42.863
−6.556
24.359


84
CA
GLN
12
42.021
−7.514
23.640


85
C
GLN
12
40.698
−7.758
24.363


86
O
GLN
12
39.838
−8.491
23.856


87
CB
GLN
12
42.781
−8.827
23.516


88
CG
GLN
12
44.033
−8.645
22.670


89
CD
GLN
12
44.866
−9.921
22.663


90
OE1
GLN
12
44.338
−11.038
22.701


91
NE2
GLN
12
46.174
−9.734
22.641


92
N
LEU
13
40.541
−7.157
25.532


93
CA
LEU
13
39.319
−7.355
26.317


94
C
LEU
13
38.145
−6.575
25.735


95
O
LEU
13
38.043
−5.351
25.890


96
CB
LEU
13
39.554
−6.893
27.751


97
CG
LEU
13
38.410
−7.348
28.651


98
CD1
LEU
13
38.446
−8.863
28.809


99
CD2
LEU
13
38.463
−6.678
30.019


100
N
GLY
14
37.224
−7.307
25.136


101
CA
GLY
14
36.005
−6.697
24.608


102
C
GLY
14
34.777
−7.223
25.341


103
O
GLY
14
33.937
−7.913
24.755


104
N
LEU
15
34.693
−6.913
26.625


105
CA
LEU
15
33.578
−7.426
27.427


106
C
LEU
15
32.576
−6.360
27.863


107
O
LEU
15
31.650
−6.683
28.616


108
CB
LEU
15
34.102
−8.202
28.627


109
CG
LEU
15
34.651
−9.559
28.192


110
CD1
LEU
15
35.148
−10.367
29.385


111
CD2
LEU
15
33.598
−10.357
27.431


112
N
SER
16
32.775
−5.114
27.462


113
CA
SER
16
31.725
−4.117
27.693


114
C
SER
16
30.623
−4.364
26.676


115
O
SER
16
30.874
−4.970
25.628


116
CB
SER
16
32.248
−2.697
27.519


117
OG
SER
16
32.327
−2.425
26.128


118
N
GLN
17
29.426
−3.892
26.979


119
CA
GLN
17
28.291
−4.081
26.067


120
C
GLN
17
28.616
−3.507
24.688


121
O
GLN
17
29.367
−2.529
24.582


122
CB
GLN
17
27.064
−3.433
26.697


123
CG
GLN
17
26.717
−4.172
27.985


124
CD
GLN
17
25.440
−3.624
28.606


125
OB1
GLN
17
25.487
−2.778
29.506


126
NE2
GLN
17
24.317
−4.150
28.152


127
N
PRO
18
28.080
−4.125
23.643


128
CA
PRO
18
28.749
−4.095
22.329


129
C
PRO
18
28.801
−2.727
21.642


130
O
PRO
18
29.812
−2.434
20.996


131
CB
PRO
18
28.001
−5.078
21.481


132
CG
PRO
18
26.922
−5.750
22.315


133
CD
PRO
18
27.057
−5.174
23.713


134
N
ALA
19
27.904
−1.818
21.988


135
CA
ALA
19
27.961
−0.473
21.402


136
C
ALA
19
29.068
0.373
22.036


137
O
ALA
19
29.795
1.066
21.314


138
CB
ALA
19
26.612
0.207
21.605


139
N
ALA
20
29.401
0.053
23.277


140
CA
ALA
20
30.474
0.759
23.971


141
C
ALA
20
31.825
0.157
23.605


142
O
ALA
20
32.795
0.906
23.448


143
CB
ALA
20
30.251
0.646
25.476


144
N
ILE
21
31.812
−1.099
23.184


145
CA
ILE
21
33.039
−1.757
22.721


146
C
ILE
21
33.295
−1.479
21.233


147
O
ILE
21
34.412
−1.666
20.739


148
CB
ILE
21
32.930
−3.253
23.025


149
CG1
ILE
21
34.104
−3.709
23.887


150
CG2
ILE
21
32.821
−4.113
21.769


151
CD1
ILE
21
35.454
−3.370
23.262


152
N
GLU
22
32.319
−0.871
20.577


153
CA
GLU
22
32.496
−0.402
19.203


154
C
GLU
22
33.003
1.044
19.195


155
O
GLU
22
33.363
1.585
18.142


156
CB
GLU
22
31.142
−0.510
18.506


157
CG
GLU
22
31.223
−0.242
17.008


158
CD
GLU
22
29.836
−0.369
16.389


159
OB1
GLU
22
28.871
−0.230
17.129


160
OE2
GLU
22
29.764
−0.683
15.209


161
N
GLY
23
33.065
1.646
20.372


162
CA
GLY
23
33.584
3.006
20.505


163
C
GLY
23
34.946
2.986
21.187


164
O
GLY
23
35.858
3.727
20.798


165
N
THR
24
35.055
2.181
22.230


166
CA
THR
24
36.326
2.044
22.950


167
C
THR
24
37.289
1.120
22.214


168
O
THR
24
37.043
−0.076
22.031


169
CB
THR
24
36.081
1.521
24.365


170
OG1
THR
24
35.447
0.248
24.296


171
CG2
THR
24
35.187
2.460
25.166


172
N
HIS
25
38.430
1.688
21.878


173
CA
HIS
25
39.479
0.946
21.180


174
C
HIS
25
40.765
0.996
21.991


175
O
HIS
25
41.564
1.924
21.817


176
CB
HIS
25
39.711
1.597
19.820


177
CG
HIS
25
38.524
1.531
18.878


178
ND1
HIS
25
37.648
2.519
18.603


179
CD2
HIS
25
38.140
0.439
18.136


180
CE1
HIS
25
36.738
2.072
17.714


181
NE2
HIS
25
37.041
0.786
17.428


182
N
PRO
26
41.021
−0.039
22.780


183
CA
PRO
26
42.041
0.047
23.840


184
C
PRO
26
43.485
0.102
23.328


185
O
PRO
26
44.320
0.789
23.925


186
CB
PRO
26
41.829
−1.177
24.679


187
CG
PRO
26
40.740
−2.039
24.059


188
CD
PRO
26
40.232
−1.274
22.851


189
N
GLN
27
43.694
−0.352
22.107


190
CA
GLN
27
45.029
−0.344
21.503


191
C
GLN
27
45.335
0.987
20.806


192
O
GLN
27
46.466
1.196
20.353


193
CB
GLN
27
45.178
−1.505
20.509


194
CG
GLN
27
44.325
−1.405
19.235


195
CD
GLN
27
42.869
−1.804
19.466


196
OE1
GLN
27
42.031
−0.960
19.809


197
NE2
GLN
27
42.627
−3.101
19.441


198
N
TYR
28
44.349
1.867
20.716


199
CA
TYR
28
44.585
3.203
20.164


200
C
TYR
28
44.409
4.242
21.265


201
O
TYR
28
45.075
5.284
21.280


202
CB
TYR
28
43.586
3.465
19.041


203
CG
TYR
28
43.643
2.439
17.914


204
CD1
TYR
28
44.825
2.242
17.212


205
CD2
TYR
28
42.509
1.705
17.590


206
CE1
TYR
28
44.879
1.300
16.194


207
CE2
TYR
28
42.560
0.762
16.572


208
CZ
TYR
28
43.747
0.560
15.880


209
OH
TYR
28
43.803
−0.390
14.884


210
N
ASN
29
43.484
3.957
22.160


211
CA
ASN
29
43.264
4.805
23.325


212
C
ASN
29
43.702
4.047
24.568


213
O
ASN
29
42.955
3.205
25.090


214
CB
ASN
29
41.784
5.170
23.393


215
CG
ASN
29
41.477
6.149
24.528


216
OD1
ASN
29
42.364
6.556
25.290


217
ND2
ASN
29
40.206
6.476
24.658


218
N
VAL
30
44.738
4.587
25.190


219
CA
VAL
30
45.383
3.945
26.341


220
C
VAL
30
44.553
4.074
27.621


221
O
VAL
30
44.571
3.152
28.448


222
CB
VAL
30
46.746
4.608
26.529


223
CG1
VAL
30
47.533
3.969
27.668


224
CG2
VAL
30
47.554
4.551
25.237


225
N
VAL
31
43.603
4.997
27.623


226
CA
VAL
31
42.705
5.121
28.771


227
C
VAL
31
41.603
4.057
28.731


228
O
VAL
31
41.159
3.618
29.798


229
CB
VAL
31
42.124
6.528
28.774


230
CG1
VAL
31
41.219
6.756
29.980


231
CG2
VAL
31
43.256
7.548
28.772


232
N
ASP
32
41.428
3.414
27.584


233
CA
ASP
32
40.503
2.279
27.502


234
C
ASP
32
41.160
1.006
28.030


235
O
ASP
32
40.460
0.159
28.596


236
CB
ASP
32
40.092
2.022
26.058


237
CG
ASP
32
39.317
3.186
25.461


238
OD1
ASP
32
38.599
3.842
26.200


239
OD2
ASP
32
39.335
3.309
24.242


240
N
VAL
33
42.485
0.976
28.072


241
CA
VAL
33
43.181
−0.173
28.664


242
C
VAL
33
43.085
−0.089
30.178


243
O
VAL
33
42.802
−1.092
30.846


244
CB
VAL
33
44.658
−0.127
28.293


245
CG1
VAL
33
45.401
−1.346
28.833


246
CG2
VAL
33
44.850
−0.014
26.793


247
N
PHE
34
43.072
1.139
30.673


248
CA
PHE
34
42.913
1.357
32.106


249
C
PHE
34
41.467
1.169
32.536


250
O
PHE
34
41.242
0.510
33.555


251
CB
PHE
34
43.371
2.764
32.447


252
CG
PHE
34
44.868
2.994
32.285


253
CD1
PHE
34
45.335
3.929
31.371


254
CD2
PHE
34
45.767
2.277
33.064


255
CE1
PHE
34
46.699
4.140
31.229


256
CE2
PHE
34
47.132
2.489
32.922


257
CZ
PHE
34
47.598
3.420
32.004


258
N
ARG
35
40.522
1.472
31.660


259
CA
ARG
35
39.112
1.211
31.968


260
C
ARG
35
38.805
−0.283
31.976


261
O
ARG
35
38.191
−0.762
32.938


262
CB
ARG
35
38.231
1.902
30.935


263
CG
ARG
35
38.229
3.413
31.123


264
CD
ARG
35
37.347
4.095
30.083


265
NE
ARG
35
37.235
5.537
30.357


266
CZ
ARG
35
37.500
6.485
29.456


267
NH1
ARG
35
37.923
6.148
28.236


268
NH2
ARG
35
37.364
7.772
29.782


269
N
ASN
36
39.469
−1.028
31.108


270
CA
ASN
36
39.302
−2.480
31.094


271
C
ASN
36
39.990
−3.153
32.281


272
O
ASN
36
39.383
−4.031
32.907


273
CB
ASN
36
39.883
−3.021
29.794


274
CG
ASN
36
38.954
−2.745
28.615


275
OD1
ASN
36
37.837
−2.238
28.776


276
ND2
ASN
36
39.430
−3.106
27.437


277
N
TYR
37
41.102
−2.595
32.733


278
CA
TYR
37
41.786
−3.145
33.910


279
C
TYR
37
41.026
−2.836
35.196


280
O
TYR
37
40.783
−3.748
35.998


281
CB
TYR
37
43.188
−2.546
33.994


282
CG
TYR
37
43.939
−2.901
35.276


283
CD1
TYR
37
44.473
−4.173
35.442


284
CD2
TYR
37
44.084
−1.949
36.279


285
CE1
TYR
37
45.143
−4.497
36.615


286
CE2
TYR
37
44.751
−2.272
37.452


287
CZ
TYR
37
45.273
−3.546
37.619


288
OH
TYR
37
45.820
−3.898
38.831


289
N
ILE
38
40.451
−1.648
35.270


290
CA
ILE
38
39.696
−1.249
36.458


291
C
ILE
38
38.373
−1.995
36.551


292
O
ILE
38
38.081
−2.565
37.611


293
CB
ILE
38
39.445
0.252
36.376


294
CG1
ILE
38
40.749
1.024
36.535


295
CG2
ILE
38
38.431
0.694
37.423


296
CD1
ILE
38
40.544
2.511
36.276


297
N
ALA
39
37.764
−2.260
35.407


298
CA
ALA
39
36.515
−3.014
35.398


299
C
ALA
39
36.746
−4.502
35.624


300
O
ALA
39
35.941
−5.132
36.315


301
CB
ALA
39
35.834
−2.806
34.056


302
N
GLU
40
37.924
−4.993
35.277


303
CA
GLU
40
38.262
−6.392
35.538


304
C
GLU
40
38.540
−6.630
37.018


305
O
GLU
40
37.953
−7.550
37.604


306
CB
GLU
40
39.509
−6.731
34.731


307
CG
GLU
40
39.987
−8.157
34.975


308
CD
GLU
40
41.272
−8.391
34.188


309
OE1
GLU
40
41.943
−7.408
33.903


310
OE2
GLU
40
41.495
−9.523
33.783


311
N
GLU
41
39.207
−5.680
37.657


312
CA
GLU
41
39.512
−5.852
39.079


313
C
GLU
41
38.243
−5.654
39.901


314
O
GLU
41
37.890
−6.540
40.687


315
CB
GLU
41
40.546
−4.831
39.555


316
CG
GLU
41
41.789
−4.689
38.678


317
CD
GLU
41
42.596
−5.978
38.549


318
OE1
GLU
41
43.550
−6.135
39.303


319
OE2
GLU
41
42.365
−6.681
37.575


320
N
LEU
42
37.433
−4.681
39.512


321
CA
LEU
42
36.215
−4.382
40.270


322
C
LEU
42
35.098
−5.387
39.995


323
O
LEU
42
34.330
−5.680
40.918


324
CB
LEU
42
35.745
−2.975
39.917


325
CG
LEU
42
34.636
−2.503
40.853


326
CD1
LEU
42
35.087
−2.568
42.309


327
CD2
LEU
42
34.182
−1.092
40.496


328
N
HIS
43
35.190
−6.112
38.893


329
CA
HIS
43
34.238
−7.184
38.616


330
C
HIS
43
34.365
−8.278
39.664


331
O
HIS
43
33.393
−8.541
40.380


332
CB
HIS
43
34.570
−7.768
37.250


333
CG
HIS
43
33.608
−8.834
36.775


334
ND1
HIS
43
32.278
−8.845
36.971


335
CD2
HIS
43
33.918
−9.965
36.061


336
CE1
HIS
43
31.750
−9.946
36.401


337
NE2
HIS
43
32.766
−10.639
35.838


338
N
ARG
44
35.600
−8.628
39.980


339
CA
ARG
44
35.849
−9.679
40.967


340
C
ARG
44
35.909
−9.179
42.416


341
O
ARG
44
35.969
−10.002
43.336


342
CB
ARG
44
37.154
−10.355
40.576


343
CG
ARG
44
37.015
−10.980
39.191


344
CD
ARG
44
38.342
−11.515
38.670


345
NE
ARG
44
39.311
−10.422
38.506


346
CZ
ARG
44
40.521
−10.433
39.065


347
NH1
ARG
44
40.899
−11.467
39.818


348
NH2
ARG
44
41.350
−9.405
38.880


349
N
ILE
45
35.832
−7.874
42.632


350
CA
ILE
45
35.831
−7.348
44.005


351
C
ILE
45
34.434
−6.910
44.466


352
O
ILE
45
34.110
−6.995
45.658


353
CB
ILE
45
36.804
−6.168
44.058


354
CG1
ILE
45
38.222
−6.625
43.740


355
CG2
ILE
45
36.786
−5.479
45.417


356
CD1
ILE
45
39.187
−5.447
43.706


357
N
SER
46
33.593
−6.518
43.523


358
CA
SER
46
32.255
−6.031
43.877


359
C
SER
46
31.144
−6.949
43.389


360
O
SER
46
29.975
−6.722
43.721


361
CB
SER
46
32.048
−4.684
43.200


362
OG
SER
46
31.856
−4.931
41.815


363
N
SER
47
31.501
−7.832
42.467


364
CA
SER
47
30.579
−8.761
41.796


365
C
SER
47
29.612
−8.047
40.831


366
O
SER
47
28.627
−8.645
40.383


367
CB
SER
47
29.830
−9.571
42.856


368
OG
SER
47
29.130
−10.633
42.222


369
N
VAL
48
29.908
−6.808
40.459


370
CA
VAL
48
29.100
−6.132
39.446


371
C
VAL
48
29.708
−6.410
38.078


372
O
VAL
48
30.934
−6.494
37.950


373
CB
VAL
48
29.101
−4.635
39.760


374
CG1
VAL
48
28.203
−3.845
38.814


375
CG2
VAL
48
28.665
−4.392
41.199


376
N
ASP
49
28.848
−6.684
37.109


377
CA
ASP
49
29.269
−6.946
35.727


378
C
ASP
49
30.137
−5.793
35.221


379
O
ASP
49
29.687
−4.643
35.134


380
CB
ASP
49
27.993
−7.073
34.889


381
CG
ASP
49
28.221
−7.645
33.486


382
OD1
ASP
49
29.348
−7.594
33.013


383
OD2
ASP
49
27.231
−7.986
32.858


384
N
LYS
50
31.309
−6.138
34.712


385
CA
LYS
50
32.262
−5.119
34.267


386
C
LYS
50
31.901
−4.475
32.930


387
O
LYS
50
32.336
−3.345
32.679


388
CB
LYS
50
33.657
−5.715
34.221


389
CG
LYS
50
33.780
−6.952
33.348


390
CD
LYS
50
35.216
−7.458
33.407


391
CE
LYS
50
35.399
−8.764
32.647


392
NZ
LYS
50
36.789
−9.238
32.770


393
N
SER
51
30.893
−5.018
32.263


394
CA
SER
51
30.344
−4.371
31.072


395
C
SER
51
29.557
−3.105
31.415


396
O
SER
51
29.479
−2.208
30.568


397
CB
SER
51
29.410
−5.352
30.370


398
OG
SER
51
28.284
−5.589
31.205


399
N
ILE
52
29.085
−2.973
32.649


400
CA
ILE
52
28.427
−1.733
33.060


401
C
ILE
52
29.351
−0.910
33.957


402
O
ILE
52
29.221
0.319
34.004


403
CB
ILE
52
27.095
−2.030
33.753


404
CG1
ILE
52
27.269
−2.789
35.063


405
CG2
ILE
52
26.171
−2.801
32.816


406
CD1
ILE
52
25.925
−3.057
35.729


407
N
ILE
53
30.425
−1.534
34.424


408
CA
ILE
53
31.415
−0.810
35.227


409
C
ILE
53
32.280
0.071
34.337


410
O
ILE
53
32.505
1.231
34.699


411
CB
ILE
53
32.290
−1.800
35.988


412
CG1
ILE
53
31.468
−2.593
36.995


413
CG2
ILE
53
33.436
−1.084
36.694


414
CD1
ILE
53
32.346
−3.556
37.784


415
N
ILE
54
32.434
−0.329
33.082


416
CA
ILE
54
33.139
0.518
32.112


417
C
ILE
54
32.318
1.750
31.717


418
O
ILE
54
32.896
2.837
31.599


419
CB
ILE
54
33.455
−0.322
30.876


420
CG1
ILE
54
34.442
−1.424
31.229


421
CG2
ILE
54
34.009
0.536
29.743


422
CD1
ILE
54
34.778
−2.300
30.029


423
N
GLN
55
31.003
1.663
31.860


424
CA
GLN
55
30.132
2.803
31.553


425
C
GLN
55
29.976
3.738
32.758


426
O
GLN
55
29.479
4.862
32.617


427
CB
GLN
55
28.764
2.269
31.144


428
CG
GLN
55
28.859
1.383
29.906


429
CD
GLN
55
27.482
0.828
29.549


430
OE1
GLN
55
26.575
1.569
29.153


431
NE2
GLN
55
27.357
−0.481
29.672


432
N
ALA
56
30.449
3.295
33.912


433
CA
ALA
56
30.423
4.116
35.123


434
C
ALA
56
31.786
4.739
35.429


435
O
ALA
56
31.918
5.466
36.421


436
CB
ALA
56
29.974
3.247
36.291


437
N
LEU
57
32.781
4.470
34.595


438
CA
LEU
57
34.126
5.034
34.807


439
C
LEU
57
34.269
6.410
34.155


440
O
LEU
57
34.884
6.550
33.092


441
CB
LEU
57
35.160
4.085
34.209


442
CG
LEU
57
35.144
2.721
34.890


443
CD1
LEU
57
36.098
1.749
34.207


444
CD2
LEU
57
35.459
2.823
36.378


445
N
ASP
58
33.821
7.430
34.866


446
CA
ASP
58
33.799
8.789
34.322


447
C
ASP
58
35.045
9.585
34.710


448
O
ASP
58
35.354
9.753
35.894


449
CB
ASP
58
32.559
9.482
34.877


450
CG
ASP
58
32.347
10.831
34.198


451
OD1
ASP
58
31.831
11.723
34.856


452
OD2
ASP
58
32.581
10.896
32.998


453
N
THR
59
35.740
10.094
33.709


454
CA
THR
59
36.901
10.959
33.957


455
C
THR
59
36.456
12.280
34.590


456
O
THR
59
35.476
12.885
34.146


457
CB
THR
59
37.602
11.196
32.618


458
OG1
THR
59
38.157
9.954
32.209


459
CG2
THR
59
38.750
12.197
32.708


460
N
PRO
60
37.053
12.604
35.728


461
CA
PRO
60
36.763
13.858
36.425


462
C
PRO
60
37.243
15.075
35.643


463
O
PRO
60
37.980
14.970
34.656


464
CB
PRO
60
37.480
13.765
37.734


465
CG
PRO
60
38.337
12.513
37.739


466
CD
PRO
60
38.052
11.801
36.430


467
N
LYS
61
36.810
16.235
36.107


468
CA
LYS
61
37.212
17.499
35.483


469
C
LYS
61
38.518
18.017
36.087


470
O
LYS
61
39.216
18.843
35.490


471
CB
LYS
61
36.105
18.510
35.755


472
CG
LYS
61
34.742
17.952
35.360


473
CD
LYS
61
33.615
18.893
35.769


474
CE
LYS
61
32.250
18.301
35.437


475
NZ
LYS
61
31.167
19.201
35.864


476
N
VAL
62
38.837
17.513
37.268


477
CA
VAL
62
40.065
17.903
37.964


478
C
VAL
62
40.952
16.675
38.150


479
O
VAL
62
40.498
15.688
38.741


480
CB
VAL
62
39.662
18.465
39.331


481
CG1
VAL
62
40.842
19.090
40.067


482
CG2
VAL
62
38.545
19.495
39.202


483
N
LEU
63
42.241
16.814
37.868


484
CA
LEU
63
43.188
15.692
38.033


485
C
LEU
63
43.533
15.394
39.496


486
O
LEU
63
43.960
14.281
39.818


487
CB
LEU
63
44.474
15.999
37.276


488
CG
LEU
63
44.256
16.002
35.769


489
CD1
LEU
63
45.543
16.372
35.042


490
CD2
LEU
63
43.741
14.647
35.292


491
N
ASP
64
43.122
16.275
40.396


492
CA
ASP
64
43.245
16.024
41.838


493
C
ASP
64
42.113
15.127
42.350


494
O
ASP
64
42.071
14.802
43.539


495
CB
ASP
64
43.198
17.354
42.583


496
CG
ASP
64
44.337
18.261
42.129


497
OD1
ASP
64
45.447
18.055
42.597


498
OD2
ASP
64
44.098
19.071
41.243


499
N
GLN
65
41.183
14.802
41.464


500
CA
GLN
65
40.118
13.843
41.730


501
C
GLN
65
40.378
12.524
40.989


502
O
GLN
65
39.503
11.651
40.959


503
CB
GLN
65
38.804
14.460
41.255


504
CG
GLN
65
38.432
15.724
42.029


505
CD
GLN
65
37.924
15.373
43.426


506
OE1
GLN
65
38.702
15.093
44.346


507
NE2
GLN
65
36.611
15.353
43.564


508
N
GLY
66
41.515
12.422
40.318


509
CA
GLY
66
41.869
11.184
39.613


510
C
GLY
66
41.734
11.309
38.099


511
O
GLY
66
41.492
12.392
37.556


512
N
ASP
67
41.963
10.195
37.423


513
CA
ASP
67
41.751
10.130
35.974


514
C
ASP
67
40.432
9.438
35.685


515
O
ASP
67
39.778
9.706
34.670


516
CB
ASP
67
42.837
9.288
35.323


517
CG
ASP
67
44.217
9.901
35.483


518
OD1
ASP
67
44.547
10.757
34.674


519
OD2
ASP
67
44.975
9.369
36.287


520
N
ILE
68
40.067
8.533
36.577


521
CA
ILE
68
38.802
7.801
36.450


522
C
ILE
68
38.066
7.754
37.786


523
O
ILE
68
38.619
7.289
38.789


524
CB
ILE
68
39.107
6.380
35.966


525
CG1
ILE
68
39.541
6.357
34.505


526
CG2
ILE
68
37.909
5.462
36.157


527
CD1
ILE
68
38.421
6.841
33.590


528
N
ILE
69
36.873
8.320
37.814


529
CA
ILE
69
36.003
8.230
38.987


530
C
ILE
69
34.761
7.392
38.697


531
O
ILE
69
34.004
7.676
37.761


532
CB
ILE
69
35.580
9.637
39.405


533
CG1
ILE
69
36.701
10.359
40.130


534
CG2
ILE
69
34.325
9.631
40.270


535
CD1
ILE
69
36.206
11.695
40.675


536
N
VAL
70
34.586
6.328
39.460


537
CA
VAL
70
33.313
5.602
39.390


538
C
VAL
70
32.435
5.971
40.587


539
O
VAL
70
32.812
5.754
41.747


540
CB
VAL
70
33.542
4.093
39.293


541
CG1
VAL
70
34.481
3.566
40.367


542
CG2
VAL
70
32.226
3.321
39.309


543
N
PRO
71
31.382
6.721
40.299


544
CA
PRO
71
30.313
6.920
41.272


545
C
PRO
71
29.582
5.607
41.510


546
O
PRO
71
28.801
5.134
40.672


547
CB
PRO
71
29.414
7.951
40.666


548
CG
PRO
71
29.868
8.217
39.239


549
CD
PRO
71
31.061
7.309
38.999


550
N
ILE
72
29.728
5.112
42.724


551
CA
ILE
72
29.193
3.792
43.084


552
C
ILE
72
27.663
3.573
42.964


553
O
ILE
72
27.316
2.498
42.455


554
CB
ILE
72
29.677
3.481
44.492


555
CG1
ILE
72
31.200
3.489
44.538


556
CG2
ILE
72
29.157
2.127
44.936


557
CD1
ILE
72
31.799
2.425
43.625


558
N
PRO
73
26.759
4.520
43.238


559
CA
PRO
73
25.334
4.238
42.984


560
C
PRO
73
24.924
4.087
41.508


561
O
PRO
73
23.825
3.574
41.274


562
CB
PRO
73
24.571
5.364
43.611


563
CG
PRO
73
25.544
6.421
44.094


564
CD
PRO
73
26.926
5.865
43.812


565
N
LYS
74
25.820
4.299
40.549


566
CA
LYS
74
25.496
4.027
39.141


567
C
LYS
74
25.577
2.536
38.803


568
O
LYS
74
25.163
2.125
37.714


569
CB
LYS
74
26.450
4.786
38.230


570
CG
LYS
74
26.208
6.288
38.281


571
CD
LYS
74
27.060
7.000
37.238


572
CE
LYS
74
26.780
8.498
37.214


573
NZ
LYS
74
27.630
9.172
36.220


574
N
LEU
75
26.037
1.733
39.752


575
CA
LEU
75
25.997
0.274
39.614


576
C
LEU
75
24.667
−0.290
40.127


577
O
LEU
75
24.408
−1.492
39.987


578
CB
LEU
75
27.150
−0.313
40.423


579
CG
LEU
75
28.498
0.251
39.982


580
CD1
LEU
75
29.612
−0.197
40.922


581
CD2
LEU
75
28.824
−0.127
38.539


582
N
ARG
76
23.841
0.590
40.685


583
CA
ARG
76
22.501
0.267
41.196


584
C
ARG
76
22.492
−0.912
42.158


585
O
ARG
76
21.882
−1.955
41.897


586
CB
ARG
76
21.569
0.012
40.013


587
CG
ARG
76
21.382
1.240
39.113


588
CD
ARG
76
20.314
2.228
39.598


589
NE
ARG
76
20.706
2.983
40.802


590
CZ
ARG
76
19.836
3.607
41.598


591
NH1
ARG
76
18.534
3.600
41.301


592
NH2
ARG
76
20.271
4.253
42.682


593
N
LEU
77
23.137
−0.713
43.294


594
CA
LEU
77
23.181
−1.757
44.321


595
C
LEU
77
22.359
−1.325
45.528


596
O
LEU
77
22.839
−0.587
46.394


597
CB
LEU
77
24.629
−1.991
44.733


598
CG
LEU
77
25.494
−2.425
43.554


599
CD1
LEU
77
26.967
−2.439
43.941


600
CD2
LEU
77
25.060
−3.785
43.015


601
N
LYS
78
21.132
−1.807
45.583


602
CA
LYS
78
20.202
−1.392
46.643


603
C
LYS
78
20.625
−1.885
48.026


604
O
LYS
78
21.265
−2.934
48.170


605
CB
LYS
78
18.816
−1.936
46.315


606
CG
LYS
78
18.322
−1.418
44.970


607
CD
LYS
78
18.187
0.101
44.967


608
CE
LYS
78
17.811
0.603
43.580


609
NZ
LYS
78
18.813
0.169
42.594


610
N
GLY
79
20.371
−1.048
49.019


611
CA
GLY
79
20.567
−1.447
50.418


612
C
GLY
79
21.856
−0.912
51.037


613
O
GLY
79
21.821
−0.143
52.006


614
N
ILE
80
22.976
−1.385
50.520


615
CA
ILE
80
24.290
−1.029
51.074


616
C
ILE
80
24.609
0.442
50.779


617
O
ILE
80
24.143
1.003
49.781


618
CB
ILE
80
25.319
−1.984
50.454


619
CG1
ILE
80
24.879
−3.435
50.635


620
CG2
ILE
80
26.709
−1.813
51.064


621
CD1
ILE
80
24.910
−3.854
52.104


622
N
ASN
81
25.256
1.098
51.728


623
CA
ASN
81
25.636
2.504
51.562


624
C
ASN
81
26.889
2.646
50.691


625
O
ASN
81
27.934
2.050
50.986


626
CB
ASN
81
25.863
3.067
52.965


627
CG
ASN
81
26.500
4.449
52.927


628
OD1
ASN
81
27.733
4.570
52.888


629
ND2
ASN
81
25.661
5.469
52.895


630
N
PRO
82
26.820
3.541
49.711


631
CA
PRO
82
27.879
3.670
48.693


632
C
PRO
82
29.237
4.179
49.197


633
O
PRO
82
30.252
3.757
48.632


634
CB
PRO
82
27.324
4.612
47.669


635
CG
PRO
82
25.957
5.102
48.115


636
CD
PRO
82
25.661
4.390
49.423


637
N
ASN
83
29.287
4.791
50.373


638
CA
ASN
83
30.567
5.256
50.917


639
C
ASN
83
31.310
4.089
51.557


640
O
ASN
83
32.536
3.971
51.425


641
CB
ASN
83
30.318
6.296
52.003


642
CG
ASN
83
29.369
7.405
51.558


643
OD1
ASN
83
29.424
7.911
50.431


644
ND2
ASN
83
28.534
7.817
52.496


645
N
GLU
84
30.537
3.116
52.013


646
CA
GLU
84
31.110
1.903
52.592


647
C
GLU
84
31.636
1.015
51.480


648
O
GLU
84
32.778
0.547
51.567


649
CB
GLU
84
30.018
1.152
53.341


650
CG
GLU
84
29.419
1.988
54.462


651
CD
GLU
84
28.213
1.261
55.048


652
OE1
GLU
84
27.460
0.695
54.263


653
OE2
GLU
84
27.974
1.421
56.236


654
N
LYS
85
30.944
1.042
50.351


655
CA
LYS
85
31.376
0.277
49.182


656
C
LYS
85
32.652
0.864
48.594


657
O
LYS
85
33.624
0.124
48.405


658
CB
LYS
85
30.309
0.376
48.108


659
CG
LYS
85
28.922
−0.041
48.566


660
CD
LYS
85
27.961
0.239
47.422


661
CE
LYS
85
26.498
0.137
47.804


662
NZ
LYS
85
25.670
0.767
46.766


663
N
SER
86
32.730
2.186
48.546


664
CA
SER
86
33.920
2.849
48.005


665
C
SER
86
35.140
2.607
48.882


666
O
SER
86
36.171
2.146
48.373


667
CB
SER
86
33.678
4.351
47.934


668
OG
SER
86
32.581
4.607
47.069


669
N
LYS
87
34.942
2.653
50.188


670
CA
LYS
87
36.041
2.416
51.125


671
C
LYS
87
36.555
0.978
51.063


672
O
LYS
87
37.755
0.774
50.834


673
CB
LYS
87
35.515
2.716
52.522


674
CG
LYS
87
36.553
2.437
53.603


675
CD
LYS
87
35.965
2.680
54.987


676
CE
LYS
87
34.760
1.783
55.247


677
NZ
LYS
87
35.140
0.361
55.232


678
N
GLU
88
35.639
0.029
50.957


679
CA
GLU
88
36.024
−1.385
50.931


680
C
GLU
88
36.639
−1.796
49.600


681
O
GLU
88
37.678
−2.471
49.594


682
CB
GLU
88
34.767
−2.213
51.161


683
CG
GLU
88
34.185
−1.949
52.542


684
CD
GLU
88
32.733
−2.412
52.598


685
OE1
GLU
88
32.102
−2.440
51.550


686
OE2
GLU
88
32.246
−2.618
53.701


687
N
TRP
89
36.154
−1.224
48.513


688
CA
TRP
89
36.650
−1.640
47.204


689
C
TRP
89
37.934
−0.923
46.818


690
O
TRP
89
38.793
−1.566
46.210


691
CB
TRP
89
35.575
−1.416
46.148


692
CG
TRP
89
34.339
−2.279
46.349


693
CD1
TRP
89
34.272
−3.485
47.003


694
CD2
TRP
89
33.008
−1.997
45.875


695
NE1
TRP
89
32.994
−3.923
46.975


696
CE2
TRP
89
32.198
−3.059
46.319


697
CE3
TRP
89
32.454
−0.953
45.145


698
CZ2
TRP
89
30.837
−3.050
46.045


699
CZ3
TRP
89
31.095
−0.964
44.863


700
CH2
TRP
89
30.290
−2.002
45.312


701
N
ALA
90
38.189
0.248
47.380


702
CA
ALA
90
39.470
0.912
47.113


703
C
ALA
90
40.571
0.349
48.000


704
O
ALA
90
41.684
0.114
47.516


705
CB
ALA
90
39.336
2.403
47.379


706
N
GLU
91
40.165
−0.159
49.153


707
CA
GLU
91
41.100
−0.780
50.098


708
C
GLU
91
41.574
−2.157
49.633


709
O
GLU
91
42.681
−2.585
49.977


710
CB
GLU
91
40.333
−0.940
51.407


711
CG
GLU
91
41.105
−1.703
52.475


712
CD
GLU
91
40.160
−2.016
53.628


713
OE1
GLU
91
40.144
−1.245
54.577


714
OE2
GLU
91
39.363
−2.933
53.467


715
N
ASN
92
40.778
−2.800
48.796


716
CA
ASN
92
41.121
−4.132
48.299


717
C
ASN
92
41.426
−4.120
46.801


718
O
ASN
92
41.624
−5.184
46.199


719
CB
ASN
92
39.964
−5.086
48.607


720
CG
ASN
92
39.899
−5.408
50.105


721
OD1
ASN
92
40.686
−6.217
50.608


722
ND2
ASN
92
38.969
−4.775
50.803


723
N
PHE
93
41.478
−2.937
46.211


724
CA
PHE
93
41.672
−2.843
44.763


725
C
PHE
93
43.145
−2.979
44.404


726
O
PHE
93
44.001
−2.234
44.892


727
CB
PHE
93
41.151
−1.496
44.281


728
CG
PHE
93
40.575
−1.501
42.866


729
CD1
PHE
93
41.414
−1.494
41.759


730
CD2
PHE
93
39.198
−1.518
42.690


731
CE1
PHE
93
40.879
−1.486
40.479


732
CE2
PHE
93
38.662
−1.514
41.410


733
CZ
PHE
93
39.501
−1.495
40.305


734
N
ASN
94
43.422
−3.958
43.562


735
CA
ASN
94
44.782
−4.165
43.066


736
C
ASN
94
45.188
−3.024
42.137


737
O
ASN
94
44.486
−2.693
41.173


738
CB
ASN
94
44.818
−5.499
42.330


739
CG
ASN
94
46.223
−5.806
41.822


740
OD1
ASN
94
47.225
−5.447
42.455


741
ND2
ASN
94
46.273
−6.341
40.616


742
N
LYS
95
46.333
−2.438
42.446


743
CA
LYS
95
46.858
−1.315
41.666


744
C
LYS
95
47.678
−1.868
40.506


745
O
LYS
95
47.622
−1.373
39.365


746
CB
LYS
95
47.820
−0.522
42.552


747
CG
LYS
95
47.409
−0.442
44.020


748
CD
LYS
95
46.233
0.486
44.304


749
CE
LYS
95
45.920
0.456
45.798


750
NZ
LYS
95
44.783
1.320
46.143


751
N
GLY
96
48.280
−3.015
40.783


752
CA
GLY
96
49.169
−3.685
39.835


753
C
GLY
96
50.327
−2.774
39.458


754
O
GLY
96
51.161
−2.395
40.286


755
N
LYS
97
50.345
−2.432
38.185


756
CA
LYS
97
51.343
−1.519
37.636


757
C
LYS
97
50.662
−0.500
36.734


758
O
LYS
97
51.319
0.205
35.960


759
CB
LYS
97
52.393
−2.317
36.863


760
CG
LYS
97
51.767
−3.397
35.982


761
CD
LYS
97
52.059
−4.795
36.526


762
CE
LYS
97
51.300
−5.870
35.763


763
NZ
LYS
97
51.637
−7.205
36.277


764
N
PHE
98
49.341
−0.457
36.814


765
CA
PHE
98
48.579
0.419
35.925


766
C
PHE
98
47.944
1.560
36.700


767
O
PHE
98
47.703
2.641
36.147


768
CB
PHE
98
47.504
−0.407
35.227


769
CG
PHE
98
48.067
−1.503
34.329


770
CD1
PHE
98
48.028
−2.831
34.735


771
CD2
PHE
98
48.615
−1.170
33.097


772
CE1
PHE
98
48.548
−3.822
33.915


773
CE2
PHE
98
49.133
−2.162
32.276


774
CZ
PHE
98
49.101
−3.489
32.686


775
N
ILE
99
47.692
1.322
37.974


776
CA
ILE
99
47.181
2.383
38.843


777
C
ILE
99
48.199
2.614
39.958


778
O
ILE
99
48.731
1.652
40.521


779
CB
ILE
99
45.830
1.953
39.416


780
CG1
ILE
99
44.863
1.506
38.323


781
CG2
ILE
99
45.193
3.100
40.186


782
CD1
ILE
99
44.415
2.676
37.449


783
N
SER
100
48.504
3.871
40.232


784
CA
SER
100
49.491
4.189
41.266


785
C
SER
100
48.824
4.439
42.617


786
O
SER
100
49.440
4.200
43.662


787
CB
SER
100
50.283
5.424
40.841


788
OG
SER
100
49.393
6.528
40.736


789
N
GLU
101
47.565
4.850
42.597


790
CA
GLU
101
46.825
5.064
43.853


791
C
GLU
101
45.322
5.198
43.619


792
O
GLU
101
44.872
5.760
42.612


793
CB
GLU
101
47.355
6.302
44.574


794
CG
GLU
101
47.332
7.535
43.683


795
CD
GLU
101
47.769
8.772
44.457


796
OE1
GLU
101
46.909
9.382
45.077


797
OE2
GLU
101
48.942
9.108
44.381


798
N
ILE
102
44.564
4.632
44.543


799
CA
ILE
102
43.095
4.669
44.479


800
C
ILE
102
42.504
5.124
45.808


801
O
ILE
102
42.574
4.403
46.810


802
CB
ILE
102
42.588
3.265
44.177


803
CG1
ILE
102
43.206
2.754
42.889


804
CG2
ILE
102
41.066
3.244
44.080


805
CD1
ILE
102
42.947
1.275
42.679


806
N
LYS
103
41.904
6.300
45.813


807
CA
LYS
103
41.301
6.785
47.058


808
C
LYS
103
39.775
6.819
46.993


809
O
LYS
103
39.168
7.137
45.960


810
CB
LYS
103
41.872
8.155
47.406


811
CG
LYS
103
41.651
9.174
46.300


812
CD
LYS
103
42.254
10.519
46.687


813
CE
LYS
103
43.750
10.388
46.950


814
NZ
LYS
103
44.342
11.688
47.303


815
N
PRO
104
39.172
6.307
48.052


816
CA
PRO
104
37.730
6.441
48.245


817
C
PRO
104
37.350
7.845
48.707


818
O
PRO
104
37.620
8.230
49.851


819
CB
PRO
104
37.409
5.448
49.317


820
CG
PRO
104
38.704
4.996
49.978


821
CD
PRO
104
39.824
5.649
49.186


822
N
GLN
105
36.634
8.564
47.864


823
CA
GLN
105
36.124
9.878
48.263


824
C
GLN
105
34.606
9.801
48.391


825
O
GLN
105
33.851
10.282
47.533


826
CB
GLN
105
36.538
10.950
47.258


827
CG
GLN
105
36.223
12.350
47.790


828
CD
GLN
105
36.660
13.426
46.798


829
OE1
GLN
105
35.887
13.832
45.919


830
NE2
GLN
105
37.902
13.862
46.934


831
N
GLY
106
34.180
9.123
49.445


832
CA
GLY
106
32.752
8.958
49.749


833
C
GLY
106
32.046
8.033
48.763


834
O
GLY
106
32.164
6.809
48.831


835
N
VAL
107
31.355
8.651
47.821


836
CA
VAL
107
30.597
7.937
46.791


837
C
VAL
107
31.461
7.634
45.561


838
O
VAL
107
31.132
6.754
44.752


839
CB
VAL
107
29.446
8.874
46.416


840
CG1
VAL
107
28.726
8.485
45.132


841
CG2
VAL
107
28.455
9.032
47.563


842
N
PHE
108
32.600
8.301
45.475


843
CA
PHE
108
33.447
8.186
44.284


844
C
PHE
108
34.723
7.389
44.524


845
O
PHE
108
35.465
7.652
45.476


846
CB
PHE
108
33.849
9.597
43.870


847
CG
PHE
108
32.684
10.555
43.649


848
CD1
PHE
108
32.605
11.725
44.392


849
CD2
PHE
108
31.706
10.262
42.707


850
CE1
PHE
108
31.545
12.601
44.196


851
CE2
PHE
108
30.647
11.138
42.512


852
CZ
PHE
108
30.566
12.307
43.256


853
N
LEU
109
34.987
6.436
43.649


854
CA
LEU
109
36.306
5.789
43.648


855
C
LEU
109
37.226
6.472
42.647


856
O
LEU
109
36.928
6.511
41.446


857
CB
LEU
109
36.176
4.314
43.304


858
CG
LEU
109
35.660
3.532
44.498


859
CD1
LEU
109
35.553
2.046
44.173


860
CD2
LEU
109
36.599
3.755
45.673


861
N
GLN
110
38.315
7.025
43.156


862
CA
GLN
110
39.251
7.782
42.317


863
C
GLN
110
40.512
6.999
41.979


864
O
GLN
110
41.355
6.714
42.840


865
CB
GLN
110
39.641
9.041
43.065


866
CG
GLN
110
38.397
9.763
43.548


867
CD
GLN
110
38.784
11.130
44.080


868
OE1
GLN
110
39.901
11.344
44.564


869
NE2
GLN
110
37.845
12.047
43.965


870
N
PHE
111
40.650
6.733
40.694


871
CA
PHE
111
41.792
5.987
40.159


872
C
PHE
111
42.805
6.910
39.487


873
O
PHE
111
42.501
7.598
38.499


874
CB
PHE
111
41.268
4.985
39.134


875
CG
PHE
111
40.209
4.028
39.678


876
CD1
PHE
111
40.594
2.939
40.447


877
CD2
PHE
111
38.864
4.237
39.402


878
CE1
PHE
111
39.635
2.071
40.953


879
CE2
PHE
111
37.906
3.371
39.908


880
CZ
PHE
111
38.291
2.288
40.687


881
N
TYR
112
43.990
6.950
40.071


882
CA
TYR
112
45.111
7.723
39.521


883
C
TYR
112
46.079
6.768
38.836


884
O
TYR
112
46.685
5.915
39.501


885
CB
TYR
112
45.852
8.434
40.653


886
CG
TYR
112
45.050
9.431
41.493


887
CD1
TYR
112
45.204
10.794
41.279


888
CD2
TYR
112
44.212
8.980
42.507


889
CE1
TYR
112
44.486
11.704
42.043


890
CE2
TYR
112
43.487
9.887
43.265


891
CZ
TYR
112
43.620
11.245
43.027


892
OH
TYR
112
42.810
12.128
43.704


893
N
PHE
113
46.299
6.990
37.552


894
CA
PHE
113
47.091
6.060
36.734


895
C
PHE
113
48.562
6.061
37.133


896
O
PHE
113
49.010
6.899
37.926


897
CB
PHE
113
46.989
6.434
35.257


898
CG
PHE
113
45.602
6.363
34.614


899
CD1
PHE
113
44.598
5.569
35.155


900
CD2
PHE
113
45.356
7.097
33.460


901
CE1
PHE
113
43.346
5.526
34.555


902
CE2
PHE
113
44.107
7.049
32.856


903
CZ
PHE
113
43.100
6.265
33.405


904
N
ALA
114
49.249
5.006
36.737


905
CA
ALA
114
50.687
4.923
36.984


906
C
ALA
114
51.422
5.824
36.005


907
O
ALA
114
51.470
5.556
34.798


908
CB
ALA
114
51.150
3.481
36.827


909
N
LYS
115
52.150
6.773
36.565


910
CA
LYS
115
52.830
7.790
35.757


911
C
LYS
115
53.955
7.221
34.896


912
O
LYS
115
54.017
7.567
33.713


913
CB
LYS
115
53.406
8.842
36.698


914
CG
LYS
115
52.321
9.510
37.537


915
CD
LYS
115
52.583
9.303
39.026


916
CE
LYS
115
53.984
9.771
39.409


917
NZ
LYS
115
54.251
9.512
40.832


918
N
THR
116
54.613
6.166
35.352


919
CA
THR
116
55.696
5.577
34.549


920
C
THR
116
55.166
4.681
33.428


921
O
THR
116
55.728
4.709
32.325


922
CB
THR
116
56.629
4.799
35.470


923
OG1
THR
116
57.219
5.733
36.365


924
CG2
THR
116
57.758
4.124
34.697


925
N
LEU
117
53.929
4.233
33.580


926
CA
LEU
117
53.295
3.412
32.550


927
C
LEU
117
52.773
4.341
31.455


928
O
LEU
117
52.902
4.028
30.263


929
CB
LEU
117
52.157
2.637
33.221


930
CG
LEU
117
51.679
1.411
32.442


931
CD1
LEU
117
50.737
1.748
31.290


932
CD2
LEU
117
52.849
0.547
31.983


933
N
LEU
118
52.466
5.567
31.853


934
CA
LEU
118
52.061
6.599
30.900


935
C
LEU
118
53.264
7.172
30.163


936
O
LEU
118
53.178
7.383
28.949


937
CB
LEU
118
51.376
7.730
31.659


938
CG
LEU
118
50.064
7.286
32.290


939
CD1
LEU
118
49.518
8.369
33.210


940
CD2
LEU
118
49.044
6.921
31.218


941
N
TYR
119
54.414
7.220
30.811


942
CA
TYR
119
55.608
7.728
30.128


943
C
TYR
119
56.072
6.716
29.091


944
O
TYR
119
56.220
7.063
27.912


945
CB
TYR
119
56.740
7.944
31.124


946
CG
TYR
119
56.465
8.870
32.302


947
CD1
TYR
119
55.725
10.036
32.145


948
CD2
TYR
119
57.002
8.552
33.541


949
CE1
TYR
119
55.478
10.851
33.240


950
CE2
TYR
119
56.756
9.365
34.639


951
CZ
TYR
119
55.983
10.506
34.485


952
OH
TYR
119
55.665
11.259
35.591


953
N
ASN
120
56.075
5.453
29.483


954
CA
ASN
120
56.421
4.376
28.557


955
C
ASN
120
55.479
4.313
27.366


956
O
ASN
120
55.837
4.766
26.268


957
CB
ASN
120
56.346
3.052
29.304


958
CG
ASN
120
57.717
2.675
29.851


959
OD1
ASN
120
57.985
2.760
31.057


960
ND2
ASN
120
58.589
2.298
28.933


961
N
LEU
121
54.230
3.982
27.639


962
CA
LEU
121
53.280
3.703
26.559


963
C
LEU
121
52.951
4.940
25.736


964
O
LEU
121
53.222
4.948
24.528


965
CB
LEU
121
51.984
3.143
27.145


966
CG
LEU
121
51.874
1.618
27.057


967
CD1
LEU
121
52.932
0.893
27.883


968
CD2
LEU
121
50.483
1.161
27.484


969
N
VAL
122
52.661
6.041
26.403


970
CA
VAL
122
52.158
7.202
25.676


971
C
VAL
122
53.262
8.004
25.002


972
O
VAL
122
53.082
8.316
23.821


973
CB
VAL
122
51.364
8.083
26.632


974
CG1
VAL
122
50.845
9.338
25.941


975
CG2
VAL
122
50.216
7.292
27.246


976
N
ILE
123
54.461
8.065
25.561


977
CA
ILE
123
55.484
8.878
24.890


978
C
ILE
123
56.129
8.120
23.732


979
O
ILE
123
56.302
8.714
22.659


980
CB
ILE
123
56.537
9.342
25.889


981
CG1
ILE
123
55.902
10.216
26.966


982
CG2
ILE
123
57.654
10.103
25.185


983
CD1
ILE
123
56.945
10.715
27.959


984
N
GLU
124
56.146
6.799
23.820


985
CA
GLU
124
56.640
6.012
22.687


986
C
GLU
124
55.595
5.965
21.574


987
O
GLU
124
55.938
6.181
20.406


988
CB
GLU
124
56.935
4.594
23.160


989
CG
GLU
124
58.051
4.559
24.197


990
CD
GLU
124
58.126
3.165
24.813


991
OE1
GLU
124
57.785
2.219
24.117


992
OE2
GLU
124
58.401
3.071
26.006


993
N
ASP
125
54.331
6.005
21.965


994
CA
ASP
125
53.239
5.947
20.995


995
C
ASP
125
53.047
7.280
20.273


996
O
ASP
125
52.986
7.287
19.036


997
CB
ASP
125
51.971
5.590
21.759


998
CG
ASP
125
50.910
5.061
20.808


999
OD1
ASP
125
51.281
4.306
19.921


1000
OD2
ASP
125
49.741
5.313
21.061


1001
N
VAL
126
53.240
8.383
20.981


1002
CA
VAL
126
53.104
9.705
20.357


1003
C
VAL
126
54.297
10.021
19.467


1004
O
VAL
126
54.108
10.525
18.356


1005
CB
VAL
126
53.015
10.780
21.441


1006
CG1
VAL
126
53.015
12.178
20.837


1007
CG2
VAL
126
51.790
10.605
22.325


1008
N
LEU
127
55.457
9.494
19.817


1009
CA
LEU
127
56.657
9.769
19.022


1010
C
LEU
127
56.813
8.816
17.837


1011
O
LEU
127
57.680
9.043
16.986


1012
CB
LEU
127
57.893
9.679
19.916


1013
CG
LEU
127
58.371
11.028
20.462


1014
CD1
LEU
127
57.360
11.706
21.379


1015
CD2
LEU
127
59.688
10.859
21.205


1016
N
LYS
128
55.986
7.784
17.769


1017
CA
LYS
128
56.039
6.872
16.628


1018
C
LYS
128
54.856
7.082
15.680


1019
O
LYS
128
55.013
6.972
14.458


1020
CB
LYS
128
56.034
5.449
17.173


1021
CG
LYS
128
56.285
4.415
16.083


1022
CD
LYS
128
56.288
3.004
16.659


1023
CE
LYS
128
56.594
1.964
15.587


1024
NZ
LYS
128
56.587
0.606
16.153


1025
N
ARG
129
53.707
7.452
16.228


1026
CA
ARG
129
52.525
7.678
15.388


1027
C
ARG
129
52.346
9.143
15.005


1028
O
ARG
129
51.630
9.428
14.036


1029
CB
ARG
129
51.274
7.222
16.130


1030
CG
ARG
129
51.338
5.752
16.518


1031
CD
ARG
129
50.028
5.298
17.153


1032
NE
ARG
129
48.930
5.275
16.173


1033
CZ
ARG
129
47.645
5.162
16.520


1034
NH1
ARG
129
47.297
5.197
17.808


1035
NH2
ARG
129
46.703
5.095
15.576


1036
N
LYS
130
53.081
10.025
15.666


1037
CA
LYS
130
53.012
11.482
15.458


1038
C
LYS
130
51.592
12.023
15.300


1039
O
LYS
130
50.783
11.969
16.238


1040
CB
LYS
130
53.860
11.825
14.242


1041
CG
LYS
130
55.304
11.407
14.490


1042
CD
LYS
130
56.207
11.753
13.315


1043
CE
LYS
130
57.646
11.332
13.594


1044
NZ
LYS
130
58.526
11.680
12.468


1045
N
SER
131
51.257
12.453
14.093


1046
CA
SER
131
49.934
13.052
13.842


1047
C
SER
131
48.799
12.026
13.877


1048
O
SER
131
47.714
12.330
14.387


1049
CB
SER
131
49.950
13.721
12.470


1050
OG
SER
131
50.103
12.708
11.481


1051
N
ASP
132
49.168
10.769
13.697


1052
CA
ASP
132
48.221
9.658
13.658


1053
C
ASP
132
47.957
9.077
15.046


1054
O
ASP
132
47.169
8.133
15.155


1055
CB
ASP
132
48.797
8.560
12.768


1056
CG
ASP
132
49.209
9.115
11.404


1057
OD1
ASP
132
48.512
9.983
10.896


1058
OD2
ASP
132
50.218
8.654
10.889


1059
N
TYR
133
48.460
9.715
16.096


1060
CA
TYR
133
48.271
9.208
17.467


1061
C
TYR
133
46.823
9.316
17.968


1062
O
TYR
133
46.441
8.583
18.886


1063
CB
TYR
133
49.206
9.983
18.400


1064
CG
TYR
133
48.922
9.798
19.892


1065
CD1
TYR
133
49.184
8.583
20.517


1066
CD2
TYR
133
48.381
10.850
20.621


1067
CE1
TYR
133
48.899
8.422
21.868


1068
CE2
TYR
133
48.097
10.690
21.971


1069
CZ
TYR
133
48.354
9.475
22.591


1070
OH
TYR
133
48.039
9.311
23.923


1071
N
GLY
134
45.985
10.079
17.289


1072
CA
GLY
134
44.571
10.104
17.659


1073
C
GLY
134
43.682
9.543
16.549


1074
O
GLY
134
42.482
9.843
16.511


1075
N
TYR
135
44.254
8.723
15.682


1076
CA
TYR
135
43.508
8.216
14.526


1077
C
TYR
135
42.803
6.889
14.797


1078
O
TYR
135
43.413
5.895
15.206


1079
CB
TYR
135
44.466
8.055
13.347


1080
CG
TYR
135
43.798
7.509
12.089


1081
CD1
TYR
135
42.923
8.312
11.369


1082
CD2
TYR
135
44.057
6.209
11.670


1083
CE1
TYR
135
42.289
7.810
10.240


1084
CE2
TYR
135
43.424
5.707
10.540


1085
CZ
TYR
135
42.535
6.506
9.835


1086
OH
TYR
135
41.785
5.959
8.817


1087
N
LEU
136
41.512
6.890
14.519


1088
CA
LEU
136
40.721
5.660
14.527


1089
C
LEU
136
40.533
5.171
13.093


1090
O
LEU
136
40.203
5.959
12.199


1091
CB
LEU
136
39.359
5.938
15.154


1092
CG
LEU
136
39.481
6.377
16.606


1093
CD1
LEU
136
38.126
6.806
17.153


1094
CD2
LEU
136
40.091
5.276
17.466


1095
N
PRO
137
40.997
3.963
12.840


1096
CA
PRO
137
40.671
3.283
11.591


1097
C
PRO
137
39.273
2.669
11.636


1098
O
PRO
137
38.995
1.798
12.469


1099
CB
PRO
137
41.706
2.207
11.487


1100
CG
PRO
137
42.366
2.036
12.849


1101
CD
PRO
137
41.782
3.122
13.740


1102
N
LEU
138
38.399
3.129
10.757


1103
CA
LEU
138
37.101
2.461
10.607


1104
C
LEU
138
37.258
1.153
9.845


1105
O
LEU
138
38.204
0.973
9.068


1106
CB
LEU
138
36.110
3.344
9.857


1107
CG
LEU
138
35.524
4.441
10.735


1108
CD1
LEU
138
34.437
5.187
9.973


1109
CD2
LEU
138
34.949
3.858
12.021


1110
N
GLY
139
36.383
0.214
10.156


1111
CA
GLY
139
36.336
−1.041
9.406


1112
C
GLY
139
35.714
−0.744
8.049


1113
O
GLY
139
36.390
−0.741
7.014


1114
N
VAL
140
34.421
−0.480
8.078


1115
CA
VAL
140
33.722
−0.013
6.881


1116
C
VAL
140
33.971
1.481
6.675


1117
O
VAL
140
33.987
2.263
7.635


1118
CB
VAL
140
32.235
−0.304
7.045


1119
CG1
VAL
140
31.945
−1.789
6.855


1120
CG2
VAL
140
31.727
0.173
8.401


1121
N
GLY
141
34.212
1.851
5.429


1122
CA
GLY
141
34.465
3.256
5.081


1123
C
GLY
141
33.170
4.059
4.983


1124
O
GLY
141
32.463
4.023
3.969


1125
N
LYS
142
32.841
4.729
6.074


1126
CA
LYS
142
31.615
5.530
6.132


1127
C
LYS
142
31.810
6.909
5.510


1128
O
LYS
142
32.734
7.644
5.877


1129
CB
LYS
142
31.216
5.682
7.593


1130
CG
LYS
142
30.804
4.359
8.226


1131
CD
LYS
142
29.557
3.797
7.553


1132
CE
LYS
142
29.048
2.552
8.266


1133
NZ
LYS
142
27.861
2.005
7.589


1134
N
LYS
143
30.919
7.270
4.602


1135
CA
LYS
143
30.988
8.592
3.965


1136
C
LYS
143
30.178
9.643
4.729


1137
O
LYS
143
29.003
9.436
5.071


1138
CB
LYS
143
30.504
8.472
2.520


1139
CG
LYS
143
30.574
9.810
1.788


1140
CD
LYS
143
30.372
9.659
0.289


1141
CE
LYS
143
31.487
8.824
−0.330


1142
NZ
LYS
143
31.339
8.745
−1.792


1143
N
ALA
144
30.830
10.765
4.987


1144
CA
ALA
144
30.205
11.897
5.678


1145
C
ALA
144
30.326
13.181
4.860


1146
O
ALA
144
31.402
13.786
4.776


1147
CB
ALA
144
30.893
12.104
7.022


1148
N
ILE
145
29.215
13.599
4.285


1149
CA
ILE
145
29.171
14.865
3.550


1150
C
ILE
145
29.181
16.010
4.557


1151
O
ILE
145
28.563
15.896
5.619


1152
CB
ILE
145
27.897
14.875
2.708


1153
CG1
ILE
145
27.952
13.775
1.656


1154
CG2
ILE
145
27.649
16.226
2.046


1155
CD1
ILE
145
26.727
13.821
0.752


1156
N
VAL
146
30.004
17.015
4.311


1157
CA
VAL
146
30.096
18.153
5.232


1158
C
VAL
146
29.868
19.472
4.493


1159
O
VAL
146
30.761
19.974
3.798


1160
CB
VAL
146
31.476
18.165
5.893


1161
CG1
VAL
146
31.571
19.278
6.931


1162
CG2
VAL
146
31.799
16.827
6.550


1163
N
GLU
147
28.663
20.001
4.622


1164
CA
GLU
147
28.348
21.311
4.047


1165
C
GLU
147
28.625
22.413
5.065


1166
O
GLU
147
28.046
22.416
6.157


1167
CB
GLU
147
26.874
21.354
3.656


1168
CG
GLU
147
26.525
22.694
3.015


1169
CD
GLU
147
25.023
22.830
2.813


1170
OE1
GLU
147
24.400
21.843
2.454


1171
OE2
GLU
147
24.530
23.934
2.997


1172
N
PHE
148
29.480
23.351
4.692


1173
CA
PHE
148
29.819
24.460
5.595


1174
C
PHE
148
30.202
25.713
4.817


1175
O
PHE
148
30.471
25.626
3.616


1176
CB
PHE
148
30.953
24.033
6.525


1177
CG
PHE
148
32.296
23.670
5.885


1178
CD1
PHE
148
32.538
22.374
5.444


1179
CD2
PHE
148
33.296
24.629
5.791


1180
CE1
PHE
148
33.765
22.045
4.886


1181
CE2
PHE
148
34.524
24.299
5.235


1182
CZ
PHE
148
34.758
23.008
4.781


1183
N
SER
149
30.211
26.848
5.512


1184
CA
SER
149
30.536
28.197
4.992


1185
C
SER
149
29.488
28.745
4.024


1186
O
SER
149
28.754
29.676
4.374


1187
CB
SER
149
31.906
28.200
4.330


1188
OG
SER
149
32.167
29.525
3.887


1189
N
SER
150
29.470
28.182
2.826


1190
CA
SER
150
28.491
28.454
1.760


1191
C
SER
150
28.030
29.906
1.640


1192
O
SER
150
26.960
30.286
2.128


1193
CB
SER
150
27.328
27.518
2.034


1194
OG
SER
150
27.894
26.210
2.056


1195
N
PRO
151
28.858
30.707
0.991


1196
CA
PRO
151
28.587
32.133
0.839


1197
C
PRO
151
27.688
32.427
−0.354


1198
O
PRO
151
27.615
31.650
−1.315


1199
CB
PRO
151
29.934
32.736
0.598


1200
CG
PRO
151
30.887
31.627
0.175


1201
CD
PRO
151
30.126
30.329
0.366


1202
N
ASN
152
26.952
33.517
−0.244


1203
CA
ASN
152
26.294
34.093
−1.416


1204
C
ASN
152
27.374
34.711
−2.291


1205
O
ASN
152
28.255
35.401
−1.768


1206
CB
ASN
152
25.332
35.177
−0.961


1207
CG
ASN
152
24.175
34.585
−0.170


1208
OD1
ASN
152
23.460
33.706
−0.666


1209
ND2
ASN
152
23.932
35.157
0.996


1210
N
ILE
153
27.253
34.575
−3.600


1211
CA
ILE
153
28.347
34.996
−4.485


1212
C
ILE
153
28.360
36.498
−4.780


1213
O
ILE
153
29.329
37.001
−5.361


1214
CB
ILE
153
28.308
34.190
−5.779


1215
CG1
ILE
153
27.068
34.481
−6.610


1216
CG2
ILE
153
28.394
32.698
−5.471


1217
CD1
ILE
153
27.135
33.730
−7.933


1218
N
ALA
154
27.340
37.212
−4.326


1219
CA
ALA
154
27.374
38.675
−4.374


1220
C
ALA
154
27.937
39.274
−3.083


1221
O
ALA
154
28.183
40.483
−3.016


1222
CB
ALA
154
25.956
39.187
−4.590


1223
N
LYS
155
28.182
38.432
−2.091


1224
CA
LYS
155
28.649
38.924
−0.790


1225
C
LYS
155
30.109
38.556
−0.541


1226
O
LYS
155
30.483
37.378
−0.504


1227
CB
LYS
155
27.795
38.360
0.352


1228
CG
LYS
155
26.438
39.048
0.547


1229
CD
LYS
155
25.358
38.558
−0.414


1230
CE
LYS
155
23.987
39.141
−0.100


1231
NZ
LYS
155
22.965
38.540
−0.973


1232
N
PRO
156
30.911
39.584
−0.320


1233
CA
PRO
156
32.249
39.405
0.244


1234
C
PRO
156
32.164
38.862
1.669


1235
O
PRO
156
31.147
39.028
2.355


1236
CB
PRO
156
32.872
40.766
0.215


1237
CG
PRO
156
31.812
41.786
−0.173


1238
CD
PRO
156
30.538
40.995
−0.420


1239
N
PHE
157
33.226
38.201
2.092


1240
CA
PHE
157
33.253
37.590
3.425


1241
C
PHE
157
33.359
38.609
4.548


1242
O
PHE
157
34.434
39.168
4.781


1243
CB
PHE
157
34.456
36.658
3.545


1244
CG
PHE
157
34.229
35.197
3.162


1245
CD1
PHE
157
33.557
34.859
1.995


1246
CD2
PHE
157
34.715
34.198
3.996


1247
CE1
PHE
157
33.367
33.523
1.668


1248
CE2
PHE
157
34.523
32.862
3.669


1249
CZ
PHE
157
33.847
32.525
2.505


1250
N
HIS
158
32.239
38.896
5.188


1251
CA
HIS
158
32.271
39.583
6.482


1252
C
HIS
158
32.466
38.522
7.562


1253
O
HIS
158
32.359
37.323
7.262


1254
CB
HIS
158
31.012
40.424
6.705


1255
CG
HIS
158
29.697
39.674
6.801


1256
ND1
HIS
158
29.022
39.395
7.932


1257
CD2
HIS
158
28.945
39.195
5.755


1258
CE1
HIS
158
27.900
38.714
7.620


1259
NE2
HIS
158
27.852
38.593
6.275


1260
N
ALA
159
32.678
38.948
8.799


1261
CA
ALA
159
33.050
38.030
9.900


1262
C
ALA
159
32.033
36.950
10.306


1263
O
ALA
159
32.441
35.940
10.893


1264
CB
ALA
159
33.371
38.874
11.126


1265
N
GLY
160
30.808
37.033
9.812


1266
CA
GLY
160
29.794
36.002
10.069


1267
C
GLY
160
30.134
34.664
9.405


1268
O
GLY
160
29.966
33.611
10.034


1269
N
HIS
161
30.816
34.723
8.266


1270
CA
HIS
161
31.161
33.509
7.509


1271
C
HIS
161
32.298
32.710
8.146


1272
O
HIS
161
32.338
31.483
7.975


1273
CB
HIS
161
31.627
33.899
6.109


1274
CG
HIS
161
30.605
34.569
5.214


1275
ND1
HIS
161
30.443
35.893
5.039


1276
CD2
HIS
161
29.686
33.943
4.405


1277
CE1
HIS
161
29.448
36.110
4.156


1278
NE2
HIS
161
28.980
34.903
3.764


1279
N
LEU
162
33.018
33.332
9.071


1280
CA
LEU
162
34.171
32.692
9.707


1281
C
LEU
162
33.757
31.510
10.567


1282
O
LEU
162
34.361
30.436
10.433


1283
CB
LEU
162
34.835
33.714
10.623


1284
CG
LEU
162
35.986
33.103
11.417


1285
CD1
LEU
162
37.178
32.800
10.515


1286
CD2
LEU
162
36.403
34.025
12.555


1287
N
ARG
163
32.580
31.611
11.165


1288
CA
ARG
163
32.117
30.577
12.084


1289
C
ARG
163
31.910
29.246
11.378


1290
O
ARG
163
32.659
28.304
11.660


1291
CB
ARG
163
30.800
31.022
12.690


1292
CG
ARG
163
30.889
32.426
13.269


1293
CD
ARG
163
29.691
32.680
14.168


1294
NE
ARG
163
29.701
31.683
15.246


1295
CZ
ARG
163
29.527
31.990
16.531


1296
NH1
ARG
163
29.141
33.221
16.873


1297
NH2
ARG
163
29.621
31.035
17.457


1298
N
SER
164
31.166
29.251
10.284


1299
CA
SER
164
30.882
27.976
9.621


1300
C
SER
164
32.038
27.496
8.762


1301
O
SER
164
32.192
26.282
8.580


1302
CB
SER
164
29.670
28.112
8.724


1303
OG
SER
164
29.398
26.814
8.210


1304
N
THR
165
32.931
28.399
8.398


1305
CA
THR
165
34.097
27.990
7.628


1306
C
THR
165
35.051
27.203
8.518


1307
O
THR
165
35.325
26.034
8.218


1308
CB
THR
165
34.787
29.235
7.080


1309
OG1
THR
165
33.868
29.912
6.234


1310
CG2
THR
165
36.004
28.878
6.240


1311
N
ILE
166
35.287
27.698
9.723


1312
CA
ILE
166
36.247
27.020
10.595


1313
C
ILE
166
35.625
25.897
11.428


1314
O
ILE
166
36.330
24.928
11.735


1315
CB
ILE
166
36.935
28.055
11.482


1316
CG1
ILE
166
35.976
28.724
12.460


1317
CG2
ILE
166
37.616
29.104
10.613


1318
CD1
ILE
166
36.699
29.755
13.314


1319
N
ILE
167
34.311
25.892
11.592


1320
CA
ILE
167
33.680
24.772
12.295


1321
C
ILE
167
33.496
23.598
11.341


1322
O
ILE
167
33.789
22.458
11.720


1323
CB
ILE
167
32.344
25.219
12.883


1324
CG1
ILE
167
32.563
26.260
13.976


1325
CG2
ILE
167
31.565
24.032
13.442


1326
CD1
ILE
167
31.239
26.763
14.541


1327
N
GLY
168
33.321
23.911
10.067


1328
CA
GLY
168
33.276
22.874
9.040


1329
C
GLY
168
34.666
22.289
8.841


1330
O
GLY
168
34.821
21.063
8.826


1331
N
GLY
169
35.661
23.162
8.786


1332
CA
GLY
169
37.072
22.752
8.766


1333
C
GLY
169
37.399
21.762
9.884


1334
O
GLY
169
37.709
20.598
9.595


1335
N
PHE
170
37.148
22.153
11.124


1336
CA
PHE
170
37.409
21.264
12.261


1337
C
PHE
170
36.644
19.943
12.188


1338
O
PHE
170
37.284
18.889
12.275


1339
CB
PHE
170
37.025
21.974
13.555


1340
CG
PHE
170
36.993
21.037
14.761


1341
CD1
PHE
170
38.174
20.503
15.259


1342
CD2
PHE
170
35.778
20.691
15.340


1343
CE1
PHE
170
38.143
19.640
16.346


1344
CE2
PHE
170
35.747
19.824
16.423


1345
CZ
PHE
170
36.929
19.301
16.928


1346
N
ILE
171
35.377
19.974
11.804


1347
CA
ILE
171
34.572
18.747
11.780


1348
C
ILE
171
34.929
17.812
10.622


1349
O
ILE
171
34.985
16.593
10.832


1350
CB
ILE
171
33.105
19.165
11.708


1351
CG1
ILE
171
32.667
19.797
13.024


1352
CG2
ILE
171
32.196
17.997
11.354


1353
CD1
ILE
171
31.200
20.206
12.977


1354
N
SER
172
35.449
18.364
9.540


1355
CA
SER
172
35.852
17.528
8.409


1356
C
SER
172
37.225
16.894
8.637


1357
O
SER
172
37.397
15.705
8.327


1358
CB
SER
172
35.842
18.374
7.140


1359
OG
SER
172
36.771
19.439
7.272


1360
N
ASN
173
38.057
17.552
9.433


1361
CA
ASN
173
39.339
16.957
9.820


1362
C
ASN
173
39.110
15.910
10.902


1363
O
ASN
173
39.614
14.783
10.805


1364
CB
ASN
173
40.239
18.034
10.419


1365
CG
ASN
173
40.545
19.171
9.450


1366
OD1
ASN
173
40.575
18.996
8.225


1367
ND2
ASN
173
40.815
20.326
10.031


1368
N
LEU
174
38.137
16.199
11.748


1369
CA
LEU
174
37.790
15.349
12.886


1370
C
LEU
174
37.181
14.021
12.454


1371
O
LEU
174
37.668
12.968
12.882


1372
CB
LEU
174
36.773
16.136
13.699


1373
CG
LEU
174
36.350
15.432
14.975


1374
CD1
LEU
174
37.530
15.279
15.930


1375
CD2
LEU
174
35.224
16.214
15.635


1376
N
TYR
175
36.321
14.050
11.449


1377
CA
TYR
175
35.738
12.797
10.972


1378
C
TYR
175
36.712
11.995
10.118


1379
O
TYR
175
36.753
10.771
10.291


1380
CB
TYR
175
34.449
13.068
10.208


1381
CG
TYR
175
33.213
13.204
11.094


1382
CD1
TYR
175
32.789
14.452
11.528


1383
CD2
TYR
175
32.508
12.065
11.466


1384
CE1
TYR
175
31.661
14.563
12.329


1385
CE2
TYR
175
31.378
12.174
12.267


1386
CZ
TYR
175
30.956
13.425
12.694


1387
OH
TYR
175
29.801
13.546
13.440


1388
N
GLU
176
37.689
12.646
9.505


1389
CA
GLU
176
38.715
11.885
8.781


1390
C
GLU
176
39.745
11.280
9.730


1391
O
GLU
176
40.206
10.162
9.475


1392
CB
GLU
176
39.408
12.785
7.774


1393
CG
GLU
176
38.436
13.167
6.673


1394
CD
GLU
176
39.120
14.050
5.642


1395
OE1
GLU
176
40.104
14.685
5.995


1396
OE2
GLU
176
38.636
14.081
4.519


1397
N
LYS
177
39.834
11.839
10.926


1398
CA
LYS
177
40.677
11.286
11.988


1399
C
LYS
177
39.972
10.131
12.718


1400
O
LYS
177
40.614
9.370
13.448


1401
CB
LYS
177
40.987
12.436
12.948


1402
CG
LYS
177
42.014
12.075
14.016


1403
CD
LYS
177
43.359
11.693
13.403


1404
CE
LYS
177
43.991
12.844
12.629


1405
NZ
LYS
177
45.268
12.422
12.030


1406
N
VAL
178
38.684
9.959
12.460


1407
CA
VAL
178
37.943
8.800
12.971


1408
C
VAL
178
37.799
7.720
11.887


1409
O
VAL
178
37.404
6.582
12.177


1410
CB
VAL
178
36.579
9.295
13.453


1411
CG1
VAL
178
35.731
8.176
14.052


1412
CG2
VAL
178
36.752
10.404
14.483


1413
N
GLY
179
38.175
8.057
10.662


1414
CA
GLY
179
38.177
7.081
9.566


1415
C
GLY
179
37.030
7.298
8.581


1416
O
GLY
179
36.675
6.390
7.819


1417
N
TRP
180
36.417
8.466
8.655


1418
CA
TRP
180
35.296
8.804
7.773


1419
C
TRP
180
35.763
9.485
6.497


1420
O
TRP
180
36.624
10.376
6.498


1421
CB
TRP
180
34.326
9.739
8.489


1422
CG
TRP
180
33.496
9.116
9.595


1423
CD1
TRP
180
33.926
8.715
10.839


1424
CD2
TRP
180
32.078
8.848
9.546


1425
NE1
TRP
180
32.866
8.211
11.519


1426
CE2
TRP
180
31.741
8.274
10.783


1427
CE3
TRP
180
31.105
9.047
8.578


1428
CZ2
TRP
180
30.429
7.897
11.032


1429
CZ3
TRP
180
29.793
8.671
8.837


1430
CH2
TRP
180
29.457
8.097
10.058


1431
N
ASP
181
35.072
9.136
5.427


1432
CA
ASP
181
35.314
9.709
4.103


1433
C
ASP
181
34.524
11.006
3.955


1434
O
ASP
181
33.349
10.998
3.565


1435
CB
ASP
181
34.850
8.703
3.054


1436
CG
ASP
181
35.561
7.366
3.255


1437
OD1
ASP
181
34.893
6.412
3.627


1438
OD2
ASP
181
36.759
7.324
3.016


1439
N
VAL
182
35.162
12.099
4.333


1440
CA
VAL
182
34.512
13.411
4.318


1441
C
VAL
182
34.408
14.004
2.917


1442
O
VAL
182
35.347
13.969
2.116


1443
CB
VAL
182
35.284
14.324
5.264


1444
CG1
VAL
182
35.086
15.804
4.971


1445
CG2
VAL
182
34.940
14.001
6.713


1446
N
THR
183
33.203
14.443
2.605


1447
CA
THR
183
32.946
15.115
1.336


1448
C
THR
183
32.562
16.569
1.596


1449
O
THR
183
31.383
16.875
1.811


1450
CB
THR
183
31.809
14.379
0.640


1451
OG1
THR
183
32.105
12.989
0.678


1452
CG2
THR
183
31.657
14.810
−0.814


1453
N
ARG
184
33.561
17.438
1.621


1454
CA
ARG
184
33.334
18.875
1.840


1455
C
ARG
184
32.596
19.490
0.661


1456
O
ARG
184
33.137
19.545
−0.454


1457
CB
ARG
184
34.681
19.574
1.958


1458
CG
ARG
184
35.518
19.050
3.112


1459
CD
ARG
184
36.898
19.699
3.090


1460
NE
ARG
184
37.737
19.229
4.201


1461
CZ
ARG
184
38.605
18.218
4.109


1462
NH1
ARG
184
38.765
17.571
2.952


1463
NH2
ARG
184
39.323
17.862
5.177


1464
N
ILE
185
31.395
19.978
0.925


1465
CA
ILE
185
30.548
20.534
−0.136


1466
C
ILE
185
30.007
21.919
0.222


1467
O
ILE
185
29.262
22.095
1.194


1468
CB
ILE
185
29.374
19.590
−0.406


1469
CG1
ILE
185
29.843
18.189
−0.768


1470
CG2
ILE
185
28.497
20.124
−1.530


1471
CD1
ILE
185
28.670
17.318
−1.200


1472
N
ASN
186
30.380
22.889
−0.593


1473
CA
ASN
186
29.846
24.246
−0.466


1474
C
ASN
186
28.458
24.295
−1.092


1475
O
ASN
186
28.147
23.450
−1.937


1476
CB
ASN
186
30.771
25.204
−1.208


1477
CG
ASN
186
31.117
26.351
−0.273


1478
OD1
ASN
186
30.679
26.361
0.879


1479
ND2
ASN
186
31.916
27.288
−0.744


1480
N
TYR
187
27.587
25.136
−0.565


1481
CA
TYR
187
26.281
25.353
−1.190


1482
C
TYR
187
26.087
26.847
−1.447


1483
O
TYR
187
25.443
27.559
−0.670


1484
CB
TYR
187
25.225
24.823
−0.231


1485
CG
TYR
187
23.799
24.808
−0.762


1486
CD1
TYR
187
23.468
24.007
−1.847


1487
CD2
TYR
187
22.830
25.595
−0.155


1488
CE1
TYR
187
22.161
23.983
−2.317


1489
CE2
TYR
187
21.526
25.573
−0.626


1490
CZ
TYR
187
21.193
24.762
−1.701


1491
OH
TYR
187
19.880
24.670
−2.106


1492
N
LEU
188
26.651
27.290
−2.555


1493
CA
LEU
188
26.685
28.707
−2.928


1494
C
LEU
188
25.308
29.335
−3.116


1495
O
LEU
188
24.372
28.742
−3.673


1496
CB
LEU
188
27.447
28.824
−4.243


1497
CG
LEU
188
28.884
28.334
−4.109


1498
CD1
LEU
188
29.542
28.187
−5.475


1499
CD2
LEU
188
29.697
29.254
−3.206


1500
N
GLY
189
25.224
30.568
−2.651


1501
CA
GLY
189
24.045
31.402
−2.878


1502
C
GLY
189
24.206
32.081
−4.230


1503
O
GLY
189
24.917
33.081
−4.370


1504
N
ASP
190
23.588
31.488
−5.233


1505
CA
ASP
190
23.798
31.934
−6.609


1506
C
ASP
190
22.491
31.945
−7.389


1507
O
ASP
190
22.488
32.134
−8.609


1508
CB
ASP
190
24.784
30.979
−7.280


1509
CG
ASP
190
24.124
29.645
−7.627


1510
OD1
ASP
190
23.949
28.819
−6.740


1511
OD2
ASP
190
23.799
29.468
−8.791


1512
N
TRP
191
21.399
31.658
−6.703


1513
CA
TRP
191
20.113
31.566
−7.390


1514
C
TRP
191
19.122
32.591
−6.841


1515
O
TRP
191
19.514
33.663
−6.351


1516
CB
TRP
191
19.603
30.139
−7.208


1517
CG
TRP
191
18.962
29.485
−8.424


1518
CD1
TRP
191
17.898
28.615
−8.397


1519
CD2
TRP
191
19.332
29.635
−9.816


1520
NE1
TRP
191
17.617
28.231
−9.665


1521
CE2
TRP
191
18.452
28.814
−10.546


1522
CE3
TRP
191
20.312
30.363
−10.475


1523
CZ2
TRP
191
18.570
28.730
−11.925


1524
CZ3
TRP
191
20.420
30.274
−11.858


1525
CH2
TRP
191
19.554
29.461
−12.579


1526
N
GLY
192
17.850
32.311
−7.067


1527
CA
GLY
192
16.754
33.163
−6.593


1528
C
GLY
192
16.824
34.566
−7.183


1529
O
GLY
192
17.489
34.810
−8.200


1530
N
LYS
193
16.343
35.513
−6.397


1531
CA
LYS
193
16.410
36.917
−6.803


1532
C
LYS
193
17.778
37.547
−6.545


1533
O
LYS
193
18.003
38.670
−7.005


1534
CB
LYS
193
15.330
37.727
−6.100


1535
CG
LYS
193
13.939
37.360
−6.604


1536
CD
LYS
193
12.904
38.367
−6.114


1537
CE
LYS
193
11.514
38.054
−6.654


1538
NZ
LYS
193
10.567
39.130
−6.319


1539
N
GLN
194
18.724
36.796
−5.995


1540
CA
GLN
194
20.077
37.330
−5.847


1541
C
GLN
194
20.715
37.405
−7.224


1542
O
GLN
194
21.069
38.506
−7.659


1543
CB
GLN
194
20.916
36.429
−4.943


1544
CG
GLN
194
22.356
36.937
−4.830


1545
CD
GLN
194
23.180
36.019
−3.930


1546
OE1
GLN
194
24.412
36.141
−3.836


1547
NE2
GLN
194
22.478
35.119
−3.263


1548
N
PHE
195
20.556
36.349
−8.008


1549
CA
PHE
195
21.148
36.386
−9.343


1550
C
PHE
195
20.211
37.037
−10.358


1551
O
PHE
195
20.687
37.638
−11.328


1552
CB
PHE
195
21.533
34.988
−9.791


1553
CG
PHE
195
22.827
35.022
−10.592


1554
CD1
PHE
195
24.019
34.650
−9.985


1555
CD2
PHE
195
22.823
35.452
−11.912


1556
CE1
PHE
195
25.206
34.702
−10.701


1557
CE2
PHE
195
24.011
35.508
−12.626


1558
CZ
PHE
195
25.201
35.133
−12.019


1559
N
GLY
196
18.934
37.117
−10.023


1560
CA
GLY
196
17.997
37.898
−10.837


1561
C
GLY
196
18.352
39.386
−10.788


1562
O
GLY
196
18.547
40.031
−11.830


1563
N
LEU
197
18.619
39.868
−9.584


1564
CA
LEU
197
18.978
41.272
−9.385


1565
C
LEU
197
20.437
41.545
−9.765


1566
O
LEU
197
20.742
42.655
−10.215


1567
CB
LEU
197
18.735
41.602
−7.915


1568
CG
LEU
197
18.733
43.099
−7.635


1569
CD1
LEU
197
17.688
43.813
−8.482


1570
CD2
LEU
197
18.477
43.370
−6.158


1571
N
LEU
198
21.260
40.508
−9.810


1572
CA
LEU
198
22.632
40.652
−10.322


1573
C
LEU
198
22.662
40.738
−11.846


1574
O
LEU
198
23.477
41.490
−12.398


1575
CB
LEU
198
23.464
39.437
−9.917


1576
CG
LEU
198
24.526
39.758
−8.868


1577
CD1
LEU
198
25.377
40.948
−9.297


1578
CD2
LEU
198
23.909
40.017
−7.503


1579
N
ALA
199
21.678
40.138
−12.496


1580
CA
ALA
199
21.603
40.190
−13.955


1581
C
ALA
199
21.224
41.586
−14.422


1582
O
ALA
199
22.008
42.198
−15.162


1583
CB
ALA
199
20.570
39.180
−14.439


1584
N
VAL
200
20.234
42.185
−13.775


1585
CA
VAL
200
19.863
43.558
−14.145


1586
C
VAL
200
20.852
44.576
−13.569


1587
O
VAL
200
21.142
45.582
−14.229


1588
CB
VAL
200
18.428
43.848
−13.700


1589
CG1
VAL
200
18.211
43.610
−12.214


1590
CG2
VAL
200
17.987
45.255
−14.088


1591
N
GLY
201
21.570
44.170
−12.534


1592
CA
GLY
201
22.654
44.970
−11.971


1593
C
GLY
201
23.776
45.166
−12.977


1594
O
GLY
201
24.055
46.308
−13.357


1595
N
PHE
202
24.266
44.081
−13.555


1596
CA
PHE
202
25.374
44.189
−14.511


1597
C
PHE
202
24.909
44.633
−15.903


1598
O
PHE
202
25.728
45.070
−16.716


1599
CB
PHE
202
26.083
42.842
−14.591


1600
CG
PHE
202
27.459
42.897
−15.252


1601
CD1
PHE
202
28.542
43.397
−14.541


1602
CD2
PHE
202
27.633
42.449
−16.556


1603
CE1
PHE
202
29.798
43.450
−15.132


1604
CE2
PHE
202
28.889
42.502
−17.147


1605
CZ
PHE
202
29.971
43.003
−16.435


1606
N
GLU
203
23.606
44.665
−16.128


1607
CA
GLU
203
23.082
45.257
−17.361


1608
C
GLU
203
22.865
46.766
−17.217


1609
O
GLU
203
22.698
47.465
−18.223


1610
CB
GLU
203
21.770
44.565
−17.702


1611
CG
GLU
203
22.017
43.109
−18.077


1612
CD
GLU
203
20.702
42.338
−18.087


1613
OE1
GLU
203
19.845
42.667
−17.277


1614
OE2
GLU
203
20.612
41.371
−18.830


1615
N
ARG
204
22.935
47.262
−15.992


1616
CA
ARG
204
22.814
48.697
−15.739


1617
C
ARG
204
24.189
49.305
−15.461


1618
O
ARG
204
24.481
50.439
−15.857


1619
CB
ARG
204
21.949
48.852
−14.492


1620
CG
ARG
204
21.441
50.275
−14.299


1621
CD
ARG
204
20.316
50.580
−15.280


1622
NE
ARG
204
19.214
49.616
−15.102


1623
CZ
ARG
204
18.088
49.887
−14.436


1624
NH1
ARG
204
17.884
51.105
−13.928


1625
NH2
ARG
204
17.148
48.948
−14.308


1626
N
TYR
205
25.037
48.515
−14.823


1627
CA
TYR
205
26.380
48.958
−14.431


1628
C
TYR
205
27.438
47.941
−14.851


1629
O
TYR
205
28.225
47.477
−14.014


1630
CB
TYR
205
26.428
49.081
−12.908


1631
CG
TYR
205
25.343
49.944
−12.267


1632
CD1
TYR
205
24.314
49.340
−11.553


1633
CD2
TYR
205
25.391
51.328
−12.381


1634
CE1
TYR
205
23.328
50.119
−10.963


1635
CE2
TYR
205
24.407
52.108
−11.788


1636
CZ
TYR
205
23.379
51.502
−11.079


1637
OH
TYR
205
22.454
52.277
−10.412


1638
N
GLY
206
27.492
47.640
−16.137


1639
CA
GLY
206
28.411
46.606
−16.634


1640
C
GLY
206
29.811
47.120
−16.957


1641
O
GLY
206
30.176
47.293
−18.126


1642
N
ASP
207
30.604
47.327
−15.922


1643
CA
ASP
207
31.978
47.777
−16.136


1644
C
ASP
207
32.921
46.576
−16.189


1645
O
ASP
207
33.416
46.097
−15.158


1646
CB
ASP
207
32.378
48.741
−15.026


1647
CG
ASP
207
33.669
49.449
−15.421


1648
OD1
ASP
207
34.696
48.784
−15.391


1649
OD2
ASP
207
33.570
50.534
−15.971


1650
N
GLU
208
33.373
46.305
−17.404


1651
CA
GLU
208
34.203
45.128
−17.689


1652
C
GLU
208
35.558
45.150
−16.982


1653
O
GLU
208
35.922
44.146
−16.358


1654
CB
GLU
208
34.454
45.110
−19.195


1655
CG
GLU
208
35.088
43.806
−19.672


1656
CD
GLU
208
34.010
42.752
−19.914


1657
OE1
GLU
208
32.858
43.062
−19.642


1658
OE2
GLU
208
34.322
41.781
−20.589


1659
N
SER
209
36.147
46.327
−16.839


1660
CA
SER
209
37.486
46.408
−16.245


1661
C
SER
209
37.458
46.327
−14.719


1662
O
SER
209
38.397
45.778
−14.137


1663
CB
SER
209
38.158
47.708
−16.679


1664
OG
SER
209
37.427
48.802
−16.142


1665
N
LYS
210
36.315
46.601
−14.113


1666
CA
LYS
210
36.203
46.482
−12.658


1667
C
LYS
210
35.884
45.051
−12.254


1668
O
LYS
210
36.476
44.543
−11.291


1669
CB
LYS
210
35.110
47.417
−12.166


1670
CG
LYS
210
35.590
48.858
−12.100


1671
CD
LYS
210
36.686
49.001
−11.051


1672
CE
LYS
210
37.137
50.449
−10.904


1673
NZ
LYS
210
38.162
50.572
−9.855


1674
N
LEU
211
35.247
44.333
−13.164


1675
CA
LEU
211
34.956
42.923
−12.914


1676
C
LEU
211
36.205
42.084
−13.167


1677
O
LEU
211
36.451
41.101
−12.457


1678
CB
LEU
211
33.845
42.487
−13.858


1679
CG
LEU
211
33.291
41.126
−13.461


1680
CD1
LEU
211
32.623
41.201
−12.094


1681
CD2
LEU
211
32.294
40.633
−14.496


1682
N
ALA
212
37.077
42.599
−14.020


1683
CA
ALA
212
38.372
41.961
−14.269


1684
C
ALA
212
39.448
42.367
−13.257


1685
O
ALA
212
40.499
41.719
−13.191


1686
CB
ALA
212
38.830
42.334
−15.675


1687
N
SER
213
39.182
43.389
−12.457


1688
CA
SER
213
40.112
43.751
−11.385


1689
C
SER
213
39.782
42.954
−10.133


1690
O
SER
213
40.681
42.542
−9.390


1691
CB
SER
213
39.996
45.241
−11.086


1692
OG
SER
213
40.433
45.952
−12.234


1693
N
ASP
214
38.495
42.735
−9.922


1694
CA
ASP
214
38.040
41.816
−8.875


1695
C
ASP
214
36.553
41.551
−9.053


1696
O
ASP
214
35.708
42.383
−8.688


1697
CB
ASP
214
38.312
42.372
−7.475


1698
CG
ASP
214
38.140
41.260
−6.432


1699
OD1
ASP
214
39.149
40.828
−5.896


1700
OD2
ASP
214
37.033
40.735
−6.355


1701
N
PRO
215
36.264
40.301
−9.370


1702
CA
PRO
215
34.898
39.868
−9.657


1703
C
PRO
215
33.919
40.125
−8.515


1704
O
PRO
215
33.011
40.945
−8.695


1705
CB
PRO
215
35.006
38.407
−9.948


1706
CG
PRO
215
36.465
37.981
−9.893


1707
CD
PRO
215
37.248
39.231
−9.544


1708
N
ILE
216
34.247
39.688
−7.308


1709
CA
ILE
216
33.285
39.802
−6.202


1710
C
ILE
216
33.198
41.220
−5.635


1711
O
ILE
216
32.092
41.659
−5.294


1712
CB
ILE
216
33.677
38.819
−5.105


1713
CG1
ILE
216
33.682
37.396
−5.646


1714
CG2
ILE
216
32.732
38.920
−3.911


1715
CD1
ILE
216
33.971
36.393
−4.538


1716
N
ASN
217
34.242
42.010
−5.835


1717
CA
ASN
217
34.192
43.415
−5.417


1718
C
ASN
217
33.287
44.212
−6.342


1719
O
ASN
217
32.394
44.924
−5.859


1720
CB
ASN
217
35.583
44.039
−5.463


1721
CG
ASN
217
36.445
43.618
−4.277


1722
OD1
ASN
217
36.743
42.435
−4.083


1723
ND2
ASN
217
36.923
44.615
−3.554


1724
N
HIS
218
33.325
43.892
−7.624


1725
CA
HIS
218
32.470
44.609
−8.560


1726
C
HIS
218
31.037
44.098
−8.505


1727
O
HIS
218
30.115
44.923
−8.524


1728
CB
HIS
218
33.013
44.452
−9.967


1729
CG
HIS
218
32.255
45.304
−10.959


1730
ND1
HIS
218
31.944
46.605
−10.813


1731
CD2
HIS
218
31.746
44.903
−12.170


1732
CE1
HIS
218
31.266
47.026
−11.899


1733
NE2
HIS
218
31.144
45.971
−12.737


1734
N
LEU
219
30.862
42.841
−8.129


1735
CA
LEU
219
29.510
42.292
−7.983


1736
C
LEU
219
28.816
42.804
−6.725


1737
O
LEU
219
27.621
43.109
−6.794


1738
CB
LEU
219
29.581
40.770
−7.935


1739
CG
LEU
219
30.037
40.191
−9.270


1740
CD1
LEU
219
30.215
38.679
−9.180


1741
CD2
LEU
219
29.067
40.561
−10.387


1742
N
PHE
220
29.583
43.150
−5.704


1743
CA
PHE
220
28.984
43.755
−4.513


1744
C
PHE
220
28.693
45.239
−4.727


1745
O
PHE
220
27.616
45.706
−4.337


1746
CB
PHE
220
29.943
43.591
−3.343


1747
CG
PHE
220
29.391
44.125
−2.026


1748
CD1
PHE
220
28.164
43.673
−1.556


1749
CD2
PHE
220
30.113
45.058
−1.295


1750
CE1
PHE
220
27.659
44.156
−0.356


1751
CE2
PHE
220
29.608
45.542
−0.095


1752
CZ
PHE
220
28.381
45.091
0.374


1753
N
GLU
221
29.498
45.893
−5.551


1754
CA
GLU
221
29.253
47.305
−5.882


1755
C
GLU
221
28.001
47.439
−6.737


1756
O
GLU
221
27.070
48.170
−6.370


1757
CB
GLU
221
30.439
47.825
−6.682


1758
CG
GLU
221
31.710
47.865
−5.848


1759
CD
GLU
221
32.919
47.907
−6.776


1760
OE1
GLU
221
34.027
47.774
−6.271


1761
OE2
GLU
221
32.704
47.875
−7.982


1762
N
VAL
222
27.881
46.525
−7.684


1763
CA
VAL
222
26.726
46.474
−8.576


1764
C
VAL
222
25.458
46.030
−7.857


1765
O
VAL
222
24.403
46.634
−8.082


1766
CB
VAL
222
27.076
45.488
−9.684


1767
CG1
VAL
222
25.850
44.871
−10.334


1768
CG2
VAL
222
27.992
46.123
−10.721


1769
N
TYR
223
25.609
45.225
−6.818


1770
CA
TYR
223
24.449
44.762
−6.065


1771
C
TYR
223
23.940
45.841
−5.118


1772
O
TYR
223
22.720
45.998
−4.983


1773
CB
TYR
223
24.853
43.533
−5.264


1774
CG
TYR
223
23.673
42.718
−4.753


1775
CD1
TYR
223
22.582
42.497
−5.585


1776
CD2
TYR
223
23.691
42.186
−3.470


1777
CE1
TYR
223
21.509
41.742
−5.136


1778
CE2
TYR
223
22.615
41.434
−3.019


1779
CZ
TYR
223
21.528
41.212
−3.854


1780
OH
TYR
223
20.459
40.466
−3.409


1781
N
VAL
224
24.823
46.711
−4.658


1782
CA
VAL
224
24.379
47.820
−3.816


1783
C
VAL
224
23.676
48.880
−4.655


1784
O
VAL
224
22.513
49.184
−4.360


1785
CB
VAL
224
25.582
48.429
−3.102


1786
CG1
VAL
224
25.202
49.711
−2.368


1787
CG2
VAL
224
26.205
47.430
−2.135


1788
N
LYS
225
24.197
49.124
−5.847


1789
CA
LYS
225
23.607
50.146
−6.717


1790
C
LYS
225
22.306
49.691
−7.376


1791
O
LYS
225
21.354
50.481
−7.445


1792
CB
LYS
225
24.631
50.511
−7.782


1793
CG
LYS
225
25.876
51.135
−7.166


1794
CD
LYS
225
26.923
51.441
−8.230


1795
CE
LYS
225
27.324
50.178
−8.983


1796
NZ
LYS
225
28.400
50.446
−9.950


1797
N
ILE
226
22.157
48.394
−7.595


1798
CA
ILE
226
20.894
47.912
−8.154


1799
C
ILE
226
19.833
47.724
−7.061


1800
O
ILE
226
18.654
47.969
−7.343


1801
CB
ILE
226
21.133
46.641
−8.969


1802
CG1
ILE
226
19.936
46.336
−9.860


1803
CG2
ILE
226
21.454
45.436
−8.094


1804
CD1
ILE
226
19.777
47.392
−10.949


1805
N
ASN
227
20.258
47.648
−5.805


1806
CA
ASN
227
19.308
47.678
−4.686


1807
C
ASN
227
18.854
49.103
−4.414


1808
O
ASN
227
17.676
49.319
−4.111


1809
CB
ASN
227
19.955
47.129
−3.418


1810
CG
ASN
227
19.618
45.655
−3.212


1811
OD1
ASN
227
18.512
45.307
−2.782


1812
ND2
ASN
227
20.574
44.804
−3.526


1813
N
GLN
228
19.673
50.066
−4.801


1814
CA
GLN
228
19.262
51.466
−4.709


1815
C
GLN
228
18.313
51.830
−5.848


1816
O
GLN
228
17.310
52.508
−5.593


1817
CB
GLN
228
20.516
52.325
−4.735


1818
CG
GLN
228
21.387
51.966
−3.538


1819
CD
GLN
228
22.730
52.681
−3.599


1820
OE1
GLN
228
23.646
52.269
−4.321


1821
NE2
GLN
228
22.849
53.719
−2.790


1822
N
ASP
229
18.413
51.091
−6.944


1823
CA
ASP
229
17.461
51.230
−8.053


1824
C
ASP
229
16.135
50.519
−7.765


1825
O
ASP
229
15.126
50.828
−8.412


1826
CB
ASP
229
18.066
50.615
−9.313


1827
CG
ASP
229
19.405
51.245
−9.684


1828
OD1
ASP
229
19.522
52.458
−9.580


1829
OD2
ASP
229
20.230
50.517
−10.219


1830
N
VAL
230
16.102
49.663
−6.755


1831
CA
VAL
230
14.844
49.035
−6.339


1832
C
VAL
230
14.203
49.790
−5.175


1833
O
VAL
230
12.979
49.947
−5.143


1834
CB
VAL
230
15.123
47.598
−5.899


1835
CG1
VAL
230
13.864
46.923
−5.367


1836
CG2
VAL
230
15.720
46.770
−7.027


1837
N
THR
231
15.019
50.393
−4.328


1838
CA
THR
231
14.479
51.091
−3.147


1839
C
THR
231
14.123
52.555
−3.403


1840
O
THR
231
13.278
53.115
−2.698


1841
CB
THR
231
15.495
51.019
−2.009


1842
OG1
THR
231
16.725
51.574
−2.454


1843
CG2
THR
231
15.745
49.578
−1.574


1844
N
LYS
232
14.768
53.166
−4.383


1845
CA
LYS
232
14.414
54.530
−4.783


1846
C
LYS
232
13.662
54.494
−6.115


1847
O
LYS
232
12.981
55.458
−6.493


1848
CB
LYS
232
15.693
55.352
−4.909


1849
CG
LYS
232
16.529
55.254
−3.636


1850
CD
LYS
232
17.786
56.116
−3.699


1851
CE
LYS
232
17.426
57.593
−3.869


1852
NZ
LYS
232
18.619
58.452
−3.842


1853
N
GLU
233
13.689
53.309
−6.707


1854
CA
GLU
233
13.052
52.961
−7.991


1855
C
GLU
233
13.059
54.004
−9.099


1856
O
GLU
233
12.297
54.979
−9.039


1857
CB
GLU
233
11.624
52.476
−7.780


1858
CG
GLU
233
11.561
50.971
−7.545


1859
CD
GLU
233
12.145
50.185
−8.727


1860
OE1
GLU
233
12.307
50.768
−9.796


1861
OE2
GLU
233
12.304
48.978
−8.595


1862
N
THR
234
13.618
53.554
−10.215


1863
CA
THR
234
13.707
54.302
−11.486


1864
C
THR
234
13.736
55.822
−11.355


1865
O
THR
234
14.451
56.367
−10.507


1866
CB
THR
234
12.544
53.862
−12.365


1867
OG1
THR
234
11.349
53.933
−11.593


1868
CG2
THR
234
12.721
52.416
−12.810


1869
N
SER
235
13.153
56.481
−12.342


1870
CA
SER
235
13.152
57.946
−12.364


1871
C
SER
235
11.791
58.534
−12.736


1872
O
SER
235
11.288
59.423
−12.039


1873
CB
SER
235
14.201
58.412
−13.362


1874
OG
SER
235
15.450
57.883
−12.940


1875
N
GLU
236
11.247
58.061
−13.854


1876
CA
GLU
236
9.971
58.532
−14.456


1877
C
GLU
236
9.956
60.052
−14.724


1878
O
GLU
236
10.209
60.867
−13.824


1879
CB
GLU
236
8.807
58.099
−13.566


1880
CG
GLU
236
7.444
58.429
−14.166


1881
CD
GLU
236
6.302
57.935
−13.273


1882
OE1
GLU
236
5.548
58.775
−12.805


1883
OE2
GLU
236
6.020
56.749
−13.371


1884
N
ALA
237
9.583
60.403
−15.950


1885
CA
ALA
237
9.606
61.792
−16.452


1886
C
ALA
237
9.229
62.860
−15.423


1887
O
ALA
237
8.303
62.695
−14.618


1888
CB
ALA
237
8.681
61.896
−17.663


1889
N
THR
238
10.041
63.907
−15.436


1890
CA
THR
238
10.026
65.015
−14.464


1891
C
THR
238
9.830
64.605
−13.002


1892
O
THR
238
8.912
65.110
−12.344


1893
CB
THR
238
8.987
66.055
−14.880


1894
OG1
THR
238
7.734
65.420
−15.104


1895
CG2
THR
238
9.401
66.745
−16.177


1896
N
GLY
239
10.655
63.672
−12.539


1897
CA
GLY
239
10.727
63.289
−11.123


1898
C
GLY
239
9.407
62.762
−10.569


1899
O
GLY
239
9.005
63.140
−9.461


1900
N
GLU
240
8.722
61.922
−11.327


1901
CA
GLU
240
7.379
61.533
−10.897


1902
C
GLU
240
7.312
60.073
−10.436


1903
O
GLU
240
7.886
59.157
−11.033


1904
CB
GLU
240
6.406
61.910
−12.016


1905
CG
GLU
240
6.388
63.436
−12.217


1906
CD
GLU
240
5.518
63.865
−13.405


1907
OE1
GLU
240
5.140
62.996
−14.180


1908
OE2
GLU
240
5.204
65.049
−13.502


1909
N
THR
241
6.572
59.898
−9.356


1910
CA
THR
241
6.466
58.634
−8.592


1911
C
THR
241
7.820
57.983
−8.206


1912
O
THR
241
8.039
56.809
−8.535


1913
CB
THR
241
5.639
57.640
−9.416


1914
OG1
THR
241
4.582
58.337
−10.066


1915
CG2
THR
241
5.022
56.549
−8.545


1916
N
PRO
242
8.683
58.659
−7.449


1917
CA
PRO
242
9.881
57.977
−6.935


1918
C
PRO
242
9.514
57.016
−5.800


1919
O
PRO
242
8.508
57.227
−5.109


1920
CB
PRO
242
10.779
59.081
−6.462


1921
CG
PRO
242
9.981
60.377
−6.412


1922
CD
PRO
242
8.586
60.029
−6.913


1923
N
ALA
243
10.233
55.900
−5.740


1924
CA
ALA
243
10.086
54.820
−4.727


1925
C
ALA
243
8.848
53.913
−4.867


1926
O
ALA
243
8.932
52.718
−4.556


1927
CB
ALA
243
10.169
55.408
−3.324


1928
N
GLU
244
7.767
54.435
−5.424


1929
CA
GLU
244
6.573
53.640
−5.728


1930
C
GLU
244
6.600
53.132
−7.168


1931
O
GLU
244
5.562
52.705
−7.681


1932
CB
GLU
244
5.315
54.464
−5.483


1933
CG
GLU
244
4.532
53.978
−4.256


1934
CD
GLU
244
5.333
54.158
−2.966


1935
OE1
GLU
244
6.056
55.139
−2.866


1936
OE2
GLU
244
5.156
53.341
−2.078


1937
N
THR
245
7.757
53.282
−7.802


1938
CA
THR
245
8.108
52.733
−9.126


1939
C
THR
245
7.484
53.527
−10.249


1940
O
THR
245
6.488
54.237
−10.084


1941
CB
THR
245
7.753
51.261
−9.322


1942
OG1
THR
245
6.408
51.154
−9.773


1943
CG2
THR
245
7.993
50.395
−8.090


1944
N
ILE
246
8.102
53.390
−11.404


1945
CA
ILE
246
7.553
54.061
−12.580


1946
C
ILE
246
6.316
53.270
−12.997


1947
O
ILE
246
6.293
52.041
−12.856


1948
CB
ILE
246
8.628
54.096
−13.676


1949
CG1
ILE
246
8.171
54.732
−14.981


1950
CG2
ILE
246
9.174
52.718
−13.972


1951
CD1
ILE
246
9.132
54.376
−16.113


1952
N
ASP
247
5.279
53.986
−13.402


1953
CA
ASP
247
4.039
53.383
−13.896


1954
C
ASP
247
4.308
52.377
−15.009


1955
O
ASP
247
5.267
52.527
−15.779


1956
CB
ASP
247
3.147
54.502
−14.427


1957
CG
ASP
247
2.749
55.467
−13.310


1958
OD1
ASP
247
2.128
54.991
−12.366


1959
OD2
ASP
247
2.840
56.662
−13.541


1960
N
ALA
248
3.438
51.380
−15.101


1961
CA
ALA
248
3.611
50.306
−16.090


1962
C
ALA
248
3.529
50.806
−17.527


1963
O
ALA
248
3.340
51.993
−17.809


1964
CB
ALA
248
2.593
49.199
−15.859


1965
N
SER
249
3.581
49.850
−18.435


1966
CA
SER
249
3.740
50.162
−19.856


1967
C
SER
249
2.446
50.596
−20.559


1968
O
SER
249
2.511
51.133
−21.670


1969
CB
SER
249
4.307
48.915
−20.523


1970
OG
SER
249
5.542
48.597
−19.885


1971
N
GLU
250
1.304
50.432
−19.905


1972
CA
GLU
250
0.019
50.909
−20.456


1973
C
GLU
250
−1.002
51.186
−19.355


1974
O
GLU
250
−0.658
51.683
−18.276


1975
CB
GLU
250
−0.571
49.929
−21.469


1976
CG
GLU
250
−0.164
50.279
−22.900


1977
CD
GLU
250
−1.000
49.491
−23.905


1978
OE1
GLU
250
−1.835
50.112
−24.554


1979
OE2
GLU
250
−0.786
48.293
−24.016


1980
N
GLN
251
−2.217
50.708
−19.584


1981
CA
GLN
251
−3.372
51.025
−18.733


1982
C
GLN
251
−3.477
50.106
−17.508


1983
O
GLN
251
−4.315
50.335
−16.628


1984
CB
GLN
251
−4.651
50.863
−19.567


1985
CG
GLN
251
−4.639
51.611
−20.903


1986
CD
GLN
251
−4.285
50.695
−22.082


1987
OE1
GLN
251
−3.608
49.672
−21.912


1988
NE2
GLN
251
−4.668
51.120
−23.273


1989
N
ASP
252
−2.626
49.097
−17.431


1990
CA
ASP
252
−2.715
48.141
−16.325


1991
C
ASP
252
−1.649
48.402
−15.256


1992
O
ASP
252
−0.489
48.018
−15.433


1993
CB
ASP
252
−2.558
46.720
−16.879


1994
CG
ASP
252
−3.682
46.372
−17.859


1995
OD1
ASP
252
−3.495
46.622
−19.040


1996
OD2
ASP
252
−4.708
45.874
−17.412


1997
N
GLU
253
−2.091
48.954
−14.129


1998
CA
GLU
253
−1.274
49.118
−12.895


1999
C
GLU
253
−0.097
50.107
−12.980


2000
O
GLU
253
0.550
50.288
−14.014


2001
CB
GLU
253
−0.754
47.759
−12.432


2002
CG
GLU
253
−1.889
46.817
−12.056


2003
CD
GLU
253
−1.304
45.521
−11.509


2004
OE1
GLU
253
−0.324
45.616
−10.787


2005
OE2
GLU
253
−1.896
44.481
−11.744


2006
N
LYS
254
0.199
50.707
−11.839


2007
CA
LYS
254
1.275
51.702
−11.716


2008
C
LYS
254
2.643
51.130
−11.319


2009
O
LYS
254
3.604
51.894
−11.210


2010
CB
LYS
254
0.844
52.636
−10.595


2011
CG
LYS
254
−0.591
53.120
−10.787


2012
CD
LYS
254
−1.165
53.696
−9.496


2013
CE
LYS
254
−0.292
54.825
−8.948


2014
NZ
LYS
254
−0.827
55.336
−7.675


2015
N
LYS
255
2.766
49.820
−11.201


2016
CA
LYS
255
3.899
49.273
−10.454


2017
C
LYS
255
5.085
48.697
−11.240


2018
O
LYS
255
5.214
48.843
−12.465


2019
CB
LYS
255
3.376
48.278
−9.431


2020
CG
LYS
255
2.341
47.301
−9.973


2021
CD
LYS
255
1.806
46.481
−8.807


2022
CE
LYS
255
1.220
47.394
−7.737


2023
NZ
LYS
255
0.750
46.609
−6.588


2024
N
ILE
256
5.811
47.859
−10.507


2025
CA
ILE
256
7.219
47.514
−10.753


2026
C
ILE
256
7.590
46.641
−11.952


2027
O
ILE
256
8.773
46.295
−12.056


2028
CB
ILE
256
7.715
46.796
−9.505


2029
CG1
ILE
256
6.822
47.100
−8.314


2030
CG2
ILE
256
9.146
47.196
−9.189


2031
CD1
ILE
256
7.297
46.366
−7.070


2032
N
GLN
257
6.691
46.347
−12.874


2033
CA
GLN
257
7.097
45.508
−14.008


2034
C
GLN
257
7.784
46.349
−15.082


2035
O
GLN
257
8.588
45.842
−15.875


2036
CB
GLN
257
5.884
44.794
−14.585


2037
CG
GLN
257
6.274
43.770
−15.651


2038
CD
GLN
257
7.173
42.674
−15.067


2039
OE1
GLN
257
6.721
41.848
−14.265


2040
NE2
GLN
257
8.439
42.697
−15.453


2041
N
SER
258
7.581
47.653
−14.989


2042
CA
SER
258
8.335
48.606
−15.809


2043
C
SER
258
9.542
49.136
−15.030


2044
O
SER
258
10.278
50.004
−15.512


2045
CB
SER
258
7.426
49.763
−16.201


2046
OG
SER
258
7.018
50.418
−15.009


2047
N
SER
259
9.709
48.630
−13.818


2048
CA
SER
259
10.803
49.032
−12.936


2049
C
SER
259
11.765
47.883
−12.650


2050
O
SER
259
11.523
46.720
−13.004


2051
CB
SER
259
10.240
49.559
−11.625


2052
OG
SER
259
10.048
50.964
−11.730


2053
N
THR
260
12.703
48.198
−11.775


2054
CA
THR
260
13.889
47.360
−11.569


2055
C
THR
260
13.601
46.007
−10.923


2056
O
THR
260
14.057
44.977
−11.437


2057
CB
THR
260
14.847
48.123
−10.660


2058
OG1
THR
260
15.052
49.437
−11.164


2059
CG2
THR
260
16.196
47.426
−10.563


2060
N
ASN
261
12.685
45.976
−9.969


2061
CA
ASN
261
12.448
44.735
−9.225


2062
C
ASN
261
11.767
43.636
−10.042


2063
O
ASN
261
12.285
42.511
−10.073


2064
CB
ASN
261
11.611
45.067
−7.998


2065
CG
ASN
261
11.127
43.785
−7.332


2066
OD1
ASN
261
9.934
43.468
−7.377


2067
ND2
ASN
261
12.065
43.038
−6.776


2068
N
GLU
262
10.790
43.972
−10.868


2069
CA
GLU
262
10.139
42.896
−11.615


2070
C
GLU
262
10.860
42.596
−12.929


2071
O
GLU
262
10.653
41.528
−13.520


2072
CB
GLU
262
8.660
43.202
−11.792


2073
CG
GLU
262
7.957
43.200
−10.436


2074
CD
GLU
262
6.477
43.543
−10.577


2075
OE1
GLU
262
5.963
44.177
−9.664


2076
OE2
GLU
262
5.965
43.415
−11.677


2077
N
GLU
263
11.849
43.412
−13.259


2078
CA
GLU
263
12.761
43.053
−14.345


2079
C
GLU
263
13.798
42.052
−13.832


2080
O
GLU
263
14.015
41.023
−14.484


2081
CB
GLU
263
13.444
44.319
−14.853


2082
CG
GLU
263
12.446
45.224
−15.568


2083
CD
GLU
263
13.054
46.604
−15.803


2084
OE1
GLU
263
13.765
47.070
−14.922


2085
OE2
GLU
263
12.742
47.199
−16.824


2086
N
ALA
264
14.135
42.177
−12.557


2087
CA
ALA
264
15.094
41.271
−11.917


2088
C
ALA
264
14.513
39.890
−11.637


2089
O
ALA
264
15.186
38.877
−11.868


2090
CB
ALA
264
15.501
41.891
−10.590


2091
N
ARG
265
13.223
39.827
−11.348


2092
CA
ARG
265
12.617
38.513
−11.125


2093
C
ARG
265
12.258
37.835
−12.450


2094
O
ARG
265
12.271
36.599
−12.519


2095
CB
ARG
265
11.390
38.636
−10.227


2096
CG
ARG
265
10.212
39.303
−10.919


2097
CD
ARG
265
8.993
39.365
−10.012


2098
NE
ARG
265
7.803
39.721
−10.798


2099
CZ
ARG
265
6.556
39.541
−10.359


2100
NH1
ARG
265
6.345
39.072
−9.127


2101
NH2
ARG
265
5.523
39.858
−11.141


2102
N
ARG
266
12.237
38.605
−13.529


2103
CA
ARG
266
12.017
38.014
−14.842


2104
C
ARG
266
13.335
37.489
−15.406


2105
O
ARG
266
13.331
36.472
−16.107


2106
CB
ARG
266
11.416
39.059
−15.773


2107
CG
ARG
266
10.940
38.391
−17.054


2108
CD
ARG
266
9.936
37.296
−16.718


2109
NE
ARG
266
9.581
36.504
−17.903


2110
CZ
ARG
266
8.475
35.761
−17.969


2111
NH1
ARG
266
7.613
35.759
−16.950


2112
NH2
ARG
266
8.214
35.049
−19.067


2113
N
PHE
267
14.445
37.990
−14.885


2114
CA
PHE
267
15.753
37.431
−15.245


2115
C
PHE
267
16.045
36.157
−14.457


2116
O
PHE
267
16.666
35.233
−14.999


2117
CB
PHE
267
16.838
38.469
−14.991


2118
CG
PHE
267
16.772
39.655
−15.947


2119
CD1
PHE
267
16.557
39.440
−17.302


2120
CD2
PHE
267
16.941
40.947
−15.468


2121
CE1
PHE
267
16.490
40.517
−18.175


2122
CE2
PHE
267
16.871
42.024
−16.341


2123
CZ
PHE
267
16.644
41.810
−17.694


2124
N
PHE
268
15.372
35.999
−13.328


2125
CA
PHE
268
15.446
34.732
−12.597


2126
C
PHE
268
14.660
33.652
−13.342


2127
O
PHE
268
15.215
32.574
−13.603


2128
CB
PHE
268
14.879
34.930
−11.194


2129
CG
PHE
268
14.781
33.655
−10.360


2130
CD1
PHE
268
15.785
32.695
−10.418


2131
CD2
PHE
268
13.684
33.459
−9.532


2132
CE1
PHE
268
15.680
31.534
−9.664


2133
CE2
PHE
268
13.581
32.299
−8.774


2134
CZ
PHE
268
14.578
31.335
−8.843


2135
N
ARG
269
13.541
34.041
−13.935


2136
CA
ARG
269
12.760
33.094
−14.739


2137
C
ARG
269
13.407
32.810
−16.090


2138
O
ARG
269
13.390
31.658
−16.534


2139
CB
ARG
269
11.380
33.680
−14.980


2140
CG
ARG
269
10.653
33.926
−13.670


2141
CD
ARG
269
9.269
34.495
−13.935


2142
NE
ARG
269
8.568
34.781
−12.679


2143
CZ
ARG
269
8.099
35.992
−12.378


2144
NH1
ARG
269
8.280
37.008
−13.226


2145
NH2
ARG
269
7.463
36.188
−11.223


2146
N
ARG
270
14.210
33.745
−16.563


2147
CA
ARG
270
14.944
33.584
−17.818


2148
C
ARG
270
16.065
32.553
−17.676


2149
O
ARG
270
16.190
31.659
−18.530


2150
CB
ARG
270
15.523
34.958
−18.125


2151
CG
ARG
270
16.113
35.086
−19.518


2152
CD
ARG
270
16.642
36.501
−19.713


2153
NE
ARG
270
17.103
36.726
−21.089


2154
CZ
ARG
270
18.249
37.345
−21.379


2155
NH1
ARG
270
19.066
37.731
−20.396


2156
NH2
ARG
270
18.593
37.544
−22.652


2157
N
MET
271
16.655
32.497
−16.491


2158
CA
MET
271
17.662
31.472
−16.216


2159
C
MET
271
17.019
30.116
−15.931


2160
O
MET
271
17.491
29.106
−16.465


2161
CB
MET
271
18.490
31.903
−15.014


2162
CG
MET
271
19.241
33.199
−15.288


2163
SD
MET
271
20.310
33.770
−13.949


2164
CE
MET
271
19.076
33.895
−12.633


2165
N
GLU
272
15.811
30.135
−15.385


2166
CA
GLU
272
15.072
28.887
−15.122


2167
C
GLU
272
14.433
28.293
−16.381


2168
O
GLU
272
14.119
27.099
−16.411


2169
CB
GLU
272
13.954
29.183
−14.131


2170
CG
GLU
272
14.482
29.644
−12.782


2171
CD
GLU
272
13.310
30.058
−11.898


2172
OE1
GLU
272
12.888
29.243
−11.087


2173
OE2
GLU
272
12.882
31.198
−12.016


2174
N
ASP
273
14.308
29.102
−17.422


2175
CA
ASP
273
13.788
28.631
−18.708


2176
C
ASP
273
14.899
28.093
−19.605


2177
O
ASP
273
14.615
27.512
−20.660


2178
CB
ASP
273
13.097
29.792
−19.422


2179
CG
ASP
273
11.879
30.295
−18.645


2180
OD1
ASP
273
11.228
29.479
−18.007


2181
OD2
ASP
273
11.562
31.469
−18.792


2182
N
GLY
274
16.143
28.279
−19.194


2183
CA
GLY
274
17.272
27.761
−19.966


2184
C
GLY
274
17.712
28.731
−21.056


2185
O
GLY
274
18.101
28.306
−22.151


2186
N
ASP
275
17.584
30.020
−20.791


2187
CA
ASP
275
18.077
31.003
−21.757


2188
C
ASP
275
19.597
31.031
−21.673


2189
O
ASP
275
20.156
31.540
−20.693


2190
CB
ASP
275
17.486
32.370
−21.435


2191
CG
ASP
275
18.035
33.416
−22.397


2192
OD1
ASP
275
19.128
33.899
−22.121


2193
OD2
ASP
275
17.484
33.541
−23.478


2194
N
GLU
276
20.251
30.704
−22.776


2195
CA
GLU
276
21.697
30.457
−22.724


2196
C
GLU
276
22.549
31.719
−22.555


2197
O
GLU
276
23.612
31.595
−21.944


2198
CB
GLU
276
22.099
29.686
−23.984


2199
CG
GLU
276
23.405
28.895
−23.832


2200
CD
GLU
276
24.653
29.758
−24.029


2201
OE1
GLU
276
25.711
29.338
−23.584


2202
OE2
GLU
276
24.543
30.724
−24.774


2203
N
SER
277
22.033
32.908
−22.820


2204
CA
SER
277
22.856
34.099
−22.569


2205
C
SER
277
22.756
34.534
−21.105


2206
O
SER
277
23.772
34.924
−20.516


2207
CB
SER
277
22.442
35.234
−23.501


2208
OG
SER
277
21.097
35.590
−23.222


2209
N
ALA
278
21.660
34.155
−20.463


2210
CA
ALA
278
21.494
34.419
−19.035


2211
C
ALA
278
22.229
33.363
−18.215


2212
O
ALA
278
22.830
33.689
−17.184


2213
CB
ALA
278
20.007
34.394
−18.702


2214
N
LEU
279
22.396
32.189
−18.800


2215
CA
LEU
279
23.200
31.143
−18.158


2216
C
LEU
279
24.691
31.325
−18.445


2217
O
LEU
279
25.521
30.915
−17.626


2218
CB
LEU
279
22.765
29.752
−18.626


2219
CG
LEU
279
21.614
29.145
−17.815


2220
CD1
LEU
279
21.850
29.311
−16.319


2221
CD2
LEU
279
20.246
29.700
−18.186


2222
N
LYS
280
25.016
32.105
−19.462


2223
CA
LYS
280
26.416
32.396
−19.770


2224
C
LYS
280
26.968
33.452
−18.824


2225
O
LYS
280
28.032
33.238
−18.227


2226
CB
LYS
280
26.505
32.908
−21.202


2227
CG
LYS
280
27.930
33.315
−21.553


2228
CD
LYS
280
28.010
33.930
−22.944


2229
CE
LYS
280
29.423
34.419
−23.239


2230
NZ
LYS
280
29.848
35.413
−22.239


2231
N
ILE
281
26.141
34.428
−18.481


2232
CA
ILE
281
26.580
35.434
−17.510


2233
C
ILE
281
26.447
34.896
−16.080


2234
O
ILE
281
27.280
35.225
−15.222


2235
CB
ILE
281
25.794
36.730
−17.732


2236
CG1
ILE
281
26.248
37.830
−16.782


2237
CG2
ILE
281
24.288
36.527
−17.615


2238
CD1
ILE
281
25.448
39.110
−16.999


2239
N
TRP
282
25.631
33.863
−15.929


2240
CA
TRP
282
25.545
33.140
−14.665


2241
C
TRP
282
26.801
32.319
−14.408


2242
O
TRP
282
27.433
32.502
−13.360


2243
CB
TRP
282
24.324
32.229
−14.711


2244
CG
TRP
282
24.295
31.185
−13.615


2245
CD1
TRP
282
23.938
31.370
−12.299


2246
CD2
TRP
282
24.649
29.793
−13.755


2247
NE1
TRP
282
24.075
30.188
−11.650


2248
CE2
TRP
282
24.509
29.221
−12.481


2249
CE3
TRP
282
25.084
29.024
−14.825


2250
CZ2
TRP
282
24.822
27.884
−12.292


2251
CZ3
TRP
282
25.388
27.684
−14.628


2252
CH2
TRP
282
25.259
27.117
−13.366


2253
N
ALA
283
27.296
31.658
−15.442


2254
CA
ALA
283
28.496
30.835
−15.295


2255
C
ALA
283
29.746
31.692
−15.195


2256
O
ALA
283
30.621
31.390
−14.373


2257
CB
ALA
283
28.606
29.908
−16.500


2258
N
ARG
284
29.701
32.871
−15.793


2259
CA
ARG
284
30.822
33.798
−15.679


2260
C
ARG
284
30.975
34.293
−14.245


2261
O
ARG
284
32.001
33.986
−13.620


2262
CB
ARG
284
30.588
34.983
−16.605


2263
CG
ARG
284
31.786
35.922
−16.563


2264
CD
ARG
284
31.599
37.138
−17.458


2265
NE
ARG
284
32.802
37.980
−17.401


2266
CZ
ARG
284
32.828
39.261
−17.770


2267
NH1
ARG
284
31.712
39.855
−18.199


2268
NH2
ARG
284
33.964
39.956
−17.678


2269
N
PHE
285
29.883
34.745
−13.646


2270
CA
PHE
285
29.971
35.288
−12.283


2271
C
PHE
285
30.172
34.191
−11.247


2272
O
PHE
285
31.019
34.346
−10.358


2273
CB
PHE
285
28.692
36.039
−11.930


2274
CG
PHE
285
28.431
37.335
−12.692


2275
CD1
PHE
285
29.466
38.010
−13.326


2276
CD2
PHE
285
27.141
37.848
−12.731


2277
CE1
PHE
285
29.206
39.188
−14.011


2278
CE2
PHE
285
26.882
39.029
−13.412


2279
CZ
PHE
285
27.916
39.697
−14.054


2280
N
ARG
286
29.615
33.022
−11.513


2281
CA
ARG
286
29.772
31.896
−10.596


2282
C
ARG
286
31.199
31.357
−10.597


2283
O
ARG
286
31.797
31.281
−9.519


2284
CB
ARG
286
28.809
30.795
−11.018


2285
CG
ARG
286
29.015
29.538
−10.186


2286
CD
ARG
286
28.073
28.421
−10.616


2287
NE
ARG
286
28.404
27.177
−9.907


2288
CZ
ARG
286
27.740
26.727
−8.840


2289
NH1
ARG
286
26.653
27.366
−8.406


2290
NH2
ARG
286
28.132
25.602
−8.244


2291
N
ASP
287
31.830
31.309
−11.760


2292
CA
ASP
287
33.194
30.773
−11.840


2293
C
ASP
287
34.221
31.777
−11.328


2294
O
ASP
287
35.159
31.382
−10.623


2295
CB
ASP
287
33.519
30.424
−13.290


2296
CG
ASP
287
32.609
29.315
−13.818


2297
OD1
ASP
287
32.145
28.519
−13.010


2298
OD2
ASP
287
32.496
29.204
−15.032


2299
N
LEU
288
33.906
33.056
−11.446


2300
CA
LEU
288
34.803
34.089
−10.925


2301
C
LEU
288
34.719
34.179
−9.403


2302
O
LEU
288
35.761
34.229
−8.732


2303
CB
LEU
288
34.400
35.421
−11.542


2304
CG
LEU
288
34.703
35.485
−13.034


2305
CD1
LEU
288
34.171
36.781
−13.637


2306
CD2
LEU
288
36.198
35.342
−13.298


2307
N
SER
289
33.535
33.918
−8.872


2308
CA
SER
289
33.354
33.907
−7.423


2309
C
SER
289
33.943
32.643
−6.810


2310
O
SER
289
34.650
32.747
−5.802


2311
CB
SER
289
31.865
33.979
−7.104


2312
OG
SER
289
31.363
35.215
−7.596


2313
N
ILE
290
33.913
31.548
−7.554


2314
CA
ILE
290
34.508
30.297
−7.076


2315
C
ILE
290
36.033
30.342
−7.075


2316
O
ILE
290
36.624
29.885
−6.091


2317
CB
ILE
290
34.026
29.143
−7.954


2318
CG1
ILE
290
32.544
28.873
−7.731


2319
CG2
ILE
290
34.834
27.874
−7.700


2320
CD1
ILE
290
32.059
27.716
−8.597


2321
N
GLU
291
36.634
31.141
−7.944


2322
CA
GLU
291
38.099
31.248
−7.926


2323
C
GLU
291
38.570
32.087
−6.741


2324
O
GLU
291
39.475
31.655
−6.013


2325
CB
GLU
291
38.589
31.884
−9.223


2326
CG
GLU
291
38.205
31.065
−10.451


2327
CD
GLU
291
38.737
29.637
−10.361


2328
OE1
GLU
291
39.881
29.435
−10.744


2329
OE2
GLU
291
37.947
28.759
−10.040


2330
N
LYS
292
37.767
33.071
−6.373


2331
CA
LYS
292
38.105
33.897
−5.212


2332
C
LYS
292
37.791
33.171
−3.907


2333
O
LYS
292
38.585
33.256
−2.960


2334
CB
LYS
292
37.318
35.198
−5.289


2335
CG
LYS
292
37.718
36.011
−6.513


2336
CD
LYS
292
39.191
36.402
−6.452


2337
CE
LYS
292
39.620
37.156
−7.705


2338
NZ
LYS
292
41.040
37.530
−7.635


2339
N
TYR
293
36.840
32.252
−3.960


2340
CA
TYR
293
36.530
31.433
−2.788


2341
C
TYR
293
37.573
30.338
−2.591


2342
O
TYR
293
38.011
30.151
−1.451


2343
CB
TYR
293
35.140
30.820
−2.935


2344
CG
TYR
293
33.994
31.826
−2.845


2345
CD1
TYR
293
34.131
32.964
−2.059


2346
CD2
TYR
293
32.810
31.599
−3.537


2347
CE1
TYR
293
33.095
33.885
−1.980


2348
CE2
TYR
293
31.772
32.520
−3.460


2349
CZ
TYR
293
31.919
33.662
−2.683


2350
OH
TYR
293
30.893
34.579
−2.600


2351
N
VAL
294
38.191
29.885
−3.671


2352
CA
VAL
294
39.294
28.923
−3.557


2353
C
VAL
294
40.534
29.583
−2.960


2354
O
VAL
294
41.151
28.999
−2.062


2355
CB
VAL
294
39.633
28.373
−4.942


2356
CG1
VAL
294
40.928
27.569
−4.926


2357
CG2
VAL
294
38.497
27.530
−5.508


2358
N
ASP
295
40.691
30.875
−3.207


2359
CA
ASP
295
41.806
31.621
−2.615


2360
C
ASP
295
41.577
31.871
−1.122


2361
O
ASP
295
42.481
31.628
−0.309


2362
CB
ASP
295
41.911
32.972
−3.319


2363
CG
ASP
295
42.087
32.811
−4.827


2364
OD1
ASP
295
42.836
31.928
−5.225


2365
OD2
ASP
295
41.594
33.673
−5.544


2366
N
THR
296
40.322
32.081
−0.758


2367
CA
THR
296
39.976
32.381
0.635


2368
C
THR
296
40.022
31.133
1.510


2369
O
THR
296
40.682
31.147
2.558


2370
CB
THR
296
38.566
32.961
0.656


2371
OG1
THR
296
38.550
34.119
−0.168


2372
CG2
THR
296
38.142
33.370
2.062


2373
N
TYR
297
39.567
30.017
0.966


2374
CA
TYR
297
39.586
28.762
1.720


2375
C
TYR
297
40.987
28.157
1.730


2376
O
TYR
297
41.407
27.625
2.766


2377
CB
TYR
297
38.602
27.786
1.090


2378
CG
TYR
297
37.138
28.218
1.126


2379
CD1
TYR
297
36.536
28.614
2.315


2380
CD2
TYR
297
36.401
28.200
−0.050


2381
CE1
TYR
297
35.197
28.987
2.324


2382
CE2
TYR
297
35.067
28.578
−0.044


2383
CZ
TYR
297
34.464
28.962
1.144


2384
OH
TYR
297
33.096
29.129
1.171


2385
N
GLY
298
41.778
28.497
0.726


2386
CA
GLY
298
43.201
28.143
0.699


2387
C
GLY
298
43.943
28.776
1.873


2388
O
GLY
298
44.531
28.047
2.683


2389
N
ARG
299
43.678
30.054
2.109


2390
CA
ARG
299
44.300
30.791
3.223


2391
C
ARG
299
43.761
30.377
4.606


2392
O
ARG
299
44.431
30.589
5.623


2393
CB
ARG
299
44.011
32.273
2.982


2394
CG
ARG
299
44.687
33.182
4.003


2395
CD
ARG
299
44.338
34.649
3.771


2396
NE
ARG
299
45.017
35.507
4.756


2397
CZ
ARG
299
44.679
36.777
4.991


2398
NH1
ARG
299
43.663
37.335
4.329


2399
NH2
ARG
299
45.349
37.485
5.903


2400
N
LEU
300
42.632
29.686
4.629


2401
CA
LEU
300
42.065
29.166
5.879


2402
C
LEU
300
42.364
27.677
6.104


2403
O
LEU
300
41.808
27.086
7.040


2404
CB
LEU
300
40.558
29.394
5.842


2405
CG
LEU
300
40.236
30.884
5.812


2406
CD1
LEU
300
38.763
31.132
5.508


2407
CD2
LEU
300
40.648
31.567
7.113


2408
N
ASN
301
43.192
27.090
5.246


2409
CA
ASN
301
43.563
25.659
5.299


2410
C
ASN
301
42.369
24.742
5.043


2411
O
ASN
301
42.202
23.713
5.711


2412
CB
ASN
301
44.170
25.298
6.653


2413
CG
ASN
301
45.526
25.961
6.875


2414
OD1
ASN
301
46.369
26.023
5.973


2415
ND2
ASN
301
45.720
26.434
8.092


2416
N
ILE
302
41.565
25.101
4.056


2417
CA
ILE
302
40.360
24.332
3.725


2418
C
ILE
302
40.234
24.096
2.220


2419
O
ILE
302
40.069
25.031
1.426


2420
CB
ILE
302
39.143
25.097
4.245


2421
CG1
ILE
302
39.136
25.130
5.769


2422
CG2
ILE
302
37.843
24.496
3.724


2423
CD1
ILE
302
37.935
25.890
6.308


2424
N
LYS
303
40.307
22.833
1.837


2425
CA
LYS
303
40.127
22.474
0.427


2426
C
LYS
303
38.859
21.645
0.230


2427
O
LYS
303
38.804
20.470
0.607


2428
CB
LYS
303
41.344
21.686
−0.044


2429
CG
LYS
303
41.248
21.356
−1.530


2430
CD
LYS
303
42.470
20.584
−2.012


2431
CE
LYS
303
42.370
20.269
−3.500


2432
NZ
LYS
303
43.557
19.533
−3.964


2433
N
TYR
304
37.854
22.268
−0.363


2434
CA
TYR
304
36.589
21.579
−0.654


2435
C
TYR
304
36.756
20.510
−1.719


2436
O
TYR
304
37.715
20.513
−2.498


2437
CB
TYR
304
35.547
22.568
−1.156


2438
CG
TYR
304
34.916
23.439
−0.084


2439
CD1
TYR
304
33.768
23.006
0.566


2440
CD2
TYR
304
35.487
24.659
0.248


2441
CE1
TYR
304
33.175
23.805
1.535


2442
CE2
TYR
304
34.898
25.454
1.217


2443
CZ
TYR
304
33.740
25.032
1.849


2444
OH
TYR
304
33.065
25.911
2.661


2445
N
ASP
305
35.822
19.578
−1.722


2446
CA
ASP
305
35.821
18.547
−2.754


2447
C
ASP
305
34.779
18.912
−3.799


2448
O
ASP
305
34.976
18.702
−5.002


2449
CB
ASP
305
35.466
17.200
−2.130


2450
CG
ASP
305
36.446
16.833
−1.018


2451
OD1
ASP
305
37.530
16.372
−1.341


2452
OD2
ASP
305
36.067
16.981
0.138


2453
N
VAL
306
33.692
19.503
−3.328


2454
CA
VAL
306
32.603
19.912
−4.221


2455
C
VAL
306
32.160
21.348
−3.940


2456
O
VAL
306
31.820
21.704
−2.805


2457
CB
VAL
306
31.417
18.967
−4.005


2458
CG1
VAL
306
30.240
19.311
−4.912


2459
CG2
VAL
306
31.805
17.504
−4.197


2460
N
TYR
307
32.211
22.181
−4.965


2461
CA
TYR
307
31.607
23.516
−4.874


2462
C
TYR
307
30.219
23.490
−5.507


2463
O
TYR
307
30.073
23.751
−6.704


2464
CB
TYR
307
32.475
24.544
−5.595


2465
CG
TYR
307
33.672
25.065
−4.803


2466
CD1
TYR
307
34.899
24.415
−4.863


2467
CD2
TYR
307
33.530
26.210
−4.029


2468
CE1
TYR
307
35.981
24.906
−4.142


2469
CE2
TYR
307
34.610
26.703
−3.309


2470
CZ
TYR
307
35.833
26.048
−3.367


2471
OH
TYR
307
36.911
26.548
−2.668


2472
N
SER
308
29.227
23.135
−4.709


2473
CA
SER
308
27.843
23.047
−5.182


2474
C
SER
308
27.129
24.369
−4.892


2475
O
SER
308
27.703
25.265
−4.260


2476
CB
SER
308
27.182
21.876
−4.451


2477
OG
SER
308
25.904
21.602
−5.008


2478
N
GLY
309
25.961
24.545
−5.482


2479
CA
GLY
309
25.168
25.752
−5.255


2480
C
GLY
309
23.675
25.458
−5.286


2481
O
GLY
309
23.248
24.332
−5.568


2482
N
GLU
310
22.895
26.520
−5.168


2483
CA
GLU
310
21.428
26.414
−5.186


2484
C
GLU
310
20.883
26.175
−6.590


2485
O
GLU
310
19.761
25.686
−6.747


2486
CB
GLU
310
20.855
27.731
−4.694


2487
CG
GLU
310
21.227
28.032
−3.253


2488
CD
GLU
310
20.962
29.508
−3.010


2489
OE1
GLU
310
20.669
29.874
−1.884


2490
OE2
GLU
310
21.166
30.261
−3.959


2491
N
SER
311
21.711
26.430
−7.589


2492
CA
SER
311
21.347
26.150
−8.982


2493
C
SER
311
21.751
24.753
−9.440


2494
O
SER
311
21.719
24.491
−10.646


2495
CB
SER
311
22.110
27.114
−9.873


2496
OG
SER
311
23.481
26.731
−9.803


2497
N
GLN
312
22.293
23.938
−8.549


2498
CA
GLN
312
22.845
22.657
−8.993


2499
C
GLN
312
21.900
21.491
−8.731


2500
O
GLN
312
22.186
20.357
−9.134


2501
CB
GLN
312
24.190
22.458
−8.314


2502
CG
GLN
312
25.134
23.606
−8.660


2503
CD
GLN
312
25.557
23.575
−10.127


2504
OE1
GLN
312
26.316
22.690
−10.534


2505
NE2
GLN
312
25.135
24.576
−10.883


2506
N
VAL
313
20.785
21.776
−8.079


2507
CA
VAL
313
19.724
20.778
−7.923


2508
C
VAL
313
19.108
20.436
−9.281


2509
O
VAL
313
18.610
21.311
−9.998


2510
CB
VAL
313
18.675
21.357
−6.974


2511
CG1
VAL
313
18.386
22.825
−7.267


2512
CG2
VAL
313
17.392
20.539
−6.974


2513
N
PRO
314
19.197
19.167
−9.644


2514
CA
PRO
314
18.667
18.711
−10.926


2515
C
PRO
314
17.151
18.832
−10.967


2516
O
PRO
314
16.461
18.601
−9.965


2517
CB
PRO
314
19.108
17.286
−11.057


2518
CG
PRO
314
19.831
16.872
−9.786


2519
CD
PRO
314
19.817
18.088
−8.874


2520
N
GLN
315
16.639
18.971
−12.178


2521
CA
GLN
315
15.198
19.143
−12.382


2522
C
GLN
315
14.444
17.828
−12.194


2523
O
GLN
315
13.300
17.839
−11.726


2524
CB
GLN
315
14.990
19.674
−13.795


2525
CG
GLN
315
13.533
20.020
−14.081


2526
CD
GLN
315
13.423
20.598
−15.487


2527
OE1
GLN
315
13.627
19.891
−16.480


2528
NE2
GLN
315
13.168
21.894
−15.552


2529
N
GLU
316
15.185
16.732
−12.229


2530
CA
GLU
316
14.612
15.410
−11.968


2531
C
GLU
316
14.357
15.221
−10.476


2532
O
GLU
316
13.291
14.707
−10.119


2533
CB
GLU
316
15.557
14.302
−12.449


2534
CG
GLU
316
15.646
14.137
−13.971


2535
CD
GLU
316
16.639
15.108
−14.614


2536
OE1
GLU
316
17.383
15.731
−13.864


2537
OE2
GLU
316
16.501
15.354
−15.802


2538
N
LYS
317
15.134
15.907
−9.649


2539
CA
LYS
317
14.937
15.824
−8.200


2540
C
LYS
317
13.823
16.766
−7.778


2541
O
LYS
317
13.030
16.424
−6.894


2542
CB
LYS
317
16.222
16.224
−7.486


2543
CG
LYS
317
17.370
15.286
−7.827


2544
CD
LYS
317
17.070
13.856
−7.407


2545
CE
LYS
317
18.216
12.923
−7.769


2546
NZ
LYS
317
17.894
11.540
−7.391


2547
N
MET
318
13.600
17.790
−8.585


2548
CA
MET
318
12.492
18.709
−8.330


2549
C
MET
318
11.157
18.097
−8.743


2550
O
MET
318
10.191
18.185
−7.973


2551
CB
MET
318
12.734
19.994
−9.112


2552
CG
MET
318
13.965
20.726
−8.595


2553
SD
MET
318
13.876
21.238
−6.864


2554
CE
MET
318
12.398
22.270
−6.956


2555
N
LYS
319
11.166
17.280
−9.785


2556
CA
LYS
319
9.929
16.604
−10.185


2557
C
LYS
319
9.619
15.413
−9.285


2558
O
LYS
319
8.448
15.200
−8.950


2559
CB
LYS
319
10.038
16.132
−11.631


2560
CG
LYS
319
10.195
17.299
−12.600


2561
CD
LYS
319
9.966
16.856
−14.040


2562
CE
LYS
319
10.970
15.810
−14.502


2563
NZ
LYS
319
12.314
16.383
−14.646


2564
N
GLU
320
10.647
14.819
−8.700


2565
CA
GLU
320
10.427
13.722
−7.754


2566
C
GLU
320
10.013
14.243
−6.382


2567
O
GLU
320
9.134
13.646
−5.746


2568
CB
GLU
320
11.717
12.923
−7.639


2569
CG
GLU
320
12.021
12.204
−8.947


2570
CD
GLU
320
13.468
11.725
−8.957


2571
OE1
GLU
320
13.752
10.783
−9.684


2572
OE2
GLU
320
14.285
12.377
−8.320


2573
N
ALA
321
10.432
15.456
−6.065


2574
CA
ALA
321
10.007
16.090
−4.820


2575
C
ALA
321
8.560
16.545
−4.918


2576
O
ALA
321
7.766
16.195
−4.037


2577
CB
ALA
321
10.904
17.291
−4.543


2578
N
THR
322
8.167
17.009
−6.093


2579
CA
THR
322
6.783
17.445
−6.305


2580
C
THR
322
5.831
16.256
−6.388


2581
O
THR
322
4.762
16.286
−5.761


2582
CB
THR
322
6.733
18.238
−7.607


2583
OG1
THR
322
7.573
19.376
−7.461


2584
CG2
THR
322
5.324
18.726
−7.922


2585
N
LYS
323
6.337
15.144
−6.896


2586
CA
LYS
323
5.539
13.925
−6.968


2587
C
LYS
323
5.339
13.319
−5.585


2588
O
LYS
323
4.191
13.046
−5.215


2589
CB
LYS
323
6.259
12.931
−7.873


2590
CG
LYS
323
5.420
11.684
−8.120


2591
CD
LYS
323
4.118
12.030
−8.834


2592
CE
LYS
323
4.379
12.662
−10.198


2593
NZ
LYS
323
3.111
13.012
−10.856


2594
N
LEU
324
6.348
13.404
−4.733


2595
CA
LEU
324
6.186
12.889
−3.372


2596
C
LEU
324
5.424
13.847
−2.459


2597
O
LEU
324
4.683
13.360
−1.598


2598
CB
LEU
324
7.551
12.564
−2.785


2599
CG
LEU
324
8.149
11.339
−3.465


2600
CD1
LEU
324
9.565
11.068
−2.973


2601
CD2
LEU
324
7.263
10.116
−3.250


2602
N
PHE
325
5.344
15.119
−2.818


2603
CA
PHE
325
4.493
16.039
−2.052


2604
C
PHE
325
3.029
15.759
−2.368


2605
O
PHE
325
2.176
15.768
−1.471


2606
CB
PHE
325
4.777
17.493
−2.427


2607
CG
PHE
325
6.188
18.031
−2.194


2608
CD1
PHE
325
7.023
17.476
−1.233


2609
CD2
PHE
325
6.632
19.103
−2.958


2610
CE1
PHE
325
8.302
17.984
−1.048


2611
CE2
PHE
325
7.910
19.613
−2.771


2612
CZ
PHE
325
8.747
19.051
−1.817


2613
N
GLU
326
2.779
15.299
−3.580


2614
CA
GLU
326
1.421
14.926
−3.962


2615
C
GLU
326
1.046
13.556
−3.401


2616
O
GLU
326
0.045
13.460
−2.681


2617
CB
GLU
326
1.372
14.915
−5.483


2618
CG
GLU
326
1.683
16.306
−6.022


2619
CD
GLU
326
2.065
16.242
−7.496


2620
OE1
GLU
326
1.879
17.241
−8.176


2621
OE2
GLU
326
2.643
15.238
−7.889


2622
N
ASP
327
1.994
12.631
−3.441


2623
CA
ASP
327
1.739
11.242
−3.025


2624
C
ASP
327
1.672
11.042
−1.512


2625
O
ASP
327
1.041
10.085
−1.051


2626
CB
ASP
327
2.874
10.360
−3.543


2627
CG
ASP
327
2.964
10.365
−5.067


2628
OD1
ASP
327
1.929
10.489
−5.707


2629
OD2
ASP
327
4.060
10.130
−5.561


2630
N
LYS
328
2.281
11.934
−0.749


2631
CA
LYS
328
2.233
11.811
0.710


2632
C
LYS
328
1.168
12.712
1.334


2633
O
LYS
328
0.959
12.661
2.552


2634
CB
LYS
328
3.603
12.166
1.274


2635
CG
LYS
328
4.698
11.258
0.724


2636
CD
LYS
328
4.523
9.810
1.166


2637
CE
LYS
328
5.625
8.930
0.588


2638
NZ
LYS
328
5.490
7.540
1.051


2639
N
GLY
329
0.501
13.513
0.517


2640
CA
GLY
329
−0.526
14.426
1.032


2641
C
GLY
329
0.111
15.614
1.745


2642
O
GLY
329
−0.266
15.969
2.868


2643
N
LEU
330
1.129
16.168
1.110


2644
CA
LEU
330
1.850
17.318
1.657


2645
C
LEU
330
1.503
18.577
0.872


2646
O
LEU
330
1.757
19.699
1.327


2647
CB
LEU
330
3.348
17.058
1.521


2648
CG
LEU
330
3.804
15.791
2.236


2649
CD1
LEU
330
5.270
15.495
1.933


2650
CD2
LEU
330
3.577
15.885
3.741


2651
N
ILE
331
0.912
18.372
−0.294


2652
CA
ILE
331
0.557
19.475
−1.199


2653
C
ILE
331
−0.649
20.289
−0.715


2654
O
ILE
331
−1.815
19.919
−0.894


2655
CB
ILE
331
0.300
18.875
−2.583


2656
CG1
ILE
331
−0.259
19.910
−3.555


2657
CG2
ILE
331
−0.607
17.650
−2.499


2658
CD1
ILE
331
−0.515
19.305
−4.929


2659
N
ASP
332
−0.346
21.415
−0.091


2660
CA
ASP
332
−1.394
22.330
0.367


2661
C
ASP
332
−1.303
23.655
−0.389


2662
O
ASP
332
−0.455
24.509
−0.098


2663
CB
ASP
332
−1.205
22.585
1.858


2664
CG
ASP
332
−1.082
21.277
2.639


2665
OD1
ASP
332
−1.913
20.407
2.428


2666
OD2
ASP
332
−0.271
21.250
3.556


2667
N
ILE
333
−2.175
23.817
−1.368


2668
CA
ILE
333
−2.166
25.045
−2.174


2669
C
ILE
333
−2.952
26.159
−1.486


2670
O
ILE
333
−4.188
26.202
−1.507


2671
CB
ILE
333
−2.752
24.740
−3.547


2672
CG1
ILE
333
−1.971
23.612
−4.211


2673
CG2
ILE
333
−2.735
25.982
−4.432


2674
CD1
ILE
333
−2.536
23.284
−5.589


2675
N
ASP
334
−2.205
27.087
−0.920


2676
CA
ASP
334
−2.784
28.203
−0.182


2677
C
ASP
334
−2.744
29.466
−1.031


2678
O
ASP
334
−1.737
30.187
−1.066


2679
CB
ASP
334
−1.976
28.407
1.090


2680
CG
ASP
334
−2.602
29.513
1.930


2681
OD1
ASP
334
−3.823
29.608
1.924


2682
OD2
ASP
334
−1.855
30.218
2.591


2683
N
ARG
335
−3.881
29.739
−1.655


2684
CA
ARG
335
−4.048
30.890
−2.557


2685
C
ARG
335
−2.899
30.974
−3.557


2686
O
ARG
335
−2.054
31.876
−3.481


2687
CB
ARG
335
−4.101
32.164
−1.719


2688
CG
ARG
335
−5.252
32.128
−0.719


2689
CD
ARG
335
−5.227
33.344
0.200


2690
NE
ARG
335
−5.306
34.591
−0.574


2691
CZ
ARG
335
−4.525
35.647
−0.335


2692
NH1
ARG
335
−3.609
35.590
0.633


2693
NH2
ARG
335
−4.648
36.750
−1.075


2694
N
GLY
336
−2.805
29.965
−4.408


2695
CA
GLY
336
−1.707
29.899
−5.379


2696
C
GLY
336
−0.463
29.196
−4.825


2697
O
GLY
336
−0.100
28.108
−5.286


2698
N
ALA
337
0.145
29.805
−3.818


2699
CA
ALA
337
1.413
29.322
−3.256


2700
C
ALA
337
1.284
27.938
−2.637


2701
O
ALA
337
0.373
27.659
−1.850


2702
CB
ALA
337
1.886
30.312
−2.200


2703
N
LYS
338
2.200
27.068
−3.012


2704
CA
LYS
338
2.147
25.694
−2.515


2705
C
LYS
338
3.027
25.532
−1.282


2706
O
LYS
338
4.239
25.790
−1.322


2707
CB
LYS
338
2.596
24.751
−3.625


2708
CG
LYS
338
2.580
23.293
−3.178


2709
CD
LYS
338
3.081
22.371
−4.283


2710
CE
LYS
338
3.222
20.939
−3.782


2711
NZ
LYS
338
3.623
20.034
−4.869


2712
N
LEU
339
2.394
25.175
−0.179


2713
CA
LEU
339
3.126
24.888
1.055


2714
C
LEU
339
2.826
23.491
1.590


2715
O
LEU
339
1.866
22.829
1.179


2716
CB
LEU
339
2.825
25.967
2.099


2717
CG
LEU
339
1.342
26.285
2.296


2718
CD1
LEU
339
0.653
25.276
3.208


2719
CD2
LEU
339
1.193
27.673
2.908


2720
N
ILE
340
3.744
23.009
2.403


2721
CA
ILE
340
3.548
21.755
3.130


2722
C
ILE
340
3.309
22.043
4.606


2723
O
ILE
340
4.138
22.689
5.259


2724
CB
ILE
340
4.807
20.912
2.974


2725
CG1
ILE
340
5.064
20.594
1.510


2726
CG2
ILE
340
4.717
19.624
3.784


2727
CD1
ILE
340
6.291
19.707
1.372


2728
N
ASP
341
2.160
21.636
5.116


2729
CA
ASP
341
1.895
21.817
6.544


2730
C
ASP
341
2.617
20.746
7.355


2731
O
ASP
341
2.135
19.619
7.526


2732
CB
ASP
341
0.394
21.759
6.810


2733
CG
ASP
341
0.121
22.057
8.283


2734
OD1
ASP
341
0.179
21.125
9.073


2735
OD2
ASP
341
0.005
23.228
8.613


2736
N
LEU
342
3.685
21.181
7.997


2737
CA
LEU
342
4.512
20.298
8.812


2738
C
LEU
342
4.035
20.279
10.254


2739
O
LEU
342
4.388
19.352
10.991


2740
CB
LEU
342
5.951
20.776
8.740


2741
CG
LEU
342
6.511
20.610
7.335


2742
CD1
LEU
342
7.861
21.297
7.212


2743
CD2
LEU
342
6.615
19.135
6.960


2744
N
THR
343
3.045
21.111
10.550


2745
CA
THR
343
2.394
21.155
11.870


2746
C
THR
343
1.688
19.853
12.261


2747
O
THR
343
1.737
19.485
13.442


2748
CB
THR
343
1.369
22.287
11.843


2749
OG1
THR
343
2.081
23.510
11.793


2750
CG2
THR
343
0.490
22.322
13.088


2751
N
LYS
344
1.325
19.041
11.278


2752
CA
LYS
344
0.710
17.734
11.551


2753
C
LYS
344
1.712
16.672
12.022


2754
O
LYS
344
1.298
15.629
12.541


2755
CB
LYS
344
0.087
17.244
10.251


2756
CG
LYS
344
−0.916
18.247
9.700


2757
CD
LYS
344
−1.380
17.851
8.304


2758
CE
LYS
344
−2.330
18.891
7.721


2759
NZ
LYS
344
−2.728
18.529
6.352


2760
N
PHE
345
3.000
16.934
11.866


2761
CA
PHE
345
4.031
15.988
12.304


2762
C
PHE
345
4.845
16.634
13.419


2763
O
PHE
345
5.521
15.974
14.218


2764
CB
PHE
345
4.944
15.702
11.114


2765
CG
PHE
345
4.203
15.453
9.801


2766
CD1
PHE
345
3.420
14.317
9.642


2767
CD2
PHE
345
4.309
16.374
8.765


2768
CE1
PHE
345
2.740
14.104
8.450


2769
CE2
PHE
345
3.629
16.160
7.574


2770
CZ
PHE
345
2.843
15.026
7.416


2771
N
ASN
346
4.792
17.953
13.402


2772
CA
ASN
346
5.455
18.811
14.377


2773
C
ASN
346
4.862
20.207
14.246


2774
O
ASN
346
5.198
20.946
13.307


2775
CB
ASN
346
6.944
18.855
14.061


2776
CG
ASN
346
7.676
19.682
15.110


2777
OD1
ASN
346
7.564
20.915
15.148


2778
ND2
ASN
346
8.368
18.984
15.990


2779
N
LYS
347
4.214
20.653
15.308


2780
CA
LYS
347
3.437
21.902
15.267


2781
C
LYS
347
4.289
23.169
15.169


2782
O
LYS
347
3.856
24.138
14.530


2783
CB
LYS
347
2.573
21.999
16.526


2784
CG
LYS
347
1.593
20.835
16.676


2785
CD
LYS
347
2.066
19.801
17.697


2786
CE
LYS
347
1.115
18.614
17.774


2787
NZ
LYS
347
1.596
17.622
18.748


2788
N
LYS
348
5.563
23.050
15.504


2789
CA
LYS
348
6.468
24.198
15.526


2790
C
LYS
348
7.117
24.402
14.160


2791
O
LYS
348
7.695
25.463
13.902


2792
CB
LYS
348
7.560
23.992
16.583


2793
CG
LYS
348
7.027
23.899
18.016


2794
CD
LYS
348
6.750
22.464
18.468


2795
CE
LYS
348
6.072
22.423
19.831


2796
NZ
LYS
348
5.731
21.040
20.200


2797
N
LEU
349
6.926
23.443
13.265


2798
CA
LEU
349
7.428
23.575
11.896


2799
C
LEU
349
6.404
24.224
10.964


2800
O
LEU
349
6.667
24.337
9.757


2801
CB
LEU
349
7.832
22.205
11.370


2802
CG
LEU
349
9.005
21.635
12.157


2803
CD1
LEU
349
9.395
20.262
11.628


2804
CD2
LEU
349
10.202
22.577
12.118


2805
N
GLY
350
5.243
24.568
11.508


2806
CA
GLY
350
4.234
25.388
10.813


2807
C
GLY
350
3.925
24.936
9.392


2808
O
GLY
350
3.595
23.770
9.140


2809
N
LYS
351
3.988
25.892
8.482


2810
CA
LYS
351
3.848
25.605
7.052


2811
C
LYS
351
5.113
26.013
6.302


2812
O
LYS
351
5.635
27.121
6.477


2813
CB
LYS
351
2.648
26.364
6.499


2814
CG
LYS
351
1.346
25.877
7.123


2815
CD
LYS
351
0.146
26.632
6.566


2816
CE
LYS
351
−1.164
26.083
7.116


2817
NZ
LYS
351
−2.314
26.791
6.531


2818
N
ALA
352
5.608
25.099
5.491


2819
CA
ALA
352
6.815
25.353
4.704


2820
C
ALA
352
6.495
25.555
3.225


2821
O
ALA
352
6.081
24.616
2.535


2822
CB
ALA
352
7.741
24.157
4.876


2823
N
LEU
353
6.661
26.782
2.761


2824
CA
LEU
353
6.421
27.101
1.346


2825
C
LEU
353
7.470
26.469
0.441


2826
O
LEU
353
8.671
26.739
0.556


2827
CB
LEU
353
6.447
28.614
1.168


2828
CG
LEU
353
5.121
29.258
1.551


2829
CD1
LEU
353
5.264
30.770
1.674


2830
CD2
LEU
353
4.045
28.902
0.532


2831
N
VAL
354
6.991
25.661
−0.487


2832
CA
VAL
354
7.896
24.979
−1.401


2833
C
VAL
354
7.985
25.713
−2.739


2834
O
VAL
354
9.043
25.695
−3.387


2835
CB
VAL
354
7.455
23.519
−1.549


2836
CG1
VAL
354
7.671
22.757
−0.248


2837
CG2
VAL
354
6.019
23.352
−2.022


2838
N
GLU
355
6.958
26.483
−3.062


2839
CA
GLU
355
7.023
27.331
−4.261


2840
C
GLU
355
6.050
28.505
−4.190


2841
O
GLU
355
4.911
28.387
−3.714


2842
CB
GLU
355
6.784
26.512
−5.529


2843
CG
GLU
355
5.403
25.880
−5.582


2844
CD
GLU
355
5.231
25.111
−6.890


2845
OE1
GLU
355
4.449
25.573
−7.709


2846
OE2
GLU
355
5.672
23.970
−6.923


2847
N
LYS
356
6.501
29.605
−4.767


2848
CA
LYS
356
5.758
30.871
−4.774


2849
C
LYS
356
4.519
30.751
−5.672


2850
O
LYS
356
4.425
29.818
−6.478


2851
CB
LYS
356
6.725
31.937
−5.292


2852
CG
LYS
356
6.426
33.332
−4.749


2853
CD
LYS
356
7.414
34.361
−5.286


2854
CE
LYS
356
7.050
35.766
−4.821


2855
NZ
LYS
356
7.962
36.764
−5.402


2856
N
SER
357
3.649
31.753
−5.633


2857
CA
SER
357
2.371
31.720
−6.375


2858
C
SER
357
2.515
31.889
−7.891


2859
O
SER
357
1.539
31.728
−8.629


2860
CB
SER
357
1.488
32.854
−5.865


2861
OG
SER
357
1.246
32.657
−4.480


2862
N
ASP
358
3.713
32.214
−8.348


2863
CA
ASP
358
3.986
32.292
−9.783


2864
C
ASP
358
4.672
31.024
−10.302


2865
O
ASP
358
5.130
31.007
−11.450


2866
CB
ASP
358
4.852
33.518
−10.064


2867
CG
ASP
358
6.145
33.461
−9.257


2868
OD1
ASP
358
7.033
32.723
−9.660


2869
OD2
ASP
358
6.171
34.051
−8.187


2870
N
GLY
359
4.868
30.037
−9.441


2871
CA
GLY
359
5.476
28.781
−9.888


2872
C
GLY
359
6.869
28.533
−9.313


2873
O
GLY
359
7.139
27.431
−8.817


2874
N
THR
360
7.729
29.542
−9.391


2875
CA
THR
360
9.144
29.406
−8.995


2876
C
THR
360
9.338
28.760
−7.629


2877
O
THR
360
8.721
29.144
−6.624


2878
CB
THR
360
9.811
30.777
−8.984


2879
OG1
THR
360
9.096
31.613
−8.081


2880
CG2
THR
360
9.802
31.423
−10.365


2881
N
SER
361
10.176
27.738
−7.640


2882
CA
SER
361
10.493
26.981
−6.429


2883
C
SER
361
11.337
27.826
−5.493


2884
O
SER
361
12.068
28.727
−5.921


2885
CB
SER
361
11.273
25.731
−6.805


2886
OG
SER
361
12.563
26.141
−7.238


2887
N
LEU
362
11.184
27.565
−4.212


2888
CA
LEU
362
11.913
28.345
−3.214


2889
C
LEU
362
13.196
27.656
−2.771


2890
O
LEU
362
13.479
26.506
−3.130


2891
CB
LEU
362
10.996
28.596
−2.025


2892
CG
LEU
362
9.801
29.448
−2.440


2893
CD1
LEU
362
8.837
29.644
−1.279


2894
CD2
LEU
362
10.248
30.800
−2.990


2895
N
TYR
363
13.877
28.321
−1.851


2896
CA
TYR
363
15.153
27.842
−1.299


2897
C
TYR
363
14.995
26.539
−0.513


2898
O
TYR
363
15.790
25.613
−0.716


2899
CB
TYR
363
15.662
28.947
−0.379


2900
CG
TYR
363
16.960
28.650
0.362


2901
CD1
TYR
363
18.050
28.113
−0.312


2902
CD2
TYR
363
17.056
28.949
1.715


2903
CE1
TYR
363
19.224
27.844
0.377


2904
CE2
TYR
363
18.230
28.681
2.405


2905
CZ
TYR
363
19.309
28.120
1.735


2906
OH
TYR
363
20.427
27.737
2.442


2907
N
LEU
364
13.807
26.356
0.044


2908
CA
LEU
364
13.452
25.125
0.753


2909
C
LEU
364
13.484
23.900
−0.165


2910
O
LEU
364
14.182
22.929
0.157


2911
CB
LEU
364
12.031
25.321
1.266


2912
CG
LEU
364
11.517
24.119
2.043


2913
CD1
LEU
364
12.215
24.003
3.392


2914
CD2
LEU
364
10.016
24.244
2.245


2915
N
THR
365
13.017
24.056
−1.395


2916
CA
THR
365
13.009
22.914
−2.318


2917
C
THR
365
14.318
22.755
−3.067


2918
O
THR
365
14.639
21.634
−3.475


2919
CB
THR
365
11.886
23.028
−3.333


2920
OG1
THR
365
11.960
24.286
−3.988


2921
CG2
THR
365
10.544
22.919
−2.648


2922
N
ARG
366
15.147
23.784
−3.071


2923
CA
ARG
366
16.472
23.622
−3.658


2924
C
ARG
366
17.388
22.917
−2.664


2925
O
ARG
366
18.204
22.084
−3.072


2926
CB
ARG
366
17.016
24.988
−4.050


2927
CG
ARG
366
16.045
25.669
−5.005


2928
CD
ARG
366
16.634
26.929
−5.622


2929
NE
ARG
366
17.039
27.910
−4.604


2930
CZ
ARG
366
16.624
29.178
−4.623


2931
NH1
ARG
366
15.713
29.568
−5.518


2932
NH2
ARG
366
17.060
30.037
−3.700


2933
N
ASP
367
17.069
23.034
−1.384


2934
CA
ASP
367
17.770
22.249
−0.365


2935
C
ASP
367
17.301
20.797
−0.366


2936
O
ASP
367
18.141
19.887
−0.375


2937
CB
ASP
367
17.505
22.866
1.002


2938
CG
ASP
367
18.243
24.192
1.121


2939
OD1
ASP
367
19.462
24.162
1.037


2940
OD2
ASP
367
17.595
25.198
1.366


2941
N
VAL
368
16.010
20.594
−0.586


2942
CA
VAL
368
15.457
19.234
−0.664


2943
C
VAL
368
15.992
18.479
−1.881


2944
O
VAL
368
16.618
17.424
−1.709


2945
CB
VAL
368
13.933
19.338
−0.734


2946
CG1
VAL
368
13.275
18.014
−1.109


2947
CG2
VAL
368
13.357
19.869
0.573


2948
N
GLY
369
16.005
19.143
−3.026


2949
CA
GLY
369
16.506
18.538
−4.261


2950
C
GLY
369
18.009
18.285
−4.214


2951
O
GLY
369
18.464
17.198
−4.594


2952
N
GLU
370
18.747
19.210
−3.617


2953
CA
GLU
370
20.199
19.060
−3.501


2954
C
GLU
370
20.565
17.897
−2.582


2955
O
GLU
370
21.423
17.090
−2.956


2956
CB
GLU
370
20.764
20.333
−2.885


2957
CG
GLU
370
22.171
20.635
−3.380


2958
CD
GLU
370
22.091
21.367
−4.716


2959
OE1
GLU
370
23.087
21.374
−5.423


2960
OE2
GLU
370
21.096
22.045
−4.929


2961
N
ALA
371
19.768
17.668
−1.549


2962
CA
ALA
371
20.028
16.562
−0.624


2963
C
ALA
371
19.685
15.201
−1.228


2964
O
ALA
371
20.450
14.245
−1.040


2965
CB
ALA
371
19.189
16.784
0.629


2966
N
ILE
372
18.721
15.175
−2.136


2967
CA
ILE
372
18.382
13.916
−2.809


2968
C
ILE
372
19.416
13.596
−3.885


2969
O
ILE
372
19.852
12.440
−3.992


2970
CB
ILE
372
17.002
14.037
−3.450


2971
CG1
ILE
372
15.964
14.526
−2.450


2972
CG2
ILE
372
16.566
12.688
−4.010


2973
CD1
ILE
372
14.614
14.745
−3.123


2974
N
LYS
373
20.001
14.639
−4.455


2975
CA
LYS
373
21.068
14.456
−5.439


2976
C
LYS
373
22.357
13.989
−4.771


2977
O
LYS
373
22.985
13.044
−5.261


2978
CB
LYS
373
21.352
15.786
−6.120


2979
CG
LYS
373
22.458
15.619
−7.156


2980
CD
LYS
373
23.077
16.955
−7.542


2981
CE
LYS
373
23.718
17.619
−6.329


2982
NZ
LYS
373
24.434
18.840
−6.723


2983
N
ARG
374
22.605
14.457
−3.559


2984
CA
ARG
374
23.805
14.032
−2.838


2985
C
ARG
374
23.691
12.600
−2.331


2986
O
ARG
374
24.676
11.854
−2.413


2987
CB
ARG
374
24.033
14.959
−1.655


2988
CG
ARG
374
24.302
16.387
−2.104


2989
CD
ARG
374
24.486
17.288
−0.893


2990
NE
ARG
374
24.593
18.702
−1.276


2991
CZ
ARG
374
25.017
19.635
−0.423


2992
NH1
ARG
374
25.468
19.275
0.781


2993
NH2
ARG
374
25.067
20.914
−0.802


2994
N
TYR
375
22.482
12.139
−2.055


2995
CA
TYR
375
22.358
10.735
−1.677


2996
C
TYR
375
22.505
9.838
−2.895


2997
O
TYR
375
23.317
8.909
−2.841


2998
CB
TYR
375
21.016
10.451
−1.021


2999
CG
TYR
375
20.930
9.004
−0.547


3000
CD1
TYR
375
21.704
8.591
0.531


3001
CD2
TYR
375
20.115
8.093
−1.208


3002
CE1
TYR
375
21.643
7.274
0.968


3003
CE2
TYR
375
20.052
6.775
−0.771


3004
CZ
TYR
375
20.813
6.372
0.318


3005
OH
TYR
375
20.724
5.074
0.772


3006
N
GLU
376
22.022
10.296
−4.037


3007
CA
GLU
376
22.095
9.484
−5.253


3008
C
GLU
376
23.489
9.465
−5.888


3009
O
GLU
376
23.807
8.524
−6.623


3010
CB
GLU
376
21.103
10.077
−6.248


3011
CG
GLU
376
20.921
9.204
−7.486


3012
CD
GLU
376
20.256
7.885
−7.102


3013
OE1
GLU
376
19.454
7.915
−6.178


3014
OE2
GLU
376
20.437
6.921
−7.831


3015
N
THR
377
24.328
10.435
−5.564


3016
CA
THR
377
25.670
10.471
−6.153


3017
C
THR
377
26.785
10.084
−5.185


3018
O
THR
377
27.903
9.808
−5.635


3019
CB
THR
377
25.947
11.879
−6.675


3020
OG1
THR
377
25.859
12.790
−5.586


3021
CG2
THR
377
24.941
12.302
−7.740


3022
N
TYR
378
26.520
10.098
−3.889


3023
CA
TYR
378
27.595
9.805
−2.932


3024
C
TYR
378
27.262
8.653
−1.991


3025
O
TYR
378
28.177
8.000
−1.476


3026
CB
TYR
378
27.862
11.054
−2.095


3027
CG
TYR
378
28.245
12.294
−2.897


3028
CD1
TYR
378
29.374
12.283
−3.708


3029
CD2
TYR
378
27.466
13.440
−2.806


3030
CE1
TYR
378
29.710
13.412
−4.443


3031
CE2
TYR
378
27.800
14.569
−3.541


3032
CZ
TYR
378
28.919
14.550
−4.361


3033
OH
TYR
378
29.218
15.652
−5.130


3034
N
LYS
379
25.976
8.376
−1.836


3035
CA
LYS
379
25.479
7.384
−0.867


3036
C
LYS
379
26.075
7.636
0.516


3037
O
LYS
379
26.859
6.834
1.035


3038
CB
LYS
379
25.814
5.975
−1.352


3039
CG
LYS
379
25.188
5.691
−2.714


3040
CD
LYS
379
23.665
5.758
−2.661


3041
CE
LYS
379
23.057
5.619
−4.052


3042
NZ
LYS
379
21.593
5.758
−4.001


3043
N
PHE
380
25.714
8.774
1.084


3044
CA
PHE
380
26.260
9.171
2.382


3045
C
PHE
380
25.655
8.356
3.515


3046
O
PHE
380
24.515
7.881
3.442


3047
CB
PHE
380
26.000
10.658
2.632


3048
CG
PHE
380
24.533
11.078
2.752


3049
CD1
PHE
380
23.860
10.934
3.960


3050
CD2
PHE
380
23.876
11.628
1.659


3051
CE1
PHE
380
22.528
11.309
4.068


3052
CE2
PHE
380
22.545
12.007
1.768


3053
CZ
PHE
380
21.871
11.843
2.970


3054
N
ASP
381
26.438
8.203
4.567


3055
CA
ASP
381
25.923
7.605
5.794


3056
C
ASP
381
25.595
8.730
6.764


3057
O
ASP
381
24.705
8.605
7.612


3058
CB
ASP
381
26.978
6.688
6.399


3059
CG
ASP
381
27.354
5.589
5.411


3060
OD1
ASP
381
26.634
4.603
5.357


3061
OD2
ASP
381
28.416
5.707
4.812


3062
N
LYS
382
26.264
9.852
6.558


3063
CA
LYS
382
25.990
11.063
7.338


3064
C
LYS
382
26.088
12.297
6.439


3065
O
LYS
382
26.928
12.337
5.531


3066
CB
LYS
382
27.024
11.129
8.461


3067
CG
LYS
382
26.810
12.308
9.406


3068
CD
LYS
382
27.816
12.298
10.550


3069
CE
LYS
382
27.630
11.078
11.444


3070
NZ
LYS
382
26.290
11.070
12.054


3071
N
MET
383
25.164
13.229
6.605


3072
CA
MET
383
25.257
14.503
5.878


3073
C
MET
383
25.104
15.688
6.830


3074
O
MET
383
24.000
16.053
7.252


3075
CB
MET
383
24.224
14.557
4.759


3076
CG
MET
383
24.379
15.840
3.949


3077
SD
MET
383
23.617
15.844
2.311


3078
CE
MET
383
21.925
15.416
2.762


3079
N
ILE
384
26.241
16.293
7.120


3080
CA
ILE
384
26.361
17.408
8.061


3081
C
ILE
384
26.059
18.753
7.395


3082
O
ILE
384
26.498
19.029
6.270


3083
CB
ILE
384
27.804
17.366
8.563


3084
CG1
ILE
384
28.111
15.981
9.119


3085
CG2
ILE
384
28.086
18.426
9.620


3086
CD1
ILE
384
29.552
15.878
9.597


3087
N
TYR
385
25.269
19.551
8.091


3088
CA
TYR
385
24.905
20.901
7.649


3089
C
TYR
385
25.297
21.940
8.694


3090
O
TYR
385
24.528
22.160
9.637


3091
CB
TYR
385
23.389
20.971
7.493


3092
CG
TYR
385
22.794
20.290
6.265


3093
CD1
TYR
385
22.372
18.967
6.320


3094
CD2
TYR
385
22.651
21.017
5.091


3095
CE1
TYR
385
21.818
18.371
5.195


3096
CE2
TYR
385
22.096
20.422
3.967


3097
CZ
TYR
385
21.680
19.101
4.023


3098
OH
TYR
385
21.079
18.532
2.922


3099
N
VAL
386
26.413
22.622
8.495


3100
CA
VAL
386
26.839
23.662
9.446


3101
C
VAL
386
26.210
25.007
9.070


3102
O
VAL
386
26.788
25.824
8.342


3103
CB
VAL
386
28.361
23.756
9.446


3104
CG1
VAL
386
28.844
24.650
10.584


3105
CG2
VAL
386
28.987
22.372
9.580


3106
N
ILE
387
24.987
25.188
9.540


3107
CA
ILE
387
24.172
26.355
9.185


3108
C
ILE
387
23.870
27.166
10.452


3109
O
ILE
387
24.027
26.666
11.569


3110
CB
ILE
387
22.907
25.827
8.490


3111
CG1
ILE
387
23.281
24.854
7.378


3112
CG2
ILE
387
22.049
26.931
7.884


3113
CD1
ILE
387
22.054
24.408
6.592


3114
N
ALA
388
23.606
28.450
10.282


3115
CA
ALA
388
23.342
29.329
11.424


3116
C
ALA
388
22.113
28.896
12.210


3117
O
ALA
388
21.133
28.398
11.641


3118
CB
ALA
388
23.142
30.752
10.915


3119
N
ALA
389
22.098
29.281
13.476


3120
CA
ALA
389
20.976
28.984
14.379


3121
C
ALA
389
19.718
29.801
14.071


3122
O
ALA
389
18.600
29.362
14.376


3123
CB
ALA
389
21.422
29.268
15.806


3124
N
GLN
390
19.886
30.815
13.238


3125
CA
GLN
390
18.765
31.608
12.731


3126
C
GLN
390
17.955
30.825
11.692


3127
O
GLN
390
16.733
30.989
11.606


3128
CB
GLN
390
19.384
32.830
12.065


3129
CG
GLN
390
18.353
33.736
11.406


3130
CD
GLN
390
19.090
34.777
10.572


3131
OE1
GLN
390
19.985
34.433
9.790


3132
NE2
GLN
390
18.735
36.033
10.776


3133
N
GLN
391
18.589
29.831
11.089


3134
CA
GLN
391
17.929
28.988
10.097


3135
C
GLN
391
17.495
27.645
10.681


3136
O
GLN
391
17.067
26.771
9.917


3137
CB
GLN
391
18.861
28.798
8.908


3138
CG
GLN
391
19.115
30.141
8.233


3139
CD
GLN
391
20.040
29.994
7.029


3140
OE1
GLN
391
20.085
28.947
6.375


3141
NE2
GLN
391
20.771
31.059
6.750


3142
N
ASP
392
17.419
27.554
12.005


3143
CA
ASP
392
16.983
26.319
12.685


3144
C
ASP
392
15.605
25.846
12.232


3145
O
ASP
392
15.465
24.679
11.840


3146
CB
ASP
392
16.882
26.576
14.188


3147
CG
ASP
392
18.229
26.508
14.901


3148
OD1
ASP
392
19.236
26.342
14.228


3149
OD2
ASP
392
18.208
26.377
16.117


3150
N
LEU
393
14.693
26.787
12.037


3151
CA
LEU
393
13.332
26.439
11.619


3152
C
LEU
393
13.308
25.880
10.200


3153
O
LEU
393
12.847
24.747
10.031


3154
CB
LEU
393
12.467
27.693
11.680


3155
CG
LEU
393
11.024
27.394
11.285


3156
CD1
LEU
393
10.404
26.363
12.221


3157
CD2
LEU
393
10.187
28.668
11.267


3158
N
HIS
394
14.112
26.463
9.326


3159
CA
HIS
394
14.148
26.041
7.925


3160
C
HIS
394
14.853
24.700
7.744


3161
O
HIS
394
14.341
23.832
7.021


3162
CB
HIS
394
14.888
27.120
7.144


3163
CG
HIS
394
15.425
26.650
5.809


3164
ND1
HIS
394
14.707
26.333
4.716


3165
CD2
HIS
394
16.748
26.447
5.497


3166
CE1
HIS
394
15.545
25.948
3.733


3167
NE2
HIS
394
16.806
26.016
4.218


3168
N
CYS
395
15.851
24.446
8.574


3169
CA
CYS
395
16.582
23.186
8.482


3170
C
CYS
395
15.746
22.031
9.005


3171
O
CYS
395
15.575
21.044
8.278


3172
CB
CYS
395
17.874
23.307
9.276


3173
SG
CYS
395
19.012
24.564
8.657


3174
N
ALA
396
14.975
22.290
10.049


3175
CA
ALA
396
14.103
21.247
10.591


3176
C
ALA
396
12.853
21.060
9.733


3177
O
ALA
396
12.382
19.925
9.583


3178
CB
ALA
396
13.715
21.628
12.014


3179
N
GLN
397
12.491
22.091
8.986


3180
CA
GLN
397
11.382
21.979
8.045


3181
C
GLN
397
11.738
21.093
6.862


3182
O
GLN
397
11.020
20.112
6.640


3183
CB
GLN
397
10.978
23.361
7.545


3184
CG
GLN
397
10.226
24.142
8.615


3185
CD
GLN
397
9.873
25.532
8.099


3186
OE1
GLN
397
10.755
26.310
7.708


3187
NE2
GLN
397
8.594
25.854
8.176


3188
N
PHE
398
12.918
21.248
6.279


3189
CA
PHE
398
13.216
20.386
5.129


3190
C
PHE
398
13.732
19.011
5.557


3191
O
PHE
398
13.509
18.046
4.816


3192
CB
PHE
398
14.143
21.059
4.112


3193
CG
PHE
398
15.644
21.129
4.385


3194
CD1
PHE
398
16.470
20.095
3.959


3195
CD2
PHE
398
16.195
22.244
5.001


3196
CE1
PHE
398
17.839
20.160
4.182


3197
CE2
PHE
398
17.565
22.310
5.224


3198
CZ
PHE
398
18.386
21.267
4.818


3199
N
PHE
399
14.138
18.866
6.811


3200
CA
PHE
399
14.465
17.526
7.321


3201
C
PHE
399
13.181
16.733
7.536


3202
O
PHE
399
13.074
15.591
7.068


3203
CB
PHE
399
15.188
17.608
8.666


3204
CG
PHE
399
16.596
18.203
8.690


3205
CD1
PHE
399
17.332
18.365
7.523


3206
CD2
PHE
399
17.144
18.580
9.909


3207
CE1
PHE
399
18.608
18.912
7.575


3208
CE2
PHE
399
18.420
19.123
9.961


3209
CZ
PHE
399
19.153
19.290
8.794


3210
N
GLU
400
12.144
17.429
7.978


3211
CA
GLU
400
10.845
16.791
8.194


3212
C
GLU
400
10.128
16.536
6.875


3213
O
GLU
400
9.477
15.494
6.742


3214
CB
GLU
400
9.992
17.712
9.058


3215
CG
GLU
400
8.693
17.042
9.493


3216
CD
GLU
400
9.003
15.864
10.410


3217
OE1
GLU
400
9.874
16.018
11.254


3218
OE2
GLU
400
8.363
14.833
10.250


3219
N
ILE
401
10.420
17.338
5.865


3220
CA
ILE
401
9.858
17.100
4.536


3221
C
ILE
401
10.481
15.870
3.885


3222
O
ILE
401
9.724
14.978
3.489


3223
CB
ILE
401
10.094
18.334
3.671


3224
CG1
ILE
401
9.266
19.508
4.173


3225
CG2
ILE
401
9.769
18.053
2.211


3226
CD1
ILE
401
9.512
20.755
3.335


3227
N
LEU
402
11.776
15.669
4.080


3228
CA
LEU
402
12.437
14.483
3.520


3229
C
LEU
402
12.036
13.217
4.274


3230
O
LEU
402
11.717
12.195
3.646


3231
CB
LEU
402
13.946
14.679
3.621


3232
CG
LEU
402
14.430
15.837
2.756


3233
CD1
LEU
402
15.879
16.189
3.065


3234
CD2
LEU
402
14.254
15.532
1.275


3235
N
LYS
403
11.778
13.375
5.562


3236
CA
LYS
403
11.297
12.260
6.379


3237
C
LYS
403
9.899
11.817
5.953


3238
O
LYS
403
9.732
10.644
5.592


3239
CB
LYS
403
11.263
12.727
7.829


3240
CG
LYS
403
10.710
11.653
8.756


3241
CD
LYS
403
10.587
12.176
10.182


3242
CE
LYS
403
9.969
11.132
11.104


3243
NZ
LYS
403
9.817
11.662
12.468


3244
N
GLN
404
9.035
12.783
5.676


3245
CA
GLN
404
7.649
12.488
5.286


3246
C
GLN
404
7.488
12.154
3.800


3247
O
GLN
404
6.403
11.741
3.379


3248
CB
GLN
404
6.772
13.683
5.645


3249
CG
GLN
404
6.785
13.962
7.146


3250
CD
GLN
404
6.246
12.770
7.934


3251
OE1
GLN
404
5.318
12.083
7.493


3252
NE2
GLN
404
6.768
12.599
9.136


3253
N
MET
405
8.558
12.280
3.029


3254
CA
MET
405
8.571
11.795
1.644


3255
C
MET
405
8.980
10.323
1.575


3256
O
MET
405
8.963
9.724
0.493


3257
CB
MET
405
9.557
12.625
0.833


3258
CG
MET
405
9.080
14.061
0.664


3259
SD
MET
405
10.296
15.201
−0.035


3260
CE
MET
405
10.518
14.445
−1.658


3261
N
GLY
406
9.367
9.763
2.711


3262
CA
GLY
406
9.684
8.337
2.792


3263
C
GLY
406
11.179
8.098
2.644


3264
O
GLY
406
11.608
7.078
2.090


3265
N
PHE
407
11.971
9.043
3.118


3266
CA
PHE
407
13.416
8.902
2.948


3267
C
PHE
407
14.102
8.308
4.169


3268
O
PHE
407
14.311
8.978
5.189


3269
CB
PHE
407
14.021
10.247
2.579


3270
CG
PHE
407
13.664
10.684
1.161


3271
CD1
PHE
407
13.543
9.734
0.154


3272
CD2
PHE
407
13.472
12.027
0.872


3273
CE1
PHE
407
13.225
10.127
−1.139


3274
CE2
PHE
407
13.156
12.420
−0.421


3275
CZ
PHE
407
13.032
11.471
−1.427


3276
N
GLU
408
14.712
7.161
3.915


3277
CA
GLU
408
15.459
6.407
4.937


3278
C
GLU
408
16.877
6.942
5.168


3279
O
GLU
408
17.682
6.301
5.848


3280
CB
GLU
408
15.549
4.947
4.502


3281
CG
GLU
408
16.334
4.793
3.202


3282
CD
GLU
408
16.443
3.321
2.816


3283
OE1
GLU
408
15.461
2.620
3.012


3284
OE2
GLU
408
17.428
2.976
2.180


3285
N
TRP
409
17.212
8.029
4.497


3286
CA
TRP
409
18.484
8.700
4.710


3287
C
TRP
409
18.279
10.071
5.341


3288
O
TRP
409
19.257
10.713
5.743


3289
CB
TRP
409
19.210
8.821
3.374


3290
CG
TRP
409
18.380
9.362
2.222


3291
CD1
TRP
409
17.717
8.617
1.271


3292
CD2
TRP
409
18.137
10.748
1.894


3293
NE1
TRP
409
17.111
9.463
0.403


3294
CE2
TRP
409
17.339
10.747
0.737


3295
CE3
TRP
409
18.531
11.948
2.467


3296
CZ2
TRP
409
16.951
11.949
0.166


3297
CZ3
TRP
409
18.133
13.148
1.893


3298
CH2
TRP
409
17.346
13.148
0.748


3299
N
ALA
410
17.027
10.435
5.585


3300
CA
ALA
410
16.718
11.790
6.064


3301
C
ALA
410
17.057
12.003
7.535


3302
O
ALA
410
17.449
13.111
7.917


3303
CB
ALA
410
15.236
12.054
5.849


3304
N
HIS
411
17.182
10.909
8.268


3305
CA
HIS
411
17.587
10.974
9.676


3306
C
HIS
411
19.110
10.956
9.849


3307
O
HIS
411
19.605
11.055
10.977


3308
CB
HIS
411
16.921
9.831
10.439


3309
CG
HIS
411
16.976
8.473
9.767


3310
ND1
HIS
411
15.951
7.833
9.171


3311
CD2
HIS
411
18.073
7.647
9.672


3312
CE1
HIS
411
16.381
6.649
8.695


3313
NE2
HIS
411
17.694
6.536
9.004


3314
N
ASN
412
19.835
10.919
8.739


3315
CA
ASN
412
21.298
11.018
8.763


3316
C
ASN
412
21.741
12.467
8.552


3317
O
ASN
412
22.946
12.753
8.472


3318
CB
ASN
412
21.882
10.171
7.634


3319
CG
ASN
412
21.449
8.709
7.712


3320
OD1
ASN
412
21.035
8.203
8.763


3321
ND2
ASN
412
21.550
8.041
6.576


3322
N
LEU
413
20.770
13.347
8.364


3323
CA
LEU
413
21.046
14.773
8.155


3324
C
LEU
413
21.282
15.457
9.500


3325
O
LEU
413
20.373
15.615
10.322


3326
CB
LEU
413
19.876
15.432
7.416


3327
CG
LEU
413
19.937
15.317
5.885


3328
CD1
LEU
413
19.771
13.898
5.353


3329
CD2
LEU
413
18.880
16.201
5.240


3330
N
GLU
414
22.505
15.920
9.676


3331
CA
GLU
414
22.944
16.435
10.975


3332
C
GLU
414
23.182
17.941
10.949


3333
O
GLU
414
24.247
18.405
10.521


3334
CB
GLU
414
24.235
15.707
11.337


3335
CG
GLU
414
24.726
16.051
12.739


3336
CD
GLU
414
25.991
15.254
13.047


3337
OE1
GLU
414
26.895
15.279
12.222


3338
OE2
GLU
414
26.018
14.608
14.084


3339
N
HIS
415
22.204
18.687
11.435


3340
CA
HIS
415
22.342
20.145
11.529


3341
C
HIS
415
23.275
20.536
12.675


3342
O
HIS
415
23.087
20.147
13.833


3343
CB
HIS
415
20.967
20.766
11.753


3344
CG
HIS
415
20.957
22.283
11.799


3345
ND1
HIS
415
20.212
23.050
12.616


3346
CD2
HIS
415
21.703
23.137
11.021


3347
CE1
HIS
415
20.474
24.349
12.370


3348
NE2
HIS
415
21.399
24.403
11.387


3349
N
VAL
416
24.290
21.295
12.309


3350
CA
VAL
416
25.286
21.819
13.241


3351
C
VAL
416
25.174
23.342
13.263


3352
O
VAL
416
25.734
24.028
12.398


3353
CB
VAL
416
26.653
21.386
12.716


3354
CG1
VAL
416
27.800
21.938
13.549


3355
CG2
VAL
416
26.742
19.868
12.631


3356
N
ASN
417
24.381
23.852
14.190


3357
CA
ASN
417
24.127
25.295
14.212


3358
C
ASN
417
25.176
26.121
14.946


3359
O
ASN
417
25.689
25.765
16.015


3360
CB
ASN
417
22.730
25.602
14.745


3361
CG
ASN
417
22.448
25.097
16.159


3362
OD1
ASN
417
23.336
24.701
16.925


3363
ND2
ASN
417
21.176
25.163
16.501


3364
N
PHE
418
25.551
27.194
14.278


3365
CA
PHE
418
26.438
28.186
14.882


3366
C
PHE
418
25.672
29.476
15.160


3367
O
PHE
418
24.709
29.814
14.458


3368
CB
PHE
418
27.653
28.439
13.988


3369
CG
PHE
418
27.381
29.048
12.613


3370
CD1
PHE
418
27.361
30.428
12.459


3371
CD2
PHE
418
27.184
28.229
11.510


3372
CE1
PHE
418
27.122
30.992
11.214


3373
CE2
PHE
418
26.942
28.793
10.264


3374
CZ
PHE
418
26.910
30.173
10.114


3375
N
GLY
419
26.093
30.174
16.195


3376
CA
GLY
419
25.465
31.435
16.584


3377
C
GLY
419
25.795
32.561
15.612


3378
O
GLY
419
26.195
32.337
14.464


3379
N
MET
420
25.520
33.774
16.048


3380
CA
MET
420
25.765
34.941
15.199


3381
C
MET
420
26.866
35.844
15.742


3382
O
MET
420
27.042
35.987
16.960


3383
CB
MET
420
24.466
35.731
15.085


3384
CG
MET
420
23.357
34.883
14.470


3385
SD
MET
420
21.761
35.707
14.267


3386
CE
MET
420
22.269
37.036
13.152


3387
N
VAL
421
27.633
36.404
14.819


3388
CA
VAL
421
28.591
37.461
15.169


3389
C
VAL
421
27.837
38.785
15.281


3390
O
VAL
421
27.517
39.456
14.289


3391
CB
VAL
421
29.683
37.556
14.106


3392
CG1
VAL
421
30.668
38.675
14.432


3393
CG2
VAL
421
30.425
36.234
13.970


3394
N
GLN
422
27.558
39.143
16.520


3395
CA
GLN
422
26.716
40.299
16.814


3396
C
GLN
422
27.480
41.601
16.626


3397
O
GLN
422
28.448
41.901
17.336


3398
CB
GLN
422
26.206
40.146
18.240


3399
CG
GLN
422
25.389
38.862
18.354


3400
CD
GLN
422
25.006
38.591
19.804


3401
OE1
GLN
422
23.926
38.055
20.092


3402
NE2
GLN
422
25.923
38.921
20.696


3403
N
GLY
423
27.052
42.335
15.614


3404
CA
GLY
423
27.678
43.609
15.266


3405
C
GLY
423
27.665
43.814
13.754


3406
O
GLY
423
27.786
44.948
13.276


3407
N
MET
424
27.445
42.730
13.024


3408
CA
MET
424
27.451
42.773
11.554


3409
C
MET
424
26.143
43.314
10.969


3410
O
MET
424
25.258
42.542
10.585


3411
CB
MET
424
27.657
41.351
11.041


3412
CG
MET
424
28.964
40.714
11.511


3413
SD
MET
424
30.498
41.439
10.884


3414
CE
MET
424
30.978
42.448
12.305


3415
N
SER
425
26.024
44.628
10.910


3416
CA
SER
425
24.844
45.238
10.290


3417
C
SER
425
25.215
45.960
9.000


3418
O
SER
425
25.928
46.978
9.006


3419
CB
SER
425
24.207
46.204
11.276


3420
OG
SER
425
23.959
45.479
12.472


3421
N
THR
426
24.593
45.517
7.919


3422
CA
THR
426
24.918
46.056
6.592


3423
C
THR
426
24.409
47.481
6.402


3424
O
THR
426
25.212
48.348
6.042


3425
CB
THR
426
24.331
45.152
5.504


3426
OG1
THR
426
22.918
45.087
5.646


3427
CG2
THR
426
24.872
43.730
5.584


3428
N
ARG
427
23.263
47.777
6.997


3429
CA
ARG
427
22.643
49.099
6.846


3430
C
ARG
427
23.167
50.143
7.837


3431
O
ARG
427
22.735
51.300
7.806


3432
CB
ARG
427
21.125
48.929
6.943


3433
CG
ARG
427
20.689
47.993
8.072


3434
CD
ARG
427
20.722
48.648
9.449


3435
NE
ARG
427
20.469
47.656
10.502


3436
CZ
ARG
427
20.780
47.865
11.782


3437
NH1
ARG
427
21.323
49.026
12.154


3438
NH2
ARG
427
20.533
46.921
12.691


3439
N
LYS
428
24.079
49.735
8.703


3440
CA
LYS
428
24.693
50.675
9.634


3441
C
LYS
428
26.151
50.902
9.235


3442
O
LYS
428
26.803
51.844
9.702


3443
CB
LYS
428
24.602
50.080
11.034


3444
CG
LYS
428
24.933
51.103
12.113


3445
CD
LYS
428
23.964
52.278
12.056


3446
CE
LYS
428
24.286
53.320
13.121


3447
NZ
LYS
428
23.349
54.452
13.047


3448
N
GLY
429
26.631
50.057
8.335


3449
CA
GLY
429
28.020
50.134
7.872


3450
C
GLY
429
28.976
49.527
8.894


3451
O
GLY
429
30.121
49.970
9.034


3452
N
THR
430
28.502
48.514
9.600


3453
CA
THR
430
29.315
47.892
10.649


3454
C
THR
430
29.570
46.408
10.354


3455
O
THR
430
30.051
45.648
11.205


3456
CB
THR
430
28.664
48.178
12.004


3457
OG1
THR
430
29.353
47.495
13.044


3458
CG2
THR
430
27.203
47.774
12.055


3459
N
VAL
431
29.284
46.007
9.125


3460
CA
VAL
431
29.680
44.667
8.657


3461
C
VAL
431
31.168
44.600
8.315


3462
O
VAL
431
31.556
44.691
7.145


3463
CB
VAL
431
28.882
44.297
7.415


3464
CG1
VAL
431
27.665
43.451
7.751


3465
CG2
VAL
431
28.526
45.524
6.582


3466
N
VAL
432
31.981
44.400
9.337


3467
CA
VAL
432
33.425
44.287
9.147


3468
C
VAL
432
33.771
42.974
8.452


3469
O
VAL
432
33.340
41.884
8.854


3470
CB
VAL
432
34.100
44.383
10.510


3471
CG1
VAL
432
35.619
44.397
10.376


3472
CG2
VAL
432
33.632
45.637
11.240


3473
N
PHE
433
34.449
43.129
7.329


3474
CA
PHE
433
34.872
41.990
6.524


3475
C
PHE
433
36.000
41.219
7.191


3476
O
PHE
433
36.981
41.791
7.685


3477
CB
PHE
433
35.285
42.485
5.144


3478
CG
PHE
433
34.126
43.116
4.376


3479
CD1
PHE
433
34.202
44.435
3.949


3480
CD2
PHE
433
32.986
42.367
4.112


3481
CE1
PHE
433
33.139
45.004
3.260


3482
CE2
PHE
433
31.922
42.936
3.425


3483
CZ
PHE
433
31.998
44.255
2.999


3484
N
LEU
434
35.949
39.919
6.969


3485
CA
LEU
434
36.845
38.952
7.596


3486
C
LEU
434
38.232
39.002
6.973


3487
O
LEU
434
39.232
38.890
7.692


3488
CB
LEU
434
36.235
37.576
7.355


3489
CG
LEU
434
37.060
36.462
7.978


3490
CD1
LEU
434
37.106
36.612
9.495


3491
CD2
LEU
434
36.494
35.103
7.583


3492
N
ASP
435
38.291
39.499
5.748


3493
CA
ASP
435
39.580
39.678
5.075


3494
C
ASP
435
40.343
40.875
5.652


3495
O
ASP
435
41.552
40.759
5.885


3496
CB
ASP
435
39.317
39.910
3.589


3497
CG
ASP
435
38.471
38.777
3.006


3498
OD1
ASP
435
38.996
37.680
2.880


3499
OD2
ASP
435
37.295
39.013
2.757


3500
N
ASN
436
39.602
41.835
6.186


3501
CA
ASN
436
40.210
43.029
6.776


3502
C
ASN
436
40.639
42.739
8.210


3503
O
ASN
436
41.741
43.124
8.621


3504
CB
ASN
436
39.159
44.135
6.772


3505
CG
ASN
436
39.737
45.433
7.325


3506
OD1
ASN
436
40.566
46.078
6.674


3507
ND2
ASN
436
39.281
45.815
8.506


3508
N
ILE
437
39.912
41.828
8.837


3509
CA
ILE
437
40.240
41.398
10.197


3510
C
ILE
437
41.483
40.521
10.196


3511
O
ILE
437
42.405
40.768
10.982


3512
CB
ILE
437
39.069
40.582
10.725


3513
CG1
ILE
437
37.789
41.402
10.700


3514
CG2
ILE
437
39.351
40.080
12.136


3515
CD1
ILE
437
36.585
40.536
11.042


3516
N
LEU
438
41.618
39.718
9.155


3517
CA
LEU
438
42.774
38.831
9.029


3518
C
LEU
438
44.035
39.613
8.669


3519
O
LEU
438
45.072
39.417
9.316


3520
CB
LEU
438
42.464
37.809
7.939


3521
CG
LEU
438
42.171
36.408
8.478


3522
CD1
LEU
438
41.050
36.389
9.511


3523
CD2
LEU
438
41.850
35.451
7.335


3524
N
GLN
439
43.872
40.652
7.864


3525
CA
GLN
439
45.001
41.519
7.512


3526
C
GLN
439
45.462
42.367
8.691


3527
O
GLN
439
46.652
42.337
9.029


3528
CB
GLN
439
44.567
42.448
6.384


3529
CG
GLN
439
44.519
41.735
5.038


3530
CD
GLN
439
45.938
41.443
4.560


3531
OE1
GLN
439
46.838
42.278
4.702


3532
NE2
GLN
439
46.112
40.281
3.956


3533
N
GLU
440
44.517
42.870
9.468


3534
CA
GLU
440
44.885
43.730
10.591


3535
C
GLU
440
45.403
42.937
11.788


3536
O
GLU
440
46.317
43.414
12.470


3537
CB
GLU
440
43.674
44.556
11.004


3538
CG
GLU
440
44.074
45.677
11.958


3539
CD
GLU
440
44.999
46.666
11.248


3540
OE1
GLU
440
46.207
46.533
11.390


3541
OE2
GLU
440
44.469
47.565
10.611


3542
N
THR
441
45.024
41.676
11.896


3543
CA
THR
441
45.563
40.852
12.977


3544
C
THR
441
46.938
40.304
12.601


3545
O
THR
441
47.836
40.281
13.451


3546
CB
THR
441
44.590
39.720
13.269


3547
OG1
THR
441
43.343
40.298
13.632


3548
CG2
THR
441
45.071
38.890
14.446


3549
N
LYS
442
47.177
40.188
11.304


3550
CA
LYS
442
48.509
39.843
10.800


3551
C
LYS
442
49.488
41.000
10.998


3552
O
LYS
442
50.616
40.781
11.457


3553
CB
LYS
442
48.366
39.547
9.311


3554
CG
LYS
442
49.710
39.447
8.603


3555
CD
LYS
442
49.515
39.228
7.108


3556
CE
LYS
442
50.851
39.145
6.381


3557
NZ
LYS
442
50.654
38.867
4.950


3558
N
GLU
443
48.981
42.221
10.901


3559
CA
GLU
443
49.811
43.408
11.137


3560
C
GLU
443
50.090
43.599
12.624


3561
O
GLU
443
51.254
43.779
13.009


3562
CB
GLU
443
49.062
44.627
10.609


3563
CG
GLU
443
48.831
44.530
9.106


3564
CD
GLU
443
47.898
45.644
8.638


3565
OE1
GLU
443
46.696
45.403
8.603


3566
OE2
GLU
443
48.405
46.689
8.255


3567
N
LYS
444
49.103
43.278
13.443


3568
CA
LYS
444
49.242
43.384
14.897


3569
C
LYS
444
50.253
42.378
15.442


3570
O
LYS
444
51.223
42.788
16.095


3571
CB
LYS
444
47.868
43.114
15.502


3572
CG
LYS
444
47.867
43.212
17.022


3573
CD
LYS
444
48.267
44.604
17.496


3574
CE
LYS
444
48.156
44.713
19.012


3575
NZ
LYS
444
48.966
43.678
19.672


3576
N
MET
445
50.203
41.158
14.932


3577
CA
MET
445
51.128
40.124
15.401


3578
C
MET
445
52.517
40.268
14.784


3579
O
MET
445
53.506
39.961
15.461


3580
CB
MET
445
50.541
38.758
15.071


3581
CG
MET
445
49.178
38.587
15.734


3582
SD
MET
445
49.151
38.771
17.533


3583
CE
MET
445
47.365
38.681
17.797


3584
N
HIS
446
52.612
40.981
13.672


3585
CA
HIS
446
53.923
41.257
13.085


3586
C
HIS
446
54.626
42.364
13.856


3587
O
HIS
446
55.816
42.221
14.153


3588
CB
HIS
446
53.744
41.691
11.635


3589
CG
HIS
446
55.043
41.783
10.862


3590
ND1
HIS
446
55.809
42.877
10.692


3591
CD2
HIS
446
55.668
40.749
10.207


3592
CE1
HIS
446
56.879
42.555
9.937


3593
NE2
HIS
446
56.794
41.239
9.641


3594
N
GLU
447
53.849
43.263
14.436


3595
CA
GLU
447
54.422
44.339
15.247


3596
C
GLU
447
54.878
43.828
16.609


3597
O
GLU
447
55.964
44.208
17.063


3598
CB
GLU
447
53.359
45.413
15.420


3599
CG
GLU
447
53.004
46.032
14.074


3600
CD
GLU
447
51.705
46.822
14.190


3601
OE1
GLU
447
51.439
47.322
15.274


3602
OE2
GLU
447
50.958
46.842
13.219


3603
N
VAL
448
54.223
42.784
17.095


3604
CA
VAL
448
54.654
42.149
18.347


3605
C
VAL
448
55.885
41.270
18.120


3606
O
VAL
448
56.806
41.258
18.948


3607
CB
VAL
448
53.501
41.299
18.870


3608
CG1
VAL
448
53.888
40.554
20.143


3609
CG2
VAL
448
52.266
42.155
19.111


3610
N
MET
449
56.022
40.803
16.891


3611
CA
MET
449
57.175
40.003
16.481


3612
C
MET
449
58.407
40.881
16.252


3613
O
MET
449
59.512
40.507
16.661


3614
CB
MET
449
56.740
39.330
15.188


3615
CG
MET
449
57.743
38.349
14.615


3616
SD
MET
449
57.103
37.491
13.163


3617
CE
MET
449
55.587
36.848
13.907


3618
N
GLN
450
58.153
42.141
15.926


3619
CA
GLN
450
59.215
43.144
15.785


3620
C
GLN
450
59.694
43.688
17.131


3621
O
GLN
450
60.771
44.298
17.191


3622
CB
GLN
450
58.665
44.312
14.975


3623
CG
GLN
450
58.230
43.876
13.585


3624
CD
GLN
450
57.562
45.034
12.854


3625
OE1
GLN
450
56.335
45.063
12.675


3626
NE2
GLN
450
58.393
45.942
12.376


3627
N
LYS
451
58.973
43.387
18.202


3628
CA
LYS
451
59.404
43.812
19.535


3629
C
LYS
451
60.512
42.906
20.060


3630
O
LYS
451
61.386
43.363
20.808


3631
CB
LYS
451
58.217
43.762
20.491


3632
CG
LYS
451
57.143
44.784
20.136


3633
CD
LYS
451
55.990
44.715
21.132


3634
CE
LYS
451
54.959
45.815
20.896


3635
NZ
LYS
451
54.336
45.698
19.570


3636
N
ASN
452
60.538
41.663
19.610


3637
CA
ASN
452
61.689
40.822
19.925


3638
C
ASN
452
62.520
40.680
18.666


3639
O
ASN
452
62.416
39.675
17.951


3640
CB
ASN
452
61.266
39.448
20.429


3641
CG
ASN
452
62.499
38.706
20.953


3642
OD1
ASN
452
63.419
38.365
20.195


3643
ND2
ASN
452
62.521
38.498
22.255


3644
N
GLU
453
63.535
41.522
18.580


3645
CA
GLU
453
64.344
41.607
17.360


3646
C
GLU
453
65.315
40.439
17.174


3647
O
GLU
453
65.683
40.151
16.031


3648
CB
GLU
453
65.119
42.917
17.408


3649
CG
GLU
453
64.171
44.109
17.502


3650
CD
GLU
453
64.971
45.406
17.580


3651
OE1
GLU
453
66.102
45.398
17.114


3652
OE2
GLU
453
64.484
46.340
18.200


3653
N
GLU
454
65.504
39.627
18.203


3654
CA
GLU
454
66.412
38.484
18.092


3655
C
GLU
454
65.694
37.357
17.361


3656
O
GLU
454
66.076
37.028
16.231


3657
CB
GLU
454
66.849
38.000
19.479


3658
CG
GLU
454
67.895
38.886
20.168


3659
CD
GLU
454
67.305
40.168
20.761


3660
OE1
GLU
454
67.984
41.181
20.718


3661
OE2
GLU
454
66.136
40.139
21.131


3662
N
LYS
455
64.481
37.092
17.822


3663
CA
LYS
455
63.653
36.040
17.232


3664
C
LYS
455
63.062
36.495
15.905


3665
O
LYS
455
63.072
35.712
14.946


3666
CB
LYS
455
62.527
35.710
18.205


3667
CG
LYS
455
63.060
35.117
19.504


3668
CD
LYS
455
63.713
33.761
19.263


3669
CE
LYS
455
64.313
33.191
20.542


3670
NZ
LYS
455
64.950
31.890
20.284


3671
N
TYR
456
62.885
37.800
15.770


3672
CA
TYR
456
62.402
38.388
14.519


3673
C
TYR
456
63.443
38.292
13.404


3674
O
TYR
456
63.086
37.900
12.287


3675
CB
TYR
456
62.094
39.853
14.800


3676
CG
TYR
456
61.544
40.645
13.620


3677
CD1
TYR
456
60.331
40.283
13.050


3678
CD2
TYR
456
62.252
41.732
13.123


3679
CE1
TYR
456
59.825
41.007
11.979


3680
CE2
TYR
456
61.748
42.456
12.051


3681
CZ
TYR
456
60.535
42.091
11.483


3682
OH
TYR
456
60.014
42.831
10.445


3683
N
ALA
457
64.716
38.365
13.765


3684
CA
ALA
457
65.801
38.243
12.781


3685
C
ALA
457
66.187
36.792
12.486


3686
O
ALA
457
67.030
36.537
11.617


3687
CB
ALA
457
67.020
38.995
13.300


3688
N
GLN
458
65.572
35.853
13.188


3689
CA
GLN
458
65.789
34.433
12.906


3690
C
GLN
458
64.732
33.898
11.947


3691
O
GLN
458
64.879
32.795
11.403


3692
CB
GLN
458
65.676
33.665
14.214


3693
CG
GLN
458
66.694
34.138
15.240


3694
CD
GLN
458
66.382
33.482
16.577


3695
OE1
GLN
458
66.536
34.092
17.644


3696
NE2
GLN
458
65.871
32.266
16.494


3697
N
ILE
459
63.666
34.657
11.761


3698
CA
ILE
459
62.590
34.209
10.877


3699
C
ILE
459
62.887
34.602
9.437


3700
O
ILE
459
63.034
35.786
9.122


3701
CB
ILE
459
61.299
34.868
11.337


3702
CG1
ILE
459
61.159
34.737
12.840


3703
CG2
ILE
459
60.095
34.217
10.672


3704
CD1
ILE
459
59.967
35.534
13.336


3705
N
GLU
460
62.821
33.620
8.551


3706
CA
GLU
460
63.106
33.869
7.131


3707
C
GLU
460
61.922
34.496
6.390


3708
O
GLU
460
62.092
35.022
5.286


3709
CB
GLU
460
63.487
32.549
6.471


3710
CG
GLU
460
64.742
31.943
7.095


3711
CD
GLU
460
65.940
32.877
6.924


3712
OE1
GLU
460
66.513
32.876
5.845


3713
OE2
GLU
460
66.269
33.554
7.888


3714
N
ASP
461
60.745
34.453
6.990


3715
CA
ASP
461
59.609
35.212
6.455


3716
C
ASP
461
58.645
35.560
7.585


3717
O
ASP
461
57.615
34.894
7.770


3718
CB
ASP
461
58.900
34.406
5.367


3719
CG
ASP
461
57.926
35.288
4.579


3720
OD1
ASP
461
57.172
36.021
5.215


3721
OD2
ASP
461
57.849
35.109
3.374


3722
N
PRO
462
58.880
36.708
8.199


3723
CA
PRO
462
58.109
37.091
9.382


3724
C
PRO
462
56.669
37.511
9.073


3725
O
PRO
462
55.792
37.273
9.910


3726
CB
PRO
462
58.878
38.227
9.981


3727
CG
PRO
462
59.990
38.648
9.031


3728
CD
PRO
462
59.941
37.668
7.873


3729
N
ASP
463
56.379
37.840
7.823


3730
CA
ASP
463
55.029
38.279
7.460


3731
C
ASP
463
54.082
37.095
7.343


3732
O
ASP
463
52.972
37.151
7.883


3733
CB
ASP
463
55.081
39.017
6.126


3734
CG
ASP
463
55.878
40.311
6.260


3735
OD1
ASP
463
57.090
40.255
6.099


3736
OD2
ASP
463
55.267
41.324
6.566


3737
N
LYS
464
54.604
35.958
6.914


3738
CA
LYS
464
53.759
34.768
6.827


3739
C
LYS
464
53.622
34.081
8.180


3740
O
LYS
464
52.527
33.603
8.498


3741
CB
LYS
464
54.343
33.808
5.801


3742
CG
LYS
464
54.291
34.422
4.408


3743
CD
LYS
464
54.800
33.453
3.350


3744
CE
LYS
464
54.734
34.083
1.965


3745
NZ
LYS
464
53.356
34.482
1.636


3746
N
ILE
465
54.577
34.320
9.065


3747
CA
ILE
465
54.466
33.777
10.420


3748
C
ILE
465
53.476
34.597
11.241


3749
O
ILE
465
52.580
34.014
11.867


3750
CB
ILE
465
55.840
33.796
11.075


3751
CG1
ILE
465
56.794
32.854
10.352


3752
CG2
ILE
465
55.733
33.414
12.545


3753
CD1
ILE
465
56.339
31.402
10.458


3754
N
ALA
466
53.422
35.889
10.954


3755
CA
ALA
466
52.448
36.772
11.599


3756
C
ALA
466
51.059
36.617
10.997


3757
O
ALA
466
50.054
36.816
11.688


3758
CB
ALA
466
52.912
38.202
11.395


3759
N
ASP
467
51.001
36.075
9.793


3760
CA
ASP
467
49.720
35.757
9.172


3761
C
ASP
467
49.130
34.489
9.772


3762
O
ASP
467
47.943
34.485
10.120


3763
CB
ASP
467
49.958
35.551
7.682


3764
CG
ASP
467
48.639
35.298
6.966


3765
OD1
ASP
467
47.649
35.878
7.384


3766
OD2
ASP
467
48.661
34.593
5.966


3767
N
LEU
468
49.985
33.549
10.143


3768
CA
LEU
468
49.499
32.302
10.743


3769
C
LEU
468
49.110
32.509
12.201


3770
O
LEU
468
48.073
31.993
12.637


3771
CB
LEU
468
50.601
31.254
10.663


3772
CG
LEU
468
51.014
30.983
9.221


3773
CD1
LEU
468
52.256
30.103
9.163


3774
CD2
LEU
468
49.871
30.381
8.410


3775
N
ILE
469
49.781
33.439
12.859


3776
CA
ILE
469
49.424
33.786
14.236


3777
C
ILE
469
48.192
34.692
14.281


3778
O
ILE
469
47.292
34.468
15.103


3779
CB
ILE
469
50.630
34.486
14.847


3780
CG1
ILE
469
51.822
33.541
14.893


3781
CG2
ILE
469
50.317
35.005
16.240


3782
CD1
ILE
469
53.060
34.248
15.426


3783
N
GLY
470
48.044
35.506
13.250


3784
CA
GLY
470
46.871
36.363
13.091


3785
C
GLY
470
45.603
35.551
12.868


3786
O
GLY
470
44.659
35.630
13.668


3787
N
ILE
471
45.660
34.649
11.902


3788
CA
ILE
471
44.501
33.815
11.583


3789
C
ILE
471
44.199
32.809
12.693


3790
O
ILE
471
43.020
32.642
13.025


3791
CB
ILE
471
44.783
33.078
10.276


3792
CG1
ILE
471
45.029
34.059
9.137


3793
CG2
ILE
471
43.632
32.148
9.914


3794
CD1
ILE
471
45.352
33.322
7.843


3795
N
SER
472
45.208
32.383
13.440


3796
CA
SER
472
44.941
31.472
14.559


3797
C
SER
472
44.379
32.189
15.786


3798
O
SER
472
43.583
31.582
16.508


3799
CB
SER
472
46.201
30.698
14.941


3800
OG
SER
472
47.238
31.597
15.318


3801
N
ALA
473
44.577
33.491
15.896


3802
CA
ALA
473
43.947
34.232
16.989


3803
C
ALA
473
42.462
34.416
16.704


3804
O
ALA
473
41.628
34.002
17.523


3805
CB
ALA
473
44.615
35.596
17.106


3806
N
VAL
474
42.169
34.666
15.437


3807
CA
VAL
474
40.787
34.883
14.997


3808
C
VAL
474
39.986
33.579
14.925


3809
O
VAL
474
38.798
33.565
15.277


3810
CB
VAL
474
40.852
35.541
13.621


3811
CG1
VAL
474
39.464
35.825
13.065


3812
CG2
VAL
474
41.666
36.829
13.673


3813
N
MET
475
40.662
32.471
14.672


3814
CA
MET
475
39.977
31.180
14.690


3815
C
MET
475
39.789
30.670
16.111


3816
O
MET
475
38.661
30.302
16.464


3817
CB
MET
475
40.783
30.161
13.893


3818
CG
MET
475
40.789
30.480
12.403


3819
SD
MET
475
41.768
29.360
11.372


3820
CE
MET
475
40.958
27.796
11.775


3821
N
ILE
476
40.757
30.918
16.979


3822
CA
ILE
476
40.652
30.401
18.345


3823
C
ILE
476
39.680
31.193
19.209


3824
O
ILE
476
38.887
30.528
19.880


3825
CB
ILE
476
42.034
30.327
18.983


3826
CG1
ILE
476
42.811
29.168
18.378


3827
CG2
ILE
476
41.944
30.137
20.490


3828
CD1
ILE
476
42.108
27.850
18.682


3829
N
GLN
477
39.436
32.458
18.892


3830
CA
GLN
477
38.405
33.193
19.646


3831
C
GLN
477
36.980
32.791
19.241


3832
O
GLN
477
36.040
32.977
20.021


3833
CB
GLN
477
38.636
34.704
19.513


3834
CG
GLN
477
38.589
35.248
18.084


3835
CD
GLN
477
37.173
35.648
17.666


3836
OE1
GLN
477
36.522
36.463
18.328


3837
NE2
GLN
477
36.756
35.149
16.516


3838
N
ASP
478
36.853
32.054
18.147


3839
CA
ASP
478
35.553
31.541
17.730


3840
C
ASP
478
35.399
30.106
18.242


3841
O
ASP
478
34.324
29.728
18.723


3842
CB
ASP
478
35.542
31.567
16.204


3843
CG
ASP
478
34.131
31.587
15.623


3844
OD1
ASP
478
33.387
30.646
15.854


3845
OD2
ASP
478
33.838
32.546
14.920


3846
N
MET
479
36.531
29.427
18.365


3847
CA
MET
479
36.585
28.021
18.795


3848
C
MET
479
36.690
27.816
20.307


3849
O
MET
479
36.661
26.670
20.764


3850
CB
MET
479
37.806
27.382
18.150


3851
CG
MET
479
37.642
27.208
16.648


3852
SD
MET
479
39.097
26.543
15.810


3853
CE
MET
479
38.341
26.133
14.224


3854
N
GLN
480
36.822
28.893
21.065


3855
CA
GLN
480
36.812
28.796
22.529


3856
C
GLN
480
35.392
28.739
23.075


3857
O
GLN
480
35.182
28.379
24.239


3858
CB
GLN
480
37.519
30.018
23.103


3859
CG
GLN
480
39.004
29.977
22.780


3860
CD
GLN
480
39.691
31.287
23.149


3861
OE1
GLN
480
40.100
32.065
22.276


3862
NE2
GLN
480
39.823
31.504
24.443


3863
N
SER
481
34.430
29.060
22.230


3864
CA
SER
481
33.030
28.954
22.624


3865
C
SER
481
32.401
27.721
21.994


3866
O
SER
481
32.965
27.100
21.087


3867
CB
SER
481
32.283
30.183
22.126


3868
OG
SER
481
32.195
30.091
20.710


3869
N
LYS
482
31.256
27.345
22.536


3870
CA
LYS
482
30.408
26.358
21.870


3871
C
LYS
482
29.896
26.979
20.580


3872
O
LYS
482
29.800
28.209
20.486


3873
CB
LYS
482
29.240
26.006
22.778


3874
CG
LYS
482
29.731
25.410
24.091


3875
CD
LYS
482
28.573
25.151
25.045


3876
CE
LYS
482
27.853
26.448
25.404


3877
NZ
LYS
482
28.759
27.387
26.089


3878
N
ARG
483
29.437
26.146
19.660


3879
CA
ARG
483
29.051
26.646
18.333


3880
C
ARG
483
27.820
27.543
18.384


3881
O
ARG
483
27.792
28.564
17.695


3882
CB
ARG
483
28.685
25.470
17.446


3883
CG
ARG
483
29.624
24.287
17.596


3884
CD
ARG
483
29.083
23.150
16.747


3885
NE
ARG
483
27.614
23.129
16.854


3886
CZ
ARG
483
26.900
22.116
17.347


3887
NH1
ARG
483
27.513
21.031
17.821


3888
NH2
ARG
483
25.569
22.201
17.390


3889
N
ILE
484
26.961
27.317
19.364


3890
CA
ILE
484
25.721
28.085
19.512


3891
C
ILE
484
25.902
29.424
20.260


3892
O
ILE
484
24.937
30.185
20.403


3893
CB
ILE
484
24.746
27.159
20.246


3894
CG1
ILE
484
23.316
27.686
20.253


3895
CG2
ILE
484
25.220
26.886
21.670


3896
CD1
ILE
484
22.780
27.835
18.836


3897
N
HIS
485
27.117
29.759
20.670


3898
CA
HIS
485
27.305
30.990
21.446


3899
C
HIS
485
27.466
32.231
20.568


3900
O
HIS
485
28.499
32.418
19.919


3901
CB
HIS
485
28.538
30.832
22.328


3902
CG
HIS
485
28.921
32.102
23.064


3903
ND1
HIS
485
28.269
32.655
24.105


3904
CD2
HIS
485
29.992
32.918
22.785


3905
CE1
HIS
485
28.906
33.782
24.482


3906
NE2
HIS
485
29.971
33.945
23.665


3907
N
ASN
486
26.469
33.099
20.620


3908
CA
ASN
486
26.554
34.404
19.948


3909
C
ASN
486
27.587
35.293
20.632


3910
O
ASN
486
27.446
35.619
21.817


3911
CB
ASN
486
25.222
35.135
20.083


3912
CG
ASN
486
24.052
34.479
19.361


3913
OD1
ASN
486
24.207
33.508
18.611


3914
ND2
ASN
486
22.914
35.141
19.470


3915
N
TYR
487
28.592
35.722
19.892


3916
CA
TYR
487
29.553
36.655
20.488


3917
C
TYR
487
29.540
37.986
19.752


3918
O
TYR
487
29.232
38.051
18.557


3919
CB
TYR
487
30.959
36.055
20.545


3920
CG
TYR
487
31.736
35.871
19.241


3921
CD1
TYR
487
32.423
36.943
18.683


3922
CD2
TYR
487
31.797
34.621
18.639


3923
CE1
TYR
487
33.146
36.774
17.510


3924
CE2
TYR
487
32.522
34.449
17.467


3925
CZ
TYR
487
33.195
35.526
16.906


3926
OH
TYR
487
33.993
35.335
15.798


3927
N
GLU
488
29.749
39.054
20.501


3928
CA
GLU
488
29.811
40.385
19.891


3929
C
GLU
488
31.186
40.580
19.260


3930
O
GLU
488
32.194
40.131
19.818


3931
CB
GLU
488
29.542
41.437
20.967


3932
CG
GLU
488
29.414
42.846
20.389


3933
CD
GLU
488
29.018
43.826
21.486


3934
OE1
GLU
488
28.439
43.366
22.461


3935
OE2
GLU
488
29.294
45.007
21.335


3936
N
PHE
489
31.205
41.154
18.069


3937
CA
PHE
489
32.468
41.399
17.373


3938
C
PHE
489
33.294
42.471
18.077


3939
O
PHE
489
32.903
43.642
18.155


3940
CB
PHE
489
32.178
41.845
15.945


3941
CG
PHE
489
33.438
42.267
15.196


3942
CD1
PHE
489
34.359
41.309
14.797


3943
CD2
PHE
489
33.675
43.611
14.934


3944
CE1
PHE
489
35.517
41.694
14.135


3945
CE2
PHE
489
34.833
43.994
14.272


3946
CZ
PHE
489
35.755
43.035
13.874


3947
N
LYS
490
34.407
42.039
18.641


3948
CA
LYS
490
35.353
42.969
19.257


3949
C
LYS
490
36.735
42.778
18.651


3950
O
LYS
490
37.244
41.651
18.600


3951
CB
LYS
490
35.420
42.692
20.754


3952
CG
LYS
490
34.047
42.775
21.409


3953
CD
LYS
490
34.104
42.363
22.874


3954
CE
LYS
490
32.721
42.423
23.510


3955
NZ
LYS
490
32.174
43.786
23.433


3956
N
TRP
491
37.417
43.885
18.406


3957
CA
TRP
491
38.769
43.817
17.836


3958
C
TRP
491
39.816
43.315
18.827


3959
O
TRP
491
40.764
42.642
18.406


3960
CB
TRP
491
39.189
45.196
17.343


3961
CG
TRP
491
38.634
45.571
15.987


3962
CD1
TRP
491
37.580
46.419
15.720


3963
CD2
TRP
491
39.124
45.110
14.710


3964
NE1
TRP
491
37.420
46.492
14.373


3965
CE2
TRP
491
38.326
45.728
13.731


3966
CE3
TRP
491
40.152
44.256
14.338


3967
CZ2
TRP
491
38.571
45.486
12.389


3968
CZ3
TRP
491
40.390
44.016
12.990


3969
CH2
TRP
491
39.602
44.629
12.020


3970
N
ASP
492
39.503
43.352
20.114


3971
CA
ASP
492
40.454
42.826
21.093


3972
C
ASP
492
40.446
41.297
21.131


3973
O
ASP
492
41.521
40.724
21.334


3974
CB
ASP
492
40.155
43.369
22.483


3975
CG
ASP
492
41.299
42.961
23.410


3976
OD1
ASP
492
41.009
42.450
24.481


3977
OD2
ASP
492
42.428
42.998
22.941


3978
N
ARG
493
39.413
40.674
20.581


3979
CA
ARG
493
39.365
39.209
20.547


3980
C
ARG
493
40.241
38.663
19.420


3981
O
ARG
493
40.711
37.523
19.485


3982
CB
ARG
493
37.925
38.782
20.298


3983
CG
ARG
493
36.974
39.410
21.307


3984
CD
ARG
493
35.532
39.005
21.027


3985
NE
ARG
493
35.351
37.558
21.210


3986
CZ
ARG
493
34.384
37.044
21.970


3987
NH1
ARG
493
33.500
37.852
22.560


3988
NH2
ARG
493
34.281
35.720
22.113


3989
N
MET
494
40.600
39.540
18.495


3990
CA
MET
494
41.486
39.162
17.401


3991
C
MET
494
42.929
39.552
17.719


3992
O
MET
494
43.866
38.957
17.180


3993
CB
MET
494
41.043
39.943
16.167


3994
CG
MET
494
39.525
39.978
16.028


3995
SD
MET
494
38.705
38.390
15.759


3996
CE
MET
494
36.990
38.937
15.918


3997
N
THR
495
43.099
40.487
18.642


3998
CA
THR
495
44.429
41.057
18.907


3999
C
THR
495
45.061
40.589
20.215


4000
O
THR
495
46.223
40.921
20.483


4001
CB
THR
495
44.319
42.577
18.944


4002
OG1
THR
495
43.400
42.931
19.971


4003
CG2
THR
495
43.818
43.146
17.620


4004
N
SER
496
44.302
39.900
21.048


4005
CA
SER
496
44.868
39.395
22.300


4006
C
SER
496
45.779
38.201
22.046


4007
O
SER
496
45.360
37.162
21.528


4008
CB
SER
496
43.742
39.013
23.253


4009
OG
SER
496
43.060
40.206
23.619


4010
N
PHE
497
47.049
38.409
22.344


4011
CA
PHE
497
48.047
37.350
22.196


4012
C
PHE
497
48.391
36.696
23.532


4013
O
PHE
497
48.893
35.568
23.567


4014
CB
PHE
497
49.295
37.920
21.511


4015
CG
PHE
497
50.003
39.105
22.172


4016
CD1
PHE
497
49.640
40.409
21.854


4017
CD2
PHE
497
51.046
38.877
23.062


4018
CE1
PHE
497
50.291
41.479
22.453


4019
CE2
PHE
497
51.699
39.947
23.659


4020
CZ
PHE
497
51.319
41.249
23.358


4021
N
GLU
498
47.975
37.343
24.607


4022
CA
GLU
498
48.266
36.852
25.958


4023
C
GLU
498
47.151
35.962
26.497


4024
O
GLU
498
46.022
35.976
25.996


4025
CB
GLU
498
48.441
38.054
26.878


4026
CG
GLU
498
49.628
38.909
26.451


4027
CD
GLU
498
49.721
40.161
27.318


4028
OE1
GLU
498
48.672
40.679
27.669


4029
OE2
GLU
498
50.833
40.616
27.542


4030
N
GLY
499
47.504
35.172
27.497


4031
CA
GLY
499
46.524
34.349
28.215


4032
C
GLY
499
46.065
33.159
27.388


4033
O
GLY
499
46.858
32.511
26.703


4034
N
ASP
500
44.765
32.932
27.387


4035
CA
ASP
500
44.201
31.807
26.632


4036
C
ASP
500
43.779
32.242
25.235


4037
O
ASP
500
42.580
32.387
24.969


4038
CB
ASP
500
42.981
31.249
27.362


4039
CG
ASP
500
43.371
30.397
28.568


4040
OD1
ASP
500
43.990
30.930
29.479


4041
OD2
ASP
500
42.909
29.266
28.615


4042
N
THR
501
44.754
32.455
24.366


4043
CA
THR
501
44.458
32.873
22.989


4044
C
THR
501
45.276
32.110
21.951


4045
O
THR
501
46.254
31.422
22.272


4046
CB
THR
501
44.760
34.359
22.840


4047
OG1
THR
501
46.102
34.581
23.256


4048
CG2
THR
501
43.843
35.230
23.693


4049
N
GLY
502
44.853
32.270
20.706


4050
CA
GLY
502
45.572
31.766
19.514


4051
C
GLY
502
47.102
31.832
19.602


4052
O
GLY
502
47.750
30.779
19.661


4053
N
PRO
503
47.681
33.028
19.630


4054
CA
PRO
503
49.146
33.163
19.729


4055
C
PRO
503
49.819
32.626
21.005


4056
O
PRO
503
51.023
32.356
20.941


4057
CB
PRO
503
49.419
34.624
19.576


4058
CG
PRO
503
48.111
35.364
19.364


4059
CD
PRO
503
47.017
34.327
19.483


4060
N
TYR
504
49.077
32.262
22.039


4061
CA
TYR
504
49.693
31.586
23.185


4062
C
TYR
504
49.924
30.107
22.878


4063
O
TYR
504
50.989
29.574
23.215


4064
CB
TYR
504
48.768
31.718
24.386


4065
CG
TYR
504
49.077
30.760
25.535


4066
CD1
TYR
504
48.227
29.686
25.775


4067
CD2
TYR
504
50.188
30.962
26.345


4068
CE1
TYR
504
48.492
28.810
26.818


4069
CE2
TYR
504
50.455
30.084
27.390


4070
CZ
TYR
504
49.605
29.011
27.624


4071
OH
TYR
504
49.831
28.169
28.694


4072
N
LEU
505
49.085
29.550
22.018


4073
CA
LEU
505
49.268
28.164
21.575


4074
C
LEU
505
50.383
28.103
20.543


4075
O
LEU
505
51.249
27.219
20.606


4076
CB
LEU
505
47.976
27.691
20.922


4077
CG
LEU
505
46.808
27.746
21.894


4078
CD1
LEU
505
45.488
27.497
21.172


4079
CD2
LEU
505
47.006
26.756
23.036


4080
N
GLN
506
50.499
29.194
19.802


4081
CA
GLN
506
51.588
29.357
18.842


4082
C
GLN
506
52.930
29.462
19.557


4083
O
GLN
506
53.829
28.672
19.256


4084
CB
GLN
506
51.348
30.637
18.045


4085
CG
GLN
506
50.116
30.559
17.147


4086
CD
GLN
506
50.416
29.749
15.889


4087
OE1
GLN
506
51.382
28.986
15.837


4088
NE2
GLN
506
49.559
29.906
14.898


4089
N
TYR
507
52.962
30.196
20.660


4090
CA
TYR
507
54.207
30.374
21.414


4091
C
TYR
507
54.613
29.127
22.195


4092
O
TYR
507
55.809
28.808
22.227


4093
CB
TYR
507
54.020
31.534
22.386


4094
CG
TYR
507
55.267
31.869
23.200


4095
CD1
TYR
507
56.503
31.960
22.571


4096
CD2
TYR
507
55.163
32.094
24.567


4097
CE1
TYR
507
57.639
32.259
23.311


4098
CE2
TYR
507
56.298
32.395
25.308


4099
CZ
TYR
507
57.533
32.473
24.678


4100
OH
TYR
507
58.664
32.738
25.419


4101
N
ALA
508
53.647
28.315
22.592


4102
CA
ALA
508
53.975
27.068
23.286


4103
C
ALA
508
54.576
26.043
22.328


4104
O
ALA
508
55.646
25.494
22.628


4105
CB
ALA
508
52.705
26.511
23.918


4106
N
HIS
509
54.085
26.031
21.098


4107
CA
HIS
509
54.633
25.125
20.083


4108
C
HIS
509
56.008
25.614
19.639


4109
O
HIS
509
56.976
24.838
19.638


4110
CB
HIS
509
53.688
25.133
18.887


4111
CG
HIS
509
53.968
24.061
17.855


4112
ND1
HIS
509
54.830
24.124
16.820


4113
CD2
HIS
509
53.374
22.822
17.806


4114
CE1
HIS
509
54.783
22.965
16.132


4115
NE2
HIS
509
53.883
22.161
16.742


4116
N
SER
510
56.121
26.929
19.555


4117
CA
SER
510
57.364
27.602
19.181


4118
C
SER
510
58.520
27.287
20.114


4119
O
SER
510
59.513
26.708
19.658


4120
CB
SER
510
57.113
29.104
19.210


4121
OG
SER
510
56.270
29.425
18.112


4122
N
ARG
511
58.323
27.441
21.414


4123
CA
ARG
511
59.440
27.205
22.335


4124
C
ARG
511
59.682
25.729
22.654


4125
O
ARG
511
60.826
25.378
22.969


4126
CB
ARG
511
59.235
28.007
23.612


4127
CG
ARG
511
57.882
27.738
24.250


4128
CD
ARG
511
57.699
28.600
25.486


4129
NE
ARG
511
56.303
28.565
25.936


4130
CZ
ARG
511
55.914
29.056
27.113


4131
NH1
ARG
511
56.822
29.502
27.984


4132
NH2
ARG
511
54.622
29.040
27.442


4133
N
LEU
512
58.738
24.854
22.343


4134
CA
LEU
512
59.024
23.424
22.487


4135
C
LEU
512
59.877
22.951
21.320


4136
O
LEU
512
60.971
22.415
21.549


4137
CB
LEU
512
57.727
22.632
22.531


4138
CG
LEU
512
56.978
22.870
23.833


4139
CD1
LEU
512
55.644
22.135
23.824


4140
CD2
LEU
512
57.820
22.442
25.030


4141
N
CYS
513
59.580
23.488
20.146


4142
CA
CYS
513
60.380
23.157
18.969


4143
C
CYS
513
61.713
23.902
18.966


4144
O
CYS
513
62.705
23.342
18.492


4145
CB
CYS
513
59.599
23.502
17.710


4146
SG
CYS
513
60.418
23.024
16.173


4147
N
SER
514
61.799
24.998
19.703


4148
CA
SER
514
63.073
25.705
19.880


4149
C
SER
514
63.990
25.016
20.885


4150
O
SER
514
65.213
25.034
20.705


4151
CB
SER
514
62.783
27.114
20.381


4152
OG
SER
514
64.033
27.742
20.629


4153
N
MET
515
63.409
24.233
21.778


4154
CA
MET
515
64.209
23.455
22.724


4155
C
MET
515
64.711
22.185
22.038


4156
O
MET
515
65.847
21.748
22.263


4157
CB
MET
515
63.314
23.088
23.901


4158
CG
MET
515
64.128
22.752
25.143


4159
SD
MET
515
65.092
24.137
25.790


4160
CE
MET
515
65.717
23.382
27.306


4161
N
GLN
516
63.968
21.762
21.028


4162
CA
GLN
516
64.387
20.636
20.186


4163
C
GLN
516
65.362
21.082
19.090


4164
O
GLN
516
66.224
20.297
18.672


4165
CB
GLN
516
63.125
20.056
19.566


4166
CG
GLN
516
62.170
19.587
20.658


4167
CD
GLN
516
60.794
19.316
20.062


4168
OE1
GLN
516
59.766
19.704
20.632


4169
NE2
GLN
516
60.792
18.658
18.917


4170
N
ARG
517
65.373
22.381
18.824


4171
CA
ARG
517
66.319
23.011
17.894


4172
C
ARG
517
67.676
23.316
18.525


4173
O
ARG
517
68.549
23.888
17.862


4174
CB
ARG
517
65.711
24.280
17.302


4175
CG
ARG
517
64.841
23.937
16.095


4176
CD
ARG
517
64.356
25.173
15.339


4177
NE
ARG
517
63.177
25.805
15.957


4178
CZ
ARG
517
63.154
27.060
16.411


4179
NH1
ARG
517
64.291
27.749
16.530


4180
NH2
ARG
517
62.018
27.571
16.888


4181
N
LYS
518
67.877
22.859
19.754


4182
CA
LYS
518
69.214
22.838
20.350


4183
C
LYS
518
70.005
21.630
19.824


4184
O
LYS
518
71.225
21.568
20.013


4185
CB
LYS
518
69.078
22.750
21.867


4186
CG
LYS
518
68.243
23.899
22.427


4187
CD
LYS
518
68.901
25.256
22.200


4188
CE
LYS
518
67.999
26.387
22.689


4189
NZ
LYS
518
67.668
26.207
24.112


4190
N
SER
519
69.303
20.734
19.133


4191
CA
SER
519
69.852
19.620
18.332


4192
C
SER
519
71.030
18.864
18.937


4193
O
SER
519
72.192
19.277
18.850


4194
CB
SER
519
70.225
20.153
16.950


4195
OG
SER
519
71.131
21.239
17.100


4196
N
GLY
520
70.715
17.694
19.459


4197
CA
GLY
520
71.744
16.816
20.010


4198
C
GLY
520
71.233
15.384
20.004


4199
O
GLY
520
71.679
14.539
19.219


4200
N
ILE
521
70.293
15.134
20.898


4201
CA
ILE
521
69.672
13.814
21.021


4202
C
ILE
521
68.608
13.630
19.942


4203
O
ILE
521
67.752
14.500
19.742


4204
CB
ILE
521
69.049
13.761
22.410


4205
CG1
ILE
521
70.088
14.210
23.432


4206
CG2
ILE
521
68.558
12.355
22.734


4207
CD1
ILE
521
69.474
14.413
24.809


4208
N
SER
522
68.718
12.532
19.213


4209
CA
SER
522
67.772
12.230
18.134


4210
C
SER
522
66.447
11.718
18.685


4211
O
SER
522
66.398
11.135
19.777


4212
CB
SER
522
68.374
11.165
17.226


4213
OG
SER
522
68.346
9.926
17.925


4214
N
ILE
523
65.451
11.703
17.811


4215
CA
ILE
523
64.082
11.299
18.177


4216
C
ILE
523
63.947
9.795
18.420


4217
O
ILE
523
63.190
9.385
19.309


4218
CB
ILE
523
63.165
11.717
17.032


4219
CG1
ILE
523
63.197
13.230
16.851


4220
CG2
ILE
523
61.735
11.233
17.253


4221
CD1
ILE
523
62.248
13.678
15.746


4222
N
GLU
524
64.898
9.040
17.895


4223
CA
GLU
524
64.942
7.592
18.122


4224
C
GLU
524
65.318
7.309
19.573


4225
O
GLU
524
64.573
6.603
20.263


4226
CB
GLU
524
65.985
6.939
17.210


4227
CG
GLU
524
65.774
7.216
15.721


4228
CD
GLU
524
66.724
8.312
15.237


4229
OE1
GLU
524
67.873
7.991
14.987


4230
OE2
GLU
524
66.359
9.471
15.413


4231
N
GLU
525
66.204
8.141
20.101


4232
CA
GLU
525
66.634
8.005
21.493


4233
C
GLU
525
65.604
8.589
22.450


4234
O
GLU
525
65.487
8.099
23.576


4235
CB
GLU
525
67.941
8.761
21.676


4236
CG
GLU
525
69.049
8.204
20.796


4237
CD
GLU
525
70.259
9.125
20.876


4238
OE1
GLU
525
71.177
8.816
21.620


4239
OE2
GLU
525
70.238
10.131
20.176


4240
N
LEU
526
64.722
9.433
21.938


4241
CA
LEU
526
63.649
9.991
22.763


4242
C
LEU
526
62.498
8.994
22.887


4243
O
LEU
526
61.878
8.903
23.954


4244
CB
LEU
526
63.144
11.276
22.115


4245
CG
LEU
526
64.253
12.299
21.901


4246
CD1
LEU
526
63.713
13.547
21.212


4247
CD2
LEU
526
64.934
12.668
23.211


4248
N
GLU
527
62.423
8.071
21.940


4249
CA
GLU
527
61.445
6.978
22.012


4250
C
GLU
527
61.960
5.846
22.901


4251
O
GLU
527
61.194
4.963
23.308


4252
CB
GLU
527
61.233
6.433
20.603


4253
CG
GLU
527
60.717
7.507
19.655


4254
CD
GLU
527
60.700
6.989
18.221


4255
OE1
GLU
527
59.777
6.256
17.895


4256
OE2
GLU
527
61.659
7.257
17.509


4257
N
HIS
528
63.246
5.900
23.212


4258
CA
HIS
528
63.885
4.925
24.101


4259
C
HIS
528
64.309
5.574
25.421


4260
O
HIS
528
65.085
4.976
26.178


4261
CB
HIS
528
65.125
4.365
23.408


4262
CG
HIS
528
64.884
3.721
22.056


4263
ND1
HIS
528
63.822
2.977
21.683


4264
CD2
HIS
528
65.727
3.778
20.972


4265
CE1
HIS
528
63.975
2.588
20.403


4266
NE2
HIS
528
65.154
3.085
19.963


4267
N
ALA
529
63.805
6.772
25.681


4268
CA
ALA
529
64.204
7.567
26.851


4269
C
ALA
529
63.973
6.858
28.178


4270
O
ALA
529
63.190
5.905
28.272


4271
CB
ALA
529
63.402
8.861
26.841


4272
N
ASN
530
64.718
7.266
29.190


4273
CA
ASN
530
64.478
6.681
30.507


4274
C
ASN
530
63.313
7.396
31.175


4275
O
ASN
530
63.478
8.417
31.858


4276
CB
ASN
530
65.711
6.744
31.394


4277
CG
ASN
530
65.383
5.977
32.672


4278
OD1
ASN
530
64.801
6.529
33.614


4279
ND2
ASN
530
65.615
4.677
32.624


4280
N
PHE
531
62.200
6.687
31.196


4281
CA
PHE
531
60.939
7.231
31.703


4282
C
PHE
531
60.823
7.209
33.232


4283
O
PHE
531
59.974
7.914
33.789


4284
CB
PHE
531
59.828
6.428
31.042


4285
CG
PHE
531
59.847
6.543
29.516


4286
CD1
PHE
531
59.669
7.782
28.912


4287
CD2
PHE
531
60.054
5.417
28.731


4288
CE1
PHE
531
59.693
7.892
27.528


4289
CE2
PHE
531
60.077
5.527
27.346


4290
CZ
PHE
531
59.897
6.765
26.744


4291
N
ASP
532
61.831
6.663
33.898


4292
CA
ASP
532
61.859
6.635
35.365


4293
C
ASP
532
62.401
7.959
35.905


4294
O
ASP
532
62.178
8.301
37.072


4295
CB
ASP
532
62.792
5.518
35.826


4296
CG
ASP
532
62.481
4.201
35.125


4297
OD1
ASP
532
63.396
3.649
34.521


4298
OD2
ASP
532
61.328
3.796
35.143


4299
N
LEU
533
62.998
8.747
35.023


4300
CA
LEU
533
63.484
10.076
35.395


4301
C
LEU
533
62.417
11.157
35.224


4302
O
LEU
533
62.644
12.303
35.625


4303
CB
LEU
533
64.675
10.409
34.507


4304
CG
LEU
533
65.806
9.413
34.719


4305
CD1
LEU
533
66.880
9.557
33.648


4306
CD2
LEU
533
66.402
9.544
36.117


4307
N
LEU
534
61.245
10.787
34.730


4308
CA
LEU
534
60.180
11.765
34.471


4309
C
LEU
534
59.127
11.860
35.573


4310
O
LEU
534
58.122
12.550
35.375


4311
CB
LEU
534
59.494
11.381
33.169


4312
CG
LEU
534
60.398
11.625
31.975


4313
CD1
LEU
534
59.807
11.013
30.712


4314
CD2
LEU
534
60.644
13.117
31.797


4315
N
VAL
535
59.411
11.323
36.751


4316
CA
VAL
535
58.390
11.167
37.805


4317
C
VAL
535
58.001
12.472
38.538


4318
O
VAL
535
56.987
12.497
39.247


4319
CB
VAL
535
58.936
10.108
38.769


4320
CG1
VAL
535
58.018
9.824
39.957


4321
CG2
VAL
535
59.200
8.808
38.018


4322
N
GLU
536
58.687
13.567
38.240


4323
CA
GLU
536
58.360
14.890
38.804


4324
C
GLU
536
56.918
15.265
38.469


4325
O
GLU
536
56.464
15.014
37.348


4326
CB
GLU
536
59.294
15.957
38.213


4327
CG
GLU
536
60.734
15.892
38.731


4328
CD
GLU
536
61.673
15.192
37.747


4329
OE1
GLU
536
61.221
14.254
37.106


4330
OE2
GLU
536
62.844
15.540
37.741


4331
N
PRO
537
56.244
15.957
39.378


4332
CA
PRO
537
54.775
16.055
39.314


4333
C
PRO
537
54.224
16.841
38.117


4334
O
PRO
537
53.164
16.468
37.598


4335
CB
PRO
537
54.371
16.710
40.599


4336
CG
PRO
537
55.608
17.025
41.424


4337
CD
PRO
537
56.788
16.482
40.635


4338
N
CYS
538
55.026
17.717
37.526


4339
CA
CYS
538
54.555
18.454
36.349


4340
C
CYS
538
54.594
17.565
35.108


4341
O
CYS
538
53.610
17.526
34.360


4342
CB
CYS
538
55.441
19.672
36.116


4343
SG
CYS
538
54.740
20.882
34.974


4344
N
ALA
539
55.546
16.645
35.085


4345
CA
ALA
539
55.683
15.718
33.961


4346
C
ALA
539
54.718
14.553
34.125


4347
O
ALA
539
54.158
14.063
33.136


4348
CB
ALA
539
57.114
15.195
33.952


4349
N
SER
540
54.328
14.328
35.368


4350
CA
SER
540
53.317
13.321
35.687


4351
C
SER
540
51.955
13.755
35.163


4352
O
SER
540
51.387
13.058
34.313


4353
CB
SER
540
53.247
13.166
37.205


4354
OG
SER
540
54.543
12.817
37.680


4355
N
ALA
541
51.604
15.007
35.420


4356
CA
ALA
541
50.309
15.529
34.969


4357
C
ALA
541
50.293
15.843
33.475


4358
O
ALA
541
49.264
15.634
32.816


4359
CB
ALA
541
49.992
16.791
35.760


4360
N
LEU
542
51.463
16.094
32.912


4361
CA
LEU
542
51.553
16.315
31.472


4362
C
LEU
542
51.385
15.002
30.712


4363
O
LEU
542
50.568
14.961
29.787


4364
CB
LEU
542
52.907
16.941
31.155


4365
CG
LEU
542
53.004
17.366
29.695


4366
CD1
LEU
542
51.849
18.289
29.315


4367
CD2
LEU
542
54.343
18.037
29.414


4368
N
ALA
543
51.876
13.906
31.268


4369
CA
ALA
543
51.694
12.606
30.613


4370
C
ALA
543
50.282
12.061
30.806


4371
O
ALA
543
49.726
11.470
29.871


4372
CB
ALA
543
52.696
11.617
31.187


4373
N
ARG
544
49.634
12.470
31.886


4374
CA
ARG
544
48.235
12.098
32.106


4375
C
ARG
544
47.337
12.772
31.077


4376
O
ARG
544
46.631
12.065
30.345


4377
CB
ARG
544
47.828
12.530
33.510


4378
CG
ARG
544
48.584
11.736
34.569


4379
CD
ARG
544
48.397
12.326
35.962


4380
NE
ARG
544
46.981
12.350
36.351


4381
CZ
ARG
544
46.577
12.227
37.617


4382
NH1
ARG
544
47.476
12.088
38.594


4383
NH2
ARG
544
45.276
12.247
37.907


4384
N
THR
545
47.597
14.043
30.815


4385
CA
THR
545
46.794
14.767
29.824


4386
C
THR
545
47.160
14.409
28.384


4387
O
THR
545
46.245
14.297
27.563


4388
CB
THR
545
46.930
16.271
30.041


4389
OG1
THR
545
48.302
16.635
29.963


4390
CG2
THR
545
46.406
16.682
31.410


4391
N
LEU
546
48.377
13.941
28.147


4392
CA
LEU
546
48.742
13.492
26.794


4393
C
LEU
546
48.121
12.134
26.463


4394
O
LEU
546
47.693
11.925
25.320


4395
CB
LEU
546
50.260
13.384
26.675


4396
CG
LEU
546
50.951
14.738
26.797


4397
CD1
LEU
546
52.467
14.578
26.783


4398
CD2
LEU
546
50.497
15.700
25.707


4399
N
ALA
547
47.833
11.351
27.492


4400
CA
ALA
547
47.144
10.070
27.307


4401
C
ALA
547
45.636
10.257
27.157


4402
O
ALA
547
44.957
9.403
26.572


4403
CB
ALA
547
47.416
9.195
28.524


4404
N
GLN
548
45.165
11.443
27.508


4405
CA
GLN
548
43.753
11.792
27.379


4406
C
GLN
548
43.417
12.379
26.010


4407
O
GLN
548
42.226
12.543
25.710


4408
CB
GLN
548
43.412
12.786
28.481


4409
CG
GLN
548
43.569
12.126
29.845


4410
CD
GLN
548
43.540
13.164
30.962


4411
OE1
GLN
548
43.469
14.374
30.713


4412
NE2
GLN
548
43.710
12.683
32.181


4413
N
TYR
549
44.407
12.535
25.142


4414
CA
TYR
549
44.143
13.015
23.772


4415
C
TYR
549
43.109
12.166
23.003


4416
O
TYR
549
42.097
12.769
22.623


4417
CB
TYR
549
45.443
13.118
22.977


4418
CG
TYR
549
45.296
13.888
21.663


4419
CD1
TYR
549
44.433
14.975
21.599


4420
CD2
TYR
549
46.027
13.516
20.541


4421
CE1
TYR
549
44.294
15.688
20.415


4422
CE2
TYR
549
45.888
14.226
19.355


4423
CZ
TYR
549
45.020
15.310
19.295


4424
OH
TYR
549
44.839
15.986
18.108


4425
N
PRO
550
43.214
10.840
22.887


4426
CA
PRO
550
42.136
10.109
22.199


4427
C
PRO
550
40.775
10.140
22.918


4428
O
PRO
550
39.752
10.160
22.223


4429
CB
PRO
550
42.631
8.703
22.053


4430
CG
PRO
550
43.960
8.556
22.774


4431
CD
PRO
550
44.290
9.930
23.324


4432
N
ASP
551
40.750
10.408
24.219


4433
CA
ASP
551
39.474
10.585
24.928


4434
C
ASP
551
38.786
11.853
24.447


4435
O
ASP
551
37.666
11.786
23.925


4436
CB
ASP
551
39.717
10.780
26.422


4437
CG
ASP
551
40.368
9.571
27.070


4438
OD1
ASP
551
39.948
8.465
26.754


4439
OD2
ASP
551
41.162
9.781
27.976


4440
N
VAL
552
39.564
12.921
24.361


4441
CA
VAL
552
39.030
14.231
23.983


4442
C
VAL
552
38.716
14.331
22.492


4443
O
VAL
552
37.734
14.992
22.131


4444
CB
VAL
552
40.066
15.277
24.377


4445
CG1
VAL
552
39.635
16.678
23.964


4446
CG2
VAL
552
40.331
15.224
25.877


4447
N
ILE
553
39.334
13.486
21.682


4448
CA
ILE
553
38.977
13.444
20.262


4449
C
ILE
553
37.632
12.755
20.057


4450
O
ILE
553
36.771
13.321
19.371


4451
CB
ILE
553
40.058
12.697
19.492


4452
CG1
ILE
553
41.372
13.460
19.545


4453
CG2
ILE
553
39.641
12.465
18.043


4454
CD1
ILE
553
42.443
12.749
18.731


4455
N
LYS
554
37.343
11.758
20.878


4456
CA
LYS
554
36.055
11.069
20.774


4457
C
LYS
554
34.931
11.895
21.396


4458
O
LYS
554
33.834
11.948
20.826


4459
CB
LYS
554
36.165
9.721
21.476


4460
CG
LYS
554
37.248
8.864
20.830


4461
CD
LYS
554
37.401
7.515
21.523


4462
CE
LYS
554
38.536
6.710
20.897


4463
NZ
LYS
554
38.639
5.370
21.494


4464
N
LYS
555
35.278
12.741
22.353


4465
CA
LYS
555
34.289
13.649
22.944


4466
C
LYS
555
34.001
14.841
22.032


4467
O
LYS
555
32.836
15.243
21.901


4468
CB
LYS
555
34.842
14.144
24.275


4469
CG
LYS
555
35.092
12.979
25.223


4470
CD
LYS
555
35.842
13.423
26.473


4471
CE
LYS
555
36.169
12.235
27.371


4472
NZ
LYS
555
36.954
12.657
28.542


4473
N
ALA
556
34.977
15.202
21.213


4474
CA
ALA
556
34.782
16.270
20.233


4475
C
ALA
556
34.003
15.786
19.017


4476
O
ALA
556
33.315
16.596
18.387


4477
CB
ALA
556
36.144
16.787
19.788


4478
N
VAL
557
33.973
14.480
18.793


4479
CA
VAL
557
33.119
13.924
17.738


4480
C
VAL
557
31.693
13.749
18.248


4481
O
VAL
557
30.744
14.171
17.575


4482
CB
VAL
557
33.648
12.551
17.330


4483
CG1
VAL
557
32.782
11.929
16.239


4484
CG2
VAL
557
35.096
12.614
16.872


4485
N
LYS
558
31.584
13.410
19.523


4486
CA
LYS
558
30.286
13.116
20.142


4487
C
LYS
558
29.452
14.366
20.425


4488
O
LYS
558
28.217
14.302
20.405


4489
CB
LYS
558
30.581
12.392
21.453


4490
CG
LYS
558
29.316
12.014
22.213


4491
CD
LYS
558
29.652
11.359
23.547


4492
CE
LYS
558
28.388
11.009
24.325


4493
NZ
LYS
558
28.722
10.395
25.620


4494
N
GLY
559
30.105
15.508
20.551


4495
CA
GLY
559
29.361
16.753
20.746


4496
C
GLY
559
29.657
17.783
19.661


4497
O
GLY
559
29.181
18.921
19.762


4498
N
LEU
560
30.354
17.349
18.617


4499
CA
LEU
560
30.850
18.221
17.532


4500
C
LEU
560
31.318
19.575
18.044


4501
O
LEU
560
30.714
20.611
17.753


4502
CB
LEU
560
29.754
18.409
16.494


4503
CG
LEU
560
29.463
17.101
15.774


4504
CD1
LEU
560
28.304
17.269
14.802


4505
CD2
LEU
560
30.708
16.601
15.048


4506
N
GLU
561
32.380
19.570
18.826


4507
CA
GLU
561
32.774
20.822
19.467


4508
C
GLU
561
34.243
21.160
19.314


4509
O
GLU
561
35.114
20.541
19.942


4510
CB
GLU
561
32.398
20.779
20.945


4511
CG
GLU
561
30.973
21.271
21.188


4512
CD
GLU
561
30.884
22.759
20.859


4513
OE1
GLU
561
31.934
23.392
20.850


4514
OE2
GLU
561
29.779
23.265
20.708


4515
N
PRO
562
34.470
22.285
18.650


4516
CA
PRO
562
35.818
22.847
18.535


4517
C
PRO
562
36.396
23.263
19.888


4518
O
PRO
562
37.545
22.892
20.163


4519
CB
PRO
562
35.671
24.027
17.623


4520
CG
PRO
562
34.200
24.234
17.293


4521
CD
PRO
562
33.451
23.117
17.999


4522
N
SER
563
35.528
23.644
20.817


4523
CA
SER
563
35.966
24.012
22.168


4524
C
SER
563
36.369
22.819
23.037


4525
O
SER
563
37.081
23.024
24.023


4526
CB
SER
563
34.843
24.774
22.862


4527
OG
SER
563
33.742
23.890
23.020


4528
N
THR
564
36.133
21.597
22.585


4529
CA
THR
564
36.591
20.438
23.352


4530
C
THR
564
38.090
20.265
23.137


4531
O
THR
564
38.862
20.289
24.106


4532
CB
THR
564
35.863
19.194
22.856


4533
OG1
THR
564
34.465
19.434
22.927


4534
CG2
THR
564
36.182
17.972
23.709


4535
N
ILE
565
38.496
20.450
21.890


4536
CA
ILE
565
39.912
20.330
21.540


4537
C
ILE
565
40.668
21.611
21.885


4538
O
ILE
565
41.799
21.530
22.377


4539
CB
ILE
565
40.021
20.034
20.046


4540
CG1
ILE
565
39.387
18.689
19.712


4541
CG2
ILE
565
41.475
20.054
19.586


4542
CD1
ILE
565
40.152
17.532
20.346


4543
N
VAL
566
39.960
22.729
21.914


4544
CA
VAL
566
40.597
23.995
22.291


4545
C
VAL
566
40.805
24.120
23.802


4546
O
VAL
566
41.890
24.547
24.217


4547
CB
VAL
566
39.732
25.139
21.773


4548
CG1
VAL
566
40.221
26.489
22.274


4549
CG2
VAL
566
39.695
25.133
20.252


4550
N
THR
567
39.942
23.502
24.594


4551
CA
THR
567
40.128
23.542
26.049


4552
C
THR
567
41.148
22.501
26.497


4553
O
THR
567
41.982
22.791
27.367


4554
CB
THR
567
38.788
23.289
26.728


4555
OG1
THR
567
37.918
24.354
26.371


4556
CG2
THR
567
38.914
23.278
28.248


4557
N
TYR
568
41.257
21.430
25.728


4558
CA
TYR
568
42.300
20.440
25.992


4559
C
TYR
568
43.669
20.975
25.590


4560
O
TYR
568
44.633
20.830
26.353


4561
CB
TYR
568
41.990
19.182
25.189


4562
CG
TYR
568
43.131
18.171
25.158


4563
CD1
TYR
568
43.349
17.325
26.239


4564
CD2
TYR
568
43.958
18.104
24.044


4565
CE1
TYR
568
44.397
16.415
26.206


4566
CE2
TYR
568
45.009
17.199
24.013


4567
CZ
TYR
568
45.226
16.358
25.094


4568
OH
TYR
568
46.287
15.479
25.066


4569
N
LEU
569
43.694
21.790
24.551


4570
CA
LEU
569
44.948
22.370
24.087


4571
C
LEU
569
45.423
23.461
25.041


4572
O
LEU
569
46.590
23.428
25.450


4573
CB
LEU
569
44.699
22.941
22.698


4574
CG
LEU
569
45.997
23.256
21.973


4575
CD1
LEU
569
46.901
22.030
21.941


4576
CD2
LEU
569
45.704
23.748
20.562


4577
N
PHE
570
44.486
24.204
25.609


4578
CA
PHE
570
44.847
25.209
26.612


4579
C
PHE
570
45.343
24.575
27.901


4580
O
PHE
570
46.452
24.908
28.329


4581
CB
PHE
570
43.643
26.086
26.926


4582
CG
PHE
570
43.377
27.160
25.882


4583
CD1
PHE
570
44.444
27.815
25.282


4584
CD2
PHE
570
42.074
27.504
25.550


4585
CE1
PHE
570
44.210
28.795
24.328


4586
CE2
PHE
570
41.841
28.485
24.596


4587
CZ
PHE
570
42.907
29.127
23.981


4588
N
SER
571
44.709
23.499
28.334


4589
CA
SER
571
45.135
22.860
29.585


4590
C
SER
571
46.472
22.128
29.446


4591
O
SER
571
47.331
22.294
30.323


4592
CB
SER
571
44.045
21.903
30.065


4593
OG
SER
571
43.798
20.929
29.059


4594
N
VAL
572
46.760
21.604
28.264


4595
CA
VAL
572
48.052
20.948
28.054


4596
C
VAL
572
49.168
21.972
27.895


4597
O
VAL
572
50.200
21.826
28.561


4598
CB
VAL
572
47.972
20.052
26.822


4599
CG1
VAL
572
49.349
19.550
26.398


4600
CG2
VAL
572
47.034
18.880
27.074


4601
N
THR
573
48.860
23.117
27.306


4602
CA
THR
573
49.880
24.162
27.164


4603
C
THR
573
50.076
24.966
28.446


4604
O
THR
573
51.162
25.520
28.638


4605
CB
THR
573
49.523
25.112
26.026


4606
OG1
THR
573
48.258
25.698
26.297


4607
CG2
THR
573
49.447
24.389
24.688


4608
N
HIS
574
49.146
24.885
29.384


4609
CA
HIS
574
49.358
25.534
30.680


4610
C
HIS
574
50.315
24.698
31.508


4611
O
HIS
574
51.354
25.222
31.934


4612
CB
HIS
574
48.040
25.678
31.432


4613
CG
HIS
574
47.035
26.620
30.799


4614
ND1
HIS
574
47.295
27.649
29.972


4615
CD2
HIS
574
45.672
26.572
30.968


4616
CE1
HIS
574
46.133
28.244
29.625


4617
NE2
HIS
574
45.132
27.576
30.240


4618
N
ILE
575
50.161
23.387
31.390


4619
CA
ILE
575
51.070
22.468
32.075


4620
C
ILE
575
52.447
22.485
31.419


4621
O
ILE
575
53.446
22.584
32.141


4622
CB
ILE
575
50.486
21.061
32.032


4623
CG1
ILE
575
49.098
21.036
32.657


4624
CG2
ILE
575
51.396
20.079
32.759


4625
CD1
ILE
575
48.500
19.635
32.607


4626
N
VAL
576
52.480
22.735
30.119


4627
CA
VAL
576
53.752
22.892
29.406


4628
C
VAL
576
54.483
24.187
29.767


4629
O
VAL
576
55.698
24.121
29.981


4630
CB
VAL
576
53.476
22.866
27.904


4631
CG1
VAL
576
54.657
23.396
27.100


4632
CG2
VAL
576
53.089
21.469
27.435


4633
N
SER
577
53.756
25.234
30.132


4634
CA
SER
577
54.398
26.492
30.542


4635
C
SER
577
55.058
26.322
31.903


4636
O
SER
577
56.252
26.615
32.071


4637
CB
SER
577
53.332
27.576
30.678


4638
OG
SER
577
52.640
27.705
29.446


4639
N
GLN
578
54.360
25.596
32.761


4640
CA
GLN
578
54.851
25.321
34.109


4641
C
GLN
578
56.057
24.390
34.075


4642
O
GLN
578
57.112
24.728
34.628


4643
CB
GLN
578
53.727
24.620
34.856


4644
CG
GLN
578
52.435
25.432
34.821


4645
CD
GLN
578
51.264
24.503
35.125


4646
OE1
GLN
578
50.111
24.783
34.771


4647
NE2
GLN
578
51.599
23.346
35.669


4648
N
CYS
579
55.994
23.387
33.214


4649
CA
CYS
579
57.061
22.386
33.171


4650
C
CYS
579
58.284
22.913
32.435


4651
O
CYS
579
59.391
22.677
32.922


4652
CB
CYS
579
56.582
21.119
32.462


4653
SG
CYS
579
55.139
20.227
33.104


4654
N
TYR
580
58.094
23.860
31.530


4655
CA
TYR
580
59.213
24.445
30.780


4656
C
TYR
580
59.983
25.459
31.632


4657
O
TYR
580
61.160
25.737
31.374


4658
CB
TYR
580
58.613
25.141
29.560


4659
CG
TYR
580
59.573
25.506
28.431


4660
CD1
TYR
580
59.942
24.535
27.507


4661
CD2
TYR
580
60.062
26.803
28.313


4662
CE1
TYR
580
60.801
24.858
26.465


4663
CE2
TYR
580
60.922
27.127
27.271


4664
CZ
TYR
580
61.286
26.154
26.348


4665
OH
TYR
580
62.085
26.491
25.276


4666
N
ASP
581
59.365
25.881
32.724


4667
CA
ASP
581
60.016
26.766
33.686


4668
C
ASP
581
60.735
25.991
34.798


4669
O
ASP
581
61.591
26.561
35.489


4670
CB
ASP
581
58.911
27.636
34.282


4671
CG
ASP
581
59.457
28.627
35.303


4672
OD1
ASP
581
59.282
28.381
36.489


4673
OD2
ASP
581
59.978
29.649
34.879


4674
N
ILE
582
60.485
24.694
34.905


4675
CA
ILE
582
61.065
23.922
36.014


4676
C
ILE
582
61.993
22.800
35.543


4677
O
ILE
582
63.110
22.644
36.052


4678
CB
ILE
582
59.914
23.308
36.808


4679
CG1
ILE
582
58.950
24.379
37.306


4680
CG2
ILE
582
60.440
22.494
37.986


4681
CD1
ILE
582
57.758
23.759
38.025


4682
N
LEU
583
61.565
22.094
34.513


4683
CA
LEU
583
62.253
20.888
34.043


4684
C
LEU
583
63.132
21.192
32.835


4685
O
LEU
583
62.840
20.776
31.707


4686
CB
LEU
583
61.187
19.869
33.652


4687
CG
LEU
583
60.225
19.557
34.796


4688
CD1
LEU
583
59.066
18.692
34.312


4689
CD2
LEU
583
60.937
18.891
35.969


4690
N
TRP
584
64.213
21.904
33.091


4691
CA
TRP
584
65.121
22.319
32.019


4692
C
TRP
584
65.961
21.159
31.513


4693
O
TRP
584
66.691
20.528
32.277


4694
CB
TRP
584
66.045
23.408
32.552


4695
CG
TRP
584
65.326
24.684
32.935


4696
CD1
TRP
584
64.811
25.012
34.168


4697
CD2
TRP
584
65.057
25.807
32.065


4698
NE1
TRP
584
64.237
26.239
34.083


4699
CE2
TRP
584
64.364
26.747
32.846


4700
CE3
TRP
584
65.336
26.065
30.734


4701
CZ2
TRP
584
63.957
27.947
32.273


4702
CZ3
TRP
584
64.926
27.268
30.170


4703
CH2
TRP
584
64.240
28.203
30.937


4704
N
VAL
585
65.934
20.963
30.206


4705
CA
VAL
585
66.746
19.918
29.568


4706
C
VAL
585
68.185
20.390
29.350


4707
O
VAL
585
69.134
19.592
29.330


4708
CB
VAL
585
66.110
19.630
28.212


4709
CG1
VAL
585
66.910
18.611
27.416


4710
CG2
VAL
585
64.664
19.182
28.363


4711
N
SER
586
68.342
21.703
29.333


4712
CA
SER
586
69.657
22.317
29.132


4713
C
SER
586
70.594
22.050
30.305


4714
O
SER
586
70.229
22.216
31.477


4715
CB
SER
586
69.474
23.821
28.953


4716
OG
SER
586
68.883
24.344
30.137


4717
N
GLY
587
71.749
21.504
29.959


4718
CA
GLY
587
72.834
21.267
30.918


4719
C
GLY
587
72.535
20.139
31.901


4720
O
GLY
587
72.861
20.255
33.087


4721
N
GLN
588
71.849
19.109
31.436


4722
CA
GLN
588
71.526
17.987
32.320


4723
C
GLN
588
72.361
16.764
32.005


4724
O
GLN
588
73.204
16.770
31.100


4725
CB
GLN
588
70.057
17.623
32.157


4726
CG
GLN
588
69.165
18.808
32.482


4727
CD
GLN
588
69.416
19.294
33.905


4728
OE1
GLN
588
69.512
18.491
34.843


4729
NE2
GLN
588
69.571
20.600
34.033


4730
N
GLU
589
72.160
15.742
32.817


4731
CA
GLU
589
72.734
14.432
32.508


4732
C
GLU
589
72.113
13.945
31.210


4733
O
GLU
589
70.921
14.187
30.977


4734
CB
GLU
589
72.430
13.452
33.635


4735
CG
GLU
589
73.172
13.826
34.913


4736
CD
GLU
589
74.680
13.737
34.684


4737
OE1
GLU
589
75.083
12.890
33.898


4738
OE2
GLU
589
75.392
14.529
35.282


4739
N
LYS
590
72.858
13.157
30.453


4740
CA
LYS
590
72.419
12.777
29.103


4741
C
LYS
590
71.073
12.061
29.092


4742
O
LYS
590
70.135
12.589
28.487


4743
CB
LYS
590
73.467
11.873
28.468


4744
CG
LYS
590
73.057
11.498
27.048


4745
CD
LYS
590
74.073
10.576
26.388


4746
CE
LYS
590
73.638
10.207
24.974


4747
NZ
LYS
590
74.619
9.311
24.342


4748
N
ASP
591
70.879
11.132
30.015


4749
CA
ASP
591
69.628
10.359
30.046


4750
C
ASP
591
68.436
11.143
30.606


4751
O
ASP
591
67.304
10.899
30.170


4752
CB
ASP
591
69.853
9.111
30.895


4753
CG
ASP
591
71.005
8.287
30.325


4754
OD1
ASP
591
72.112
8.431
30.827


4755
OD2
ASP
591
70.769
7.575
29.361


4756
N
VAL
592
68.705
12.230
31.314


4757
CA
VAL
592
67.625
13.042
31.879


4758
C
VAL
592
67.185
14.077
30.850


4759
O
VAL
592
65.982
14.320
30.680


4760
CB
VAL
592
68.144
13.749
33.126


4761
CG1
VAL
592
67.036
14.551
33.797


4762
CG2
VAL
592
68.742
12.753
34.111


4763
N
ALA
593
68.124
14.438
29.992


4764
CA
ALA
593
67.827
15.337
28.883


4765
C
ALA
593
67.125
14.577
27.761


4766
O
ALA
593
66.174
15.106
27.173


4767
CB
ALA
593
69.142
15.921
28.379


4768
N
ILE
594
67.401
13.285
27.667


4769
CA
ILE
594
66.692
12.428
26.710


4770
C
ILE
594
65.248
12.216
27.152


4771
O
ILE
594
64.326
12.469
26.364


4772
CB
ILE
594
67.382
11.066
26.640


4773
CG1
ILE
594
68.826
11.172
26.174


4774
CG2
ILE
594
66.625
10.129
25.712


4775
CD1
ILE
594
69.486
9.799
26.110


4776
N
ALA
595
65.059
12.050
28.451


4777
CA
ALA
595
63.718
11.827
28.990


4778
C
ALA
595
62.836
13.067
28.885


4779
O
ALA
595
61.713
12.971
28.371


4780
CB
ALA
595
63.857
11.413
30.451


4781
N
ARG
596
63.407
14.237
29.118


4782
CA
ARG
596
62.590
15.445
29.033


4783
C
ARG
596
62.473
16.021
27.623


4784
O
ARG
596
61.458
16.669
27.339


4785
CB
ARG
596
63.096
16.473
30.031


4786
CG
ARG
596
62.845
15.945
31.436


4787
CD
ARG
596
62.944
17.030
32.494


4788
NE
ARG
596
64.319
17.504
32.688


4789
CZ
ARG
596
64.824
17.654
33.914


4790
NH1
ARG
596
64.092
17.322
34.979


4791
NH2
ARG
596
66.069
18.097
34.078


4792
N
LEU
597
63.324
15.605
26.698


4793
CA
LEU
597
63.091
15.970
25.296


4794
C
LEU
597
62.067
15.039
24.662


4795
O
LEU
597
61.283
15.486
23.817


4796
CB
LEU
597
64.382
15.914
24.492


4797
CG
LEU
597
65.230
17.162
24.679


4798
CD1
LEU
597
66.544
17.037
23.917


4799
CD2
LEU
597
64.467
18.407
24.236


4800
N
ALA
598
61.925
13.848
25.220


4801
CA
ALA
598
60.863
12.945
24.785


4802
C
ALA
598
59.518
13.460
25.275


4803
O
ALA
598
58.594
13.632
24.469


4804
CB
ALA
598
61.121
11.575
25.392


4805
N
LEU
599
59.512
13.958
26.501


4806
CA
LEU
599
58.296
14.531
27.083


4807
C
LEU
599
57.868
15.819
26.381


4808
O
LEU
599
56.708
15.921
25.958


4809
CB
LEU
599
58.581
14.847
28.545


4810
CG
LEU
599
57.367
15.457
29.234


4811
CD1
LEU
599
56.252
14.428
29.396


4812
CD2
LEU
599
57.756
16.038
30.585


4813
N
TYR
600
58.822
16.677
26.053


4814
CA
TYR
600
58.474
17.944
25.399


4815
C
TYR
600
58.135
17.775
23.925


4816
O
TYR
600
57.195
18.426
23.456


4817
CB
TYR
600
59.624
18.931
25.543


4818
CG
TYR
600
59.776
19.507
26.947


4819
CD1
TYR
600
58.655
19.692
27.747


4820
CD2
TYR
600
61.034
19.862
27.418


4821
CE1
TYR
600
58.792
20.218
29.024


4822
CE2
TYR
600
61.172
20.389
28.694


4823
CZ
TYR
600
60.051
20.564
29.493


4824
OH
TYR
600
60.189
21.086
30.755


4825
N
GLU
601
58.666
16.740
23.297


4826
CA
GLU
601
58.313
16.473
21.904


4827
C
GLU
601
56.983
15.733
21.803


4828
O
GLU
601
56.242
15.957
20.841


4829
CB
GLU
601
59.431
15.655
21.275


4830
CG
GLU
601
59.178
15.366
19.803


4831
CD
GLU
601
60.415
14.715
19.204


4832
OE1
GLU
601
61.200
15.438
18.607


4833
OE2
GLU
601
60.562
13.505
19.350


4834
N
ALA
602
56.583
15.054
22.867


4835
CA
ALA
602
55.261
14.426
22.891


4836
C
ALA
602
54.189
15.471
23.167


4837
O
ALA
602
53.168
15.506
22.467


4838
CB
ALA
602
55.234
13.367
23.988


4839
N
ALA
603
54.546
16.458
23.973


4840
CA
ALA
603
53.632
17.562
24.256


4841
C
ALA
603
53.479
18.468
23.042


4842
O
ALA
603
52.344
18.706
22.613


4843
CB
ALA
603
54.183
18.363
25.430


4844
N
ARG
604
54.574
18.699
22.334


4845
CA
ARG
604
54.533
19.531
21.130


4846
C
ARG
604
53.867
18.813
19.960


4847
O
ARG
604
53.106
19.444
19.218


4848
CB
ARG
604
55.959
19.874
20.727


4849
CG
ARG
604
55.962
20.783
19.506


4850
CD
ARG
604
57.173
20.508
18.626


4851
NE
ARG
604
57.269
19.066
18.338


4852
CZ
ARG
604
56.662
18.437
17.326


4853
NH1
ARG
604
55.910
19.118
16.458


4854
NH2
ARG
604
56.819
17.120
17.178


4855
N
GLN
605
53.950
17.492
19.934


4856
CA
GLN
605
53.278
16.728
18.884


4857
C
GLN
605
51.770
16.707
19.095


4858
O
GLN
605
51.021
16.903
18.129


4859
CB
GLN
605
53.799
15.296
18.907


4860
CG
GLN
605
53.168
14.443
17.811


4861
CD
GLN
605
53.663
14.886
16.437


4862
OE1
GLN
605
54.812
14.617
16.070


4863
NE2
GLN
605
52.767
15.467
15.660


4864
N
VAL
606
51.338
16.727
20.344


4865
CA
VAL
606
49.903
16.786
20.620


4866
C
VAL
606
49.361
18.207
20.462


4867
O
VAL
606
48.243
18.366
19.955


4868
CB
VAL
606
49.658
16.254
22.028


4869
CG1
VAL
606
48.218
16.476
22.471


4870
CG2
VAL
606
50.018
14.774
22.111


4871
N
ILE
607
50.228
19.200
20.605


4872
CA
ILE
607
49.835
20.586
20.323


4873
C
ILE
607
49.728
20.818
18.819


4874
O
ILE
607
48.711
21.358
18.362


4875
CB
ILE
607
50.873
21.532
20.922


4876
CG1
ILE
607
50.900
21.417
22.440


4877
CG2
ILE
607
50.601
22.976
20.511


4878
CD1
ILE
607
51.993
22.295
23.037


4879
N
ASN
608
50.587
20.141
18.073


4880
CA
ASN
608
50.541
20.160
16.610


4881
C
ASN
608
49.248
19.542
16.112


4882
O
ASN
608
48.479
20.213
15.414


4883
CB
ASN
608
51.706
19.316
16.091


4884
CG
ASN
608
51.736
19.277
14.563


4885
OD1
ASN
608
52.213
20.223
13.926


4886
ND2
ASN
608
51.239
18.192
13.988


4887
N
ASN
609
48.894
18.405
16.689


4888
CA
ASN
609
47.725
17.663
16.219


4889
C
ASN
609
46.425
18.336
16.652


4890
O
ASN
609
45.466
18.357
15.872


4891
CB
ASN
609
47.765
16.252
16.799


4892
CG
ASN
609
49.014
15.457
16.411


4893
OD1
ASN
609
49.819
15.842
15.545


4894
ND2
ASN
609
49.174
14.348
17.114


4895
N
GLY
610
46.459
19.041
17.771


4896
CA
GLY
610
45.305
19.820
18.227


4897
C
GLY
610
45.056
21.003
17.298


4898
O
GLY
610
43.987
21.086
16.676


4899
N
MET
611
46.109
21.765
17.040


4900
CA
MET
611
45.995
22.947
16.183


4901
C
MET
611
45.650
22.580
14.746


4902
O
MET
611
44.651
23.097
14.233


4903
CB
MET
611
47.316
23.710
16.194


4904
CG
MET
611
47.645
24.238
17.585


4905
SD
MET
611
49.116
25.279
17.705


4906
CE
MET
611
48.549
26.668
16.699


4907
N
THR
612
46.231
21.505
14.243


4908
CA
THR
612
45.976
21.087
12.860


4909
C
THR
612
44.607
20.423
12.681


4910
O
THR
612
43.975
20.633
11.638


4911
CB
THR
612
47.088
20.125
12.454


4912
OG1
THR
612
48.327
20.811
12.585


4913
CG2
THR
612
46.952
19.667
11.006


4914
N
LEU
613
44.036
19.917
13.765


4915
CA
LEU
613
42.684
19.351
13.713


4916
C
LEU
613
41.642
20.469
13.691


4917
O
LEU
613
40.632
20.369
12.982


4918
CB
LEU
613
42.488
18.507
14.968


4919
CG
LEU
613
41.226
17.657
14.913


4920
CD1
LEU
613
41.325
16.636
13.787


4921
CD2
LEU
613
41.006
16.948
16.245


4922
N
LEU
614
42.005
21.602
14.271


4923
CA
LEU
614
41.147
22.791
14.236


4924
C
LEU
614
41.317
23.578
12.933


4925
O
LEU
614
40.389
24.274
12.504


4926
CB
LEU
614
41.554
23.686
15.402


4927
CG
LEU
614
41.349
23.008
16.752


4928
CD1
LEU
614
42.137
23.712
17.850


4929
CD2
LEU
614
39.871
22.917
17.114


4930
N
GLY
615
42.454
23.410
12.278


4931
CA
GLY
615
42.714
24.092
11.005


4932
C
GLY
615
43.767
25.187
11.172


4933
O
GLY
615
43.939
26.054
10.305


4934
N
LEU
616
44.464
25.139
12.292


4935
CA
LEU
616
45.489
26.138
12.613


4936
C
LEU
616
46.859
25.687
12.129


4937
O
LEU
616
47.187
24.496
12.166


4938
CB
LEU
616
45.569
26.293
14.126


4939
CG
LEU
616
44.222
26.586
14.768


4940
CD1
LEU
616
44.320
26.438
16.280


4941
CD2
LEU
616
43.709
27.969
14.391


4942
N
THR
617
47.667
26.654
11.728


4943
CA
THR
617
49.050
26.358
11.337


4944
C
THR
617
50.020
26.837
12.419


4945
O
THR
617
50.165
28.048
12.635


4946
CB
THR
617
49.358
27.067
10.023


4947
OG1
THR
617
48.365
26.707
9.071


4948
CG2
THR
617
50.720
26.660
9.471


4949
N
PRO
618
50.594
25.884
13.138


4950
CA
PRO
618
51.557
26.187
14.203


4951
C
PRO
618
52.881
26.726
13.660


4952
O
PRO
618
53.355
26.315
12.593


4953
CB
PRO
618
51.757
24.890
14.921


4954
CG
PRO
618
51.057
23.775
14.160


4955
CD
PRO
618
50.363
24.445
12.987


4956
N
VAL
619
53.400
27.722
14.354


4957
CA
VAL
619
54.694
28.318
14.012


4958
C
VAL
619
55.764
27.909
15.019


4959
O
VAL
619
55.463
27.485
16.142


4960
CB
VAL
619
54.559
29.839
13.996


4961
CG1
VAL
619
53.478
30.281
13.013


4962
CG2
VAL
619
54.265
30.394
15.385


4963
N
ASN
620
57.008
27.988
14.579


4964
CA
ASN
620
58.142
27.713
15.467


4965
C
ASN
620
58.889
29.009
15.755


4966
O
ASN
620
58.896
29.507
16.885


4967
CB
ASN
620
59.062
26.695
14.807


4968
CG
ASN
620
58.293
25.401
14.564


4969
OD1
ASN
620
57.401
25.039
15.342


4970
ND2
ASN
620
58.629
24.730
13.476


4971
N
ARG
621
59.459
29.594
14.720


4972
CA
ARG
621
60.071
30.913
14.890


4973
C
ARG
621
59.013
32.000
14.774


4974
O
ARG
621
58.289
32.072
13.775


4975
CB
ARG
621
61.152
31.116
13.840


4976
CG
ARG
621
62.391
30.299
14.172


4977
CD
ARG
621
63.445
30.438
13.084


4978
NE
ARG
621
64.706
29.786
13.473


4979
CZ
ARG
621
65.059
28.558
13.086


4980
NH1
ARG
621
64.217
27.811
12.367


4981
NH2
ARG
621
66.233
28.053
13.472


4982
N
MET
622
58.888
32.787
15.829


4983
CA
MET
622
57.944
33.909
15.834


4984
C
MET
622
58.490
35.083
16.642


4985
O
MET
622
59.688
35.106
16.880


4986
CB
MET
622
56.618
33.452
16.429


4987
CG
MET
622
56.793
32.908
17.840


4988
SD
MET
622
55.257
32.516
18.703


4989
CE
MET
622
54.573
34.182
18.857


4990
OXT
MET
622
57.696
35.943
16.999






















TABLE 10





Atom
Atom

Residue





No.
Type
Residue
Position
X Coord.
Y Coord.
Z Coord.





















1
N
MET
1
46.935
97.191
59.424


2
CA
MET
1
47.094
97.465
57.985


3
C
MET
1
48.019
98.650
57.738


4
O
MET
1
47.573
99.746
57.382


5
CB
MET
1
45.747
97.742
57.332


6
CG
MET
1
44.891
96.482
57.263


7
SD
MET
1
45.564
95.151
56.241


8
CE
MET
1
45.637
96.017
54.653


9
N
SER
2
49.311
98.403
57.888


10
CA
SER
2
50.313
99.422
57.558


11
C
SER
2
50.751
99.268
56.106


12
O
SER
2
51.413
100.148
55.547


13
CB
SER
2
51.530
99.264
58.463


14
OG
SER
2
52.195
98.059
58.102


15
N
SER
3
50.383
98.142
55.519


16
CA
SER
3
50.665
97.882
54.111


17
C
SER
3
49.373
98.017
53.323


18
O
SER
3
48.570
97.078
53.261


19
CB
SER
3
51.214
96.468
53.980


20
OG
SER
3
52.324
96.368
54.861


21
N
VAL
4
49.187
99.179
52.725


22
CA
VAL
4
47.920
99.475
52.056


23
C
VAL
4
47.839
98.759
50.709


24
O
VAL
4
48.759
98.838
49.885


25
CB
VAL
4
47.814
100.985
51.880


26
CG1
VAL
4
46.379
101.393
51.558


27
CG2
VAL
4
48.275
101.701
53.144


28
N
ALA
5
46.792
97.965
50.562


29
CA
ALA
5
46.553
97.240
49.312


30
C
ALA
5
45.693
98.053
48.349


31
O
ALA
5
44.592
98.502
48.686


32
CB
ALA
5
45.870
95.917
49.633


33
N
SER
6
46.219
98.228
47.152


34
CA
SER
6
45.521
98.987
46.111


35
C
SER
6
44.971
98.081
45.024


36
O
SER
6
45.656
97.162
44.555


37
CB
SER
6
46.505
99.947
45.465


38
OG
SER
6
45.840
100.611
44.397


39
N
LYS
7
43.752
98.357
44.600


40
CA
LYS
7
43.218
97.627
43.458


41
C
LYS
7
43.747
98.212
42.152


42
O
LYS
7
44.023
99.411
42.051


43
CB
LYS
7
41.697
97.645
43.485


44
CG
LYS
7
41.174
96.880
44.696


45
CD
LYS
7
39.651
96.874
44.736


46
CE
LYS
7
39.090
98.285
44.877


47
NZ
LYS
7
39.512
98.900
46.147


48
N
LYS
8
44.180
97.276
41.332


49
CA
LYS
8
44.590
97.475
39.933


50
C
LYS
8
43.994
98.633
39.125


51
O
LYS
8
44.251
99.820
39.379


52
CB
LYS
8
44.248
96.165
39.230


53
CG
LYS
8
43.213
95.342
39.994


54
CD
LYS
8
42.904
94.040
39.267


55
CE
LYS
8
41.960
93.163
40.081


56
NZ
LYS
8
41.676
91.901
39.377


57
N
ILE
9
43.141
98.238
38.190


58
CA
ILE
9
42.675
99.034
37.036


59
C
ILE
9
41.731
100.227
37.310


60
O
ILE
9
41.400
100.931
36.347


61
CB
ILE
9
41.994
98.001
36.127


62
CG1
ILE
9
42.924
96.819
35.880


63
CG2
ILE
9
41.567
98.568
34.781


64
CD1
ILE
9
42.286
95.800
34.942


65
N
ILE
10
41.523
100.591
38.574


66
CA
ILE
10
40.505
101.588
38.989


67
C
ILE
10
40.470
102.795
38.042


68
O
ILE
10
41.524
103.288
37.609


69
CB
ILE
10
40.790
102.021
40.425


70
CG1
ILE
10
41.372
100.875
41.231


71
CG2
ILE
10
39.511
102.459
41.127


72
CD1
ILE
10
41.560
101.298
42.684


73
N
THR
11
39.268
103.326
37.855


74
CA
THR
11
38.880
104.048
36.629


75
C
THR
11
39.349
103.284
35.395


76
O
THR
11
38.708
102.301
35.008


77
CB
THR
11
39.296
105.523
36.567


78
OG1
THR
11
40.413
105.778
37.405


79
CG2
THR
11
38.162
106.427
37.039


80
N
PHE
12
40.374
103.778
34.727


81
CA
PHE
12
40.794
103.130
33.482


82
C
PHE
12
42.278
102.806
33.410


83
O
PHE
12
42.761
102.473
32.319


84
CB
PHE
12
40.429
104.033
32.307


85
CG
PHE
12
38.986
103.904
31.831


86
CD1
PHE
12
37.971
104.646
32.423


87
CD2
PHE
12
38.688
103.031
30.792


88
CE1
PHE
12
36.663
104.514
31.980


89
CE2
PHE
12
37.377
102.900
30.347


90
CZ
PHE
12
36.365
103.642
30.943


91
N
GLU
13
42.997
102.860
34.517


92
CA
GLU
13
44.450
102.790
34.360


93
C
GLU
13
45.219
102.067
35.458


94
O
GLU
13
44.693
101.287
36.257


95
CB
GLU
13
44.971
104.214
34.237


96
CG
GLU
13
44.473
105.111
35.365


97
CD
GLU
13
45.218
106.432
35.270


98
OE1
GLU
13
46.432
106.305
35.181


99
OE2
GLU
13
44.587
107.354
34.763


100
N
GLY
14
46.525
102.222
35.335


101
CA
GLY
14
47.466
101.712
36.324


102
C
GLY
14
48.597
102.717
36.504


103
O
GLY
14
49.028
102.954
37.639


104
N
HIS
15
48.925
103.410
35.420


105
CA
HIS
15
50.039
104.378
35.382


106
C
HIS
15
50.006
105.406
36.507


107
O
HIS
15
50.690
105.212
37.519


108
CB
HIS
15
50.003
105.109
34.047


109
CG
HIS
15
50.742
104.417
32.920


110
ND1
HIS
15
52.003
103.946
32.962


111
CD2
HIS
15
50.267
104.182
31.652


112
CE1
HIS
15
52.317
103.409
31.765


113
NE2
HIS
15
51.244
103.556
30.956


114
N
ARG
16
49.062
106.333
36.452


115
CA
ARG
16
48.985
107.390
37.470


116
C
ARG
16
48.472
106.902
38.821


117
O
ARG
16
48.874
107.472
39.842


118
CB
ARG
16
48.036
108.471
36.985


119
CG
ARG
16
48.657
109.397
35.951


120
CD
ARG
16
49.743
110.259
36.583


121
NE
ARG
16
50.126
111.359
35.685


122
CZ
ARG
16
49.969
112.641
36.023


123
NH1
ARG
16
50.186
113.602
35.123


124
NH2
ARG
16
49.480
112.953
37.225


125
N
ASN
17
47.894
105.711
38.841


126
CA
ASN
17
47.380
105.141
40.086


127
C
ASN
17
48.584
104.768
40.946


128
O
ASN
17
48.825
105.396
41.987


129
CB
ASN
17
46.558
103.878
39.784


130
CG
ASN
17
45.293
104.121
38.944


131
OD1
ASN
17
45.118
105.166
38.301


132
ND2
ASN
17
44.477
103.084
38.861


133
N
PHE
18
49.508
104.086
40.290


134
CA
PHE
18
50.738
103.627
40.926


135
C
PHE
18
51.764
104.743
41.111


136
O
PHE
18
52.397
104.804
42.172


137
CB
PHE
18
51.315
102.553
40.013


138
CG
PHE
18
52.740
102.135
40.340


139
CD1
PHE
18
53.037
101.557
41.567


140
CD2
PHE
18
53.743
102.340
39.403


141
CE1
PHE
18
54.342
101.187
41.857


142
CE2
PHE
18
55.048
101.968
39.692


143
CZ
PHE
18
55.345
101.393
40.920


144
N
ARG
19
51.761
105.732
40.231


145
CA
ARG
19
52.729
106.823
40.369


146
C
ARG
19
52.344
107.790
41.483


147
O
ARG
19
53.225
108.232
42.233


148
CB
ARG
19
52.851
107.570
39.045


149
CG
ARG
19
53.467
106.672
37.979


150
CD
ARG
19
53.871
107.448
36.730


151
NE
ARG
19
52.718
107.992
35.995


152
CZ
ARG
19
52.575
107.802
34.681


153
NH1
ARG
19
53.454
107.047
34.021


154
NH2
ARG
19
51.536
108.331
34.033


155
N
LEU
20
51.058
107.876
41.776


156
CA
LEU
20
50.633
108.728
42.881


157
C
LEU
20
50.780
107.978
44.201


158
O
LEU
20
51.153
108.585
45.212


159
CB
LEU
20
49.182
109.131
42.650


160
CG
LEU
20
48.948
110.607
42.958


161
CD1
LEU
20
49.002
110.905
44.451


162
CD2
LEU
20
49.928
111.489
42.191


163
N
ARG
21
50.778
106.657
44.134


164
CA
ARG
21
50.993
105.863
45.343


165
C
ARG
21
52.468
105.681
45.677


166
O
ARG
21
52.792
105.415
46.841


167
CB
ARG
21
50.311
104.522
45.174


168
CG
ARG
21
48.827
104.765
44.970


169
CD
ARG
21
48.044
103.466
44.990


170
NE
ARG
21
48.269
102.776
46.267


171
CZ
ARG
21
47.522
102.959
47.359


172
NH1
ARG
21
47.709
102.178
48.422


173
NH2
ARG
21
46.512
103.831
47.345


174
N
LEU
22
53.347
106.041
44.757


175
CA
LEU
22
54.770
106.109
45.092


176
C
LEU
22
55.038
107.344
45.942


177
O
LEU
22
55.712
107.237
46.977


178
CB
LEU
22
55.589
106.187
43.810


179
CG
LEU
22
55.512
104.887
43.022


180
CD1
LEU
22
56.184
105.037
41.664


181
CD2
LEU
22
56.132
103.737
43.807


182
N
VAL
23
54.263
108.389
45.684


183
CA
VAL
23
54.337
109.613
46.486


184
C
VAL
23
53.855
109.342
47.900


185
O
VAL
23
54.632
109.491
48.848


186
CB
VAL
23
53.402
110.657
45.885


187
CG1
VAL
23
53.398
111.944
46.703


188
CG2
VAL
23
53.722
110.938
44.427


189
N
LEU
24
52.722
108.667
47.992


190
CA
LEU
24
52.076
108.428
49.284


191
C
LEU
24
52.798
107.400
50.146


192
O
LEU
24
52.925
107.611
51.360


193
CB
LEU
24
50.690
107.905
48.971


194
CG
LEU
24
49.931
108.878
48.086


195
CD1
LEU
24
48.816
108.160
47.353


196
CD2
LEU
24
49.413
110.077
48.868


197
N
ALA
25
53.449
106.438
49.514


198
CA
ALA
25
54.186
105.434
50.275


199
C
ALA
25
55.414
106.046
50.918


200
O
ALA
25
55.544
105.993
52.148


201
CB
ALA
25
54.627
104.334
49.325


202
N
THR
26
56.103
106.878
50.157


203
CA
THR
26
57.319
107.519
50.666


204
C
THR
26
57.023
108.737
51.542


205
O
THR
26
57.861
109.120
52.363


206
CB
THR
26
58.153
107.949
49.467


207
OG1
THR
26
57.432
108.932
48.735


208
CG2
THR
26
58.417
106.770
48.542


209
N
LEU
27
55.800
109.234
51.476


210
CA
LEU
27
55.403
110.400
52.265


211
C
LEU
27
54.856
110.001
53.633


212
O
LEU
27
54.945
110.779
54.590


213
CB
LEU
27
54.300
111.105
51.481


214
CG
LEU
27
54.569
112.590
51.274


215
CD1
LEU
27
54.466
113.364
52.581


216
CD2
LEU
27
55.915
112.821
50.595


217
N
SER
28
54.327
108.793
53.732


218
CA
SER
28
53.763
108.346
55.007


219
C
SER
28
54.644
107.311
55.698


220
O
SER
28
54.477
107.045
56.894


221
CB
SER
28
52.404
107.724
54.720


222
OG
SER
28
51.655
108.658
53.957


223
N
GLY
29
55.554
106.725
54.940


224
CA
GLY
29
56.417
105.664
55.464


225
C
GLY
29
55.657
104.342
55.424


226
O
GLY
29
55.778
103.505
56.326


227
N
LYS
30
54.840
104.190
54.395


228
CA
LYS
30
53.970
103.015
54.293


229
C
LYS
30
54.354
102.089
53.152


230
O
LYS
30
54.538
102.512
52.003


231
CB
LYS
30
52.524
103.459
54.098


232
CG
LYS
30
51.949
104.070
55.369


233
CD
LYS
30
50.477
104.423
55.193


234
CE
LYS
30
49.892
104.990
56.481


235
NZ
LYS
30
50.016
104.019
57.580


236
N
PRO
31
54.481
100.820
53.498


237
CA
PRO
31
54.447
99.750
52.502


238
C
PRO
31
53.135
99.760
51.721


239
O
PRO
31
52.049
99.965
52.277


240
CB
PRO
31
54.595
98.481
53.281


241
CG
PRO
31
54.584
98.803
54.768


242
CD
PRO
31
54.468
100.312
54.871


243
N
ILE
32
53.258
99.574
50.420


244
CA
ILE
32
52.086
99.540
49.536


245
C
ILE
32
52.155
98.356
48.571


246
O
ILE
32
53.176
98.110
47.911


247
CB
ILE
32
51.973
100.874
48.788


248
CG1
ILE
32
51.490
101.982
49.718


249
CG2
ILE
32
51.064
100.785
47.567


250
CD1
ILE
32
51.222
103.274
48.956


251
N
LYS
33
51.072
97.595
48.561


252
CA
LYS
33
50.936
96.454
47.654


253
C
LYS
33
49.820
96.707
46.641


254
O
LYS
33
48.629
96.624
46.965


255
CB
LYS
33
50.616
95.205
48.463


256
CG
LYS
33
50.523
93.983
47.555


257
CD
LYS
33
50.079
92.746
48.323


258
CE
LYS
33
48.659
92.910
48.851


259
NZ
LYS
33
47.708
93.096
47.743


260
N
ILE
34
50.211
97.028
45.423


261
CA
ILE
34
49.218
97.302
44.379


262
C
ILE
34
49.053
96.101
43.443


263
O
ILE
34
50.018
95.582
42.865


264
CB
ILE
34
49.610
98.583
43.643


265
CG1
ILE
34
48.635
98.902
42.512


266
CG2
ILE
34
51.046
98.511
43.136


267
CD1
ILE
34
48.956
100.236
41.850


268
N
THR
35
47.821
95.625
43.385


269
CA
THR
35
47.468
94.450
42.585


270
C
THR
35
47.384
94.781
41.106


271
O
THR
35
46.986
95.886
40.735


272
CB
THR
35
46.100
93.938
43.025


273
OG1
THR
35
45.105
94.871
42.615


274
CG2
THR
35
46.012
93.762
44.536


275
N
LYS
36
47.820
93.819
40.310


276
CA
LYS
36
47.676
93.787
38.835


277
C
LYS
36
47.609
95.136
38.116


278
O
LYS
36
46.579
95.474
37.516


279
CB
LYS
36
46.419
92.997
38.503


280
CG
LYS
36
46.564
91.541
38.916


281
CD
LYS
36
45.385
90.701
38.443


282
CE
LYS
36
45.579
89.238
38.821


283
NZ
LYS
36
46.835
88.717
38.258


284
N
ILE
37
48.717
95.857
38.122


285
CA
ILE
37
48.798
97.151
37.445


286
C
ILE
37
48.638
96.943
35.947


287
O
ILE
37
49.227
96.005
35.399


288
CB
ILE
37
50.180
97.731
37.738


289
CG1
ILE
37
50.445
97.746
39.235


290
CG2
ILE
37
50.339
99.140
37.176


291
CD1
ILE
37
51.796
98.382
39.526


292
N
ARG
38
47.751
97.720
35.340


293
CA
ARG
38
47.551
97.703
33.880


294
C
ARG
38
47.341
96.296
33.331


295
O
ARG
38
48.272
95.684
32.793


296
CB
ARG
38
48.797
98.295
33.234


297
CG
ARG
38
49.014
99.740
33.653


298
CD
ARG
38
50.324
100.265
33.090


299
NE
ARG
38
50.333
100.142
31.626


300
CZ
ARG
38
51.441
99.899
30.925


301
NH1
ARG
38
52.609
99.761
31.554


302
NH2
ARG
38
51.384
99.817
29.594


303
N
SER
39
46.147
95.765
33.518


304
CA
SER
39
45.879
94.396
33.067


305
C
SER
39
44.937
94.319
31.864


306
O
SER
39
44.589
93.221
31.419


307
CB
SER
39
45.318
93.622
34.250


308
OG
SER
39
46.300
93.689
35.275


309
N
GLN
40
44.551
95.466
31.331


310
CA
GLN
40
43.627
95.486
30.185


311
C
GLN
40
44.284
96.018
28.910


312
O
GLN
40
43.599
96.464
27.982


313
CB
GLN
40
42.406
96.324
30.546


314
CG
GLN
40
42.757
97.773
30.863


315
CD
GLN
40
41.488
98.499
31.295


316
OE1
GLN
40
40.527
97.860
31.739


317
NE2
GLN
40
41.532
99.819
31.254


318
N
ASP
41
45.605
96.016
28.889


319
CA
ASP
41
46.348
96.604
27.766


320
C
ASP
41
47.394
95.637
27.220


321
O
ASP
41
47.614
94.561
27.786


322
CB
ASP
41
46.995
97.907
28.226


323
CG
ASP
41
47.769
97.687
29.520


324
OD1
ASP
41
47.178
97.895
30.574


325
OD2
ASP
41
48.886
97.199
29.426


326
N
LEU
42
48.115
96.084
26.202


327
CA
LEU
42
49.076
95.217
25.501


328
C
LEU
42
50.433
95.084
26.196


329
O
LEU
42
51.247
94.245
25.791


330
CB
LEU
42
49.288
95.780
24.101


331
CG
LEU
42
47.993
95.780
23.296


332
CD1
LEU
42
48.171
96.515
21.973


333
CD2
LEU
42
47.486
94.360
23.062


334
N
ASN
43
50.669
95.859
27.242


335
CA
ASN
43
51.919
95.719
28.003


336
C
ASN
43
51.616
95.607
29.496


337
O
ASN
43
51.866
96.560
30.242


338
CB
ASN
43
52.791
96.949
27.764


339
CG
ASN
43
53.020
97.175
26.272


340
OD1
ASN
43
52.335
97.991
25.643


341
ND2
ASN
43
53.956
96.424
25.716


342
N
PRO
44
51.184
94.432
29.933


343
CA
PRO
44
50.621
94.288
31.277


344
C
PRO
44
51.677
94.435
32.365


345
O
PRO
44
52.858
94.122
32.165


346
CB
PRO
44
49.995
92.929
31.310


347
CG
PRO
44
50.307
92.202
30.013


348
CD
PRO
44
51.093
93.185
29.163


349
N
GLY
45
51.239
94.966
33.491


350
CA
GLY
45
52.107
95.135
34.654


351
C
GLY
45
53.220
96.144
34.410


352
O
GLY
45
53.051
97.171
33.739


353
N
LEU
46
54.356
95.840
35.006


354
CA
LEU
46
55.548
96.678
34.880


355
C
LEU
46
56.221
96.627
33.514


356
O
LEU
46
57.051
95.754
33.229


357
CB
LEU
46
56.543
96.215
35.929


358
CG
LEU
46
55.979
96.447
37.317


359
CD1
LEU
46
56.819
95.749
38.376


360
CD2
LEU
46
55.842
97.938
37.593


361
N
LYS
47
55.903
97.621
32.704


362
CA
LYS
47
56.688
97.903
31.499


363
C
LYS
47
58.053
98.417
31.970


364
O
LYS
47
58.167
98.825
33.131


365
CB
LYS
47
55.942
98.976
30.710


366
CG
LYS
47
56.273
98.959
29.223


367
CD
LYS
47
55.547
100.074
28.484


368
CE
LYS
47
55.868
100.030
26.996


369
NZ
LYS
47
57.320
100.112
26.778


370
N
ASP
48
59.038
98.519
31.086


371
CA
ASP
48
60.393
98.933
31.507


372
C
ASP
48
60.463
100.380
32.010


373
O
ASP
48
61.235
100.656
32.935


374
CB
ASP
48
61.353
98.757
30.335


375
CG
ASP
48
62.773
99.128
30.765


376
OD1
ASP
48
63.208
100.214
30.411


377
OD2
ASP
48
63.355
98.355
31.512


378
N
HIS
49
59.479
101.185
31.639


379
CA
HIS
49
59.336
102.540
32.181


380
C
HIS
49
59.067
102.503
33.684


381
O
HIS
49
59.817
103.112
34.456


382
CB
HIS
49
58.134
103.164
31.479


383
CG
HIS
49
57.780
104.575
31.895


384
ND1
HIS
49
58.632
105.550
32.261


385
CD2
HIS
49
56.511
105.101
31.968


386
CE1
HIS
49
57.930
106.666
32.550


387
NE2
HIS
49
56.620
106.386
32.372


388
N
GLU
50
58.230
101.561
34.090


389
CA
GLU
50
57.846
101.451
35.493


390
C
GLU
50
58.853
100.614
36.281


391
O
GLU
50
59.076
100.904
37.459


392
CB
GLU
50
56.471
100.796
35.556


393
CG
GLU
50
55.422
101.538
34.730


394
CD
GLU
50
55.120
102.918
35.313


395
OE1
GLU
50
55.183
103.057
36.524


396
OE2
GLU
50
54.689
103.766
34.540


397
N
VAL
51
59.615
99.776
35.595


398
CA
VAL
51
60.662
98.997
36.270


399
C
VAL
51
61.859
99.875
36.620


400
O
VAL
51
62.318
99.858
37.770


401
CB
VAL
51
61.113
97.871
35.345


402
CG1
VAL
51
62.316
97.126
35.911


403
CG2
VAL
51
59.975
96.902
35.059


404
N
SER
52
62.138
100.836
35.754


405
CA
SER
52
63.240
101.769
36.000


406
C
SER
52
62.831
102.806
37.041


407
O
SER
52
63.595
103.087
37.974


408
CB
SER
52
63.576
102.476
34.690


409
OG
SER
52
63.889
101.490
33.714


410
N
PHE
53
61.539
103.087
37.058


411
CA
PHE
53
60.969
104.043
38.002


412
C
PHE
53
60.856
103.460
39.411


413
O
PHE
53
61.105
104.177
40.390


414
CB
PHE
53
59.588
104.384
37.463


415
CG
PHE
53
58.935
105.589
38.106


416
CD1
PHE
53
59.719
106.564
38.708


417
CD2
PHE
53
57.554
105.727
38.081


418
CE1
PHE
53
59.121
107.671
39.291


419
CE2
PHE
53
56.959
106.837
38.661


420
CZ
PHE
53
57.744
107.805
39.264


421
N
LEU
54
60.760
102.144
39.496


422
CA
LEU
54
60.762
101.470
40.794


423
C
LEU
54
62.123
101.548
41.452


424
O
LEU
54
62.201
102.051
42.581


425
CB
LEU
54
60.410
100.006
40.591


426
CG
LEU
54
58.908
99.822
40.496


427
CD1
LEU
54
58.576
98.456
39.925


428
CD2
LEU
54
58.264
100.025
41.861


429
N
ARG
55
63.172
101.387
40.662


430
CA
ARG
55
64.524
101.430
41.223


431
C
ARG
55
64.938
102.859
41.565


432
O
ARG
55
65.585
103.076
42.595


433
CB
ARG
55
65.486
100.859
40.194


434
CG
ARG
55
64.977
99.529
39.655


435
CD
ARG
55
65.985
98.920
38.692


436
NE
ARG
55
66.423
99.914
37.700


437
CZ
ARG
55
66.339
99.728
36.381


438
NH1
ARG
55
65.745
98.635
35.897


439
NH2
ARG
55
66.791
100.667
35.548


440
N
LEU
56
64.342
103.821
40.879


441
CA
LEU
56
64.579
105.238
41.182


442
C
LEU
56
63.959
105.608
42.528


443
O
LEU
56
64.670
106.027
43.454


444
CB
LEU
56
63.916
106.077
40.089


445
CG
LEU
56
64.856
106.549
38.977


446
CD1
LEU
56
65.664
105.433
38.321


447
CD2
LEU
56
64.081
107.324
37.920


448
N
LEU
57
62.721
105.176
42.702


449
CA
LEU
57
61.943
105.468
43.911


450
C
LEU
57
62.555
104.787
45.133


451
O
LEU
57
62.902
105.454
46.118


452
CB
LEU
57
60.539
104.916
43.630


453
CG
LEU
57
59.453
105.298
44.637


454
CD1
LEU
57
59.422
104.428
45.891


455
CD2
LEU
57
59.472
106.782
44.966


456
N
GLU
58
62.932
103.535
44.944


457
CA
GLU
58
63.434
102.714
46.043


458
C
GLU
58
64.907
102.950
46.364


459
O
GLU
58
65.333
102.601
47.471


460
CB
GLU
58
63.184
101.267
45.654


461
CG
GLU
58
61.681
101.036
45.532


462
CD
GLU
58
61.389
99.899
44.562


463
OE1
GLU
58
62.307
99.496
43.859


464
OE2
GLU
58
60.220
99.566
44.421


465
N
ALA
59
65.624
103.671
45.519


466
CA
ALA
59
66.997
104.028
45.871


467
C
ALA
59
67.038
105.320
46.677


468
O
ALA
59
67.771
105.394
47.669


469
CB
ALA
59
67.815
104.202
44.597


470
N
VAL
60
66.116
106.230
46.400


471
CA
VAL
60
66.140
107.510
47.115


472
C
VAL
60
65.359
107.437
48.427


473
O
VAL
60
65.683
108.140
49.393


474
CB
VAL
60
65.534
108.585
46.218


475
CG1
VAL
60
65.762
109.972
46.811


476
CG2
VAL
60
66.120
108.523
44.813


477
N
THR
61
64.413
106.517
48.498


478
CA
THR
61
63.652
106.354
49.736


479
C
THR
61
64.109
105.138
50.541


480
O
THR
61
63.549
104.872
51.614


481
CB
THR
61
62.173
106.223
49.395


482
OG1
THR
61
61.868
107.162
48.374


483
CG2
THR
61
61.300
106.529
50.608


484
N
ASN
62
65.125
104.438
50.054


485
CA
ASN
62
65.611
103.212
50.710


486
C
ASN
62
64.451
102.256
50.952


487
O
ASN
62
63.923
102.167
52.068


488
CB
ASN
62
66.291
103.559
52.036


489
CG
ASN
62
67.625
104.271
51.817


490
OD1
ASN
62
68.652
103.620
51.595


491
ND2
ASN
62
67.615
105.584
51.969


492
N
GLY
63
63.994
101.629
49.885


493
CA
GLY
63
62.785
100.812
49.978


494
C
GLY
63
62.940
99.397
49.441


495
O
GLY
63
63.312
99.186
48.277


496
N
SER
64
62.498
98.455
50.258


497
CA
SER
64
62.457
97.039
49.873


498
C
SER
64
61.423
96.871
48.770


499
O
SER
64
60.518
97.704
48.643


500
CB
SER
64
62.066
96.199
51.083


501
OG
SER
64
63.048
96.407
52.089


502
N
HIS
65
61.588
95.860
47.938


503
CA
HIS
65
60.710
95.759
46.772


504
C
HIS
65
60.515
94.341
46.246


505
O
HIS
65
61.401
93.722
45.644


506
CB
HIS
65
61.236
96.690
45.679


507
CG
HIS
65
62.726
96.640
45.365


508
ND1
HIS
65
63.688
97.422
45.894


509
CD2
HIS
65
63.346
95.801
44.466


510
CE1
HIS
65
64.880
97.092
45.354


511
NE2
HIS
65
64.665
96.088
44.471


512
N
ILE
66
59.323
93.836
46.497


513
CA
ILE
66
58.900
92.564
45.920


514
C
ILE
66
58.178
92.848
44.608


515
O
ILE
66
57.331
93.748
44.549


516
CB
ILE
66
57.976
91.867
46.918


517
CG1
ILE
66
58.733
91.487
48.184


518
CG2
ILE
66
57.316
90.627
46.319


519
CD1
ILE
66
59.785
90.423
47.893


520
N
GLU
67
58.593
92.148
43.563


521
CA
GLU
67
57.988
92.270
42.229


522
C
GLU
67
58.301
93.600
41.559


523
O
GLU
67
57.473
94.515
41.554


524
CB
GLU
67
56.473
92.095
42.324


525
CG
GLU
67
56.002
90.682
41.999


526
CD
GLU
67
55.886
90.520
40.488


527
OE1
GLU
67
56.926
90.525
39.838


528
OE2
GLU
67
54.762
90.495
39.998


529
N
ILE
68
59.522
93.706
41.062


530
CA
ILE
68
59.913
94.833
40.210


531
C
ILE
68
60.177
94.345
38.783


532
O
ILE
68
60.592
95.113
37.908


533
CB
ILE
68
61.126
95.542
40.821


534
CG1
ILE
68
62.267
94.591
41.188


535
CG2
ILE
68
60.701
96.351
42.041


536
CD1
ILE
68
63.200
94.284
40.019


537
N
SER
69
59.906
93.065
38.577


538
CA
SER
69
60.211
92.373
37.321


539
C
SER
69
59.398
92.907
36.155


540
O
SER
69
58.344
93.529
36.337


541
CB
SER
69
59.846
90.902
37.477


542
OG
SER
69
58.428
90.804
37.396


543
N
TYR
70
59.895
92.624
34.964


544
CA
TYR
70
59.156
92.930
33.739


545
C
TYR
70
57.836
92.173
33.752


546
O
TYR
70
57.773
91.019
34.195


547
CB
TYR
70
59.995
92.519
32.536


548
CG
TYR
70
61.294
93.308
32.410


549
CD1
TYR
70
62.518
92.648
32.392


550
CD2
TYR
70
61.249
94.692
32.317


551
CE1
TYR
70
63.697
93.375
32.279


552
CE2
TYR
70
62.426
95.420
32.205


553
CZ
TYR
70
63.647
94.759
32.186


554
OH
TYR
70
64.810
95.486
32.059


555
N
THR
71
56.787
92.923
33.450


556
CA
THR
71
55.373
92.503
33.512


557
C
THR
71
54.983
91.933
34.877


558
O
THR
71
54.149
91.022
34.972


559
CB
THR
71
55.021
91.526
32.389


560
OG1
THR
71
55.651
90.272
32.605


561
CG2
THR
71
55.430
92.060
31.020


562
N
GLY
72
55.555
92.503
35.926


563
CA
GLY
72
55.148
92.165
37.284


564
C
GLY
72
53.879
92.940
37.571


565
O
GLY
72
53.843
94.168
37.432


566
N
THR
73
52.811
92.223
37.849


567
CA
THR
73
51.540
92.919
38.001


568
C
THR
73
51.302
93.376
39.432


569
O
THR
73
50.711
94.439
39.635


570
CB
THR
73
50.404
92.010
37.538


571
OG1
THR
73
50.319
90.883
38.402


572
CG2
THR
73
50.618
91.520
36.110


573
N
THR
74
51.859
92.685
40.407


574
CA
THR
74
51.533
93.051
41.785


575
C
THR
74
52.774
93.331
42.619


576
O
THR
74
53.487
92.415
43.047


577
CB
THR
74
50.675
91.940
42.369


578
OG1
THR
74
49.502
91.880
41.561


579
CG2
THR
74
50.262
92.239
43.807


580
N
ILE
75
52.988
94.613
42.861


581
CA
ILE
75
54.200
95.080
43.550


582
C
ILE
75
53.985
95.222
45.051


583
O
ILE
75
53.016
95.849
45.496


584
CB
ILE
75
54.579
96.444
42.990


585
CG1
ILE
75
54.559
96.433
41.475


586
CG2
ILE
75
55.962
96.881
43.465


587
CD1
ILE
75
54.996
97.795
40.962


588
N
ILE
76
54.930
94.695
45.810


589
CA
ILE
76
54.905
94.797
47.273


590
C
ILE
76
56.151
95.537
47.759


591
O
ILE
76
57.166
94.922
48.118


592
CB
ILE
76
54.890
93.392
47.860


593
CG1
ILE
76
53.837
92.525
47.181


594
CG2
ILE
76
54.641
93.441
49.364


595
CD1
ILE
76
53.815
91.118
47.764


596
N
TYR
77
56.076
96.855
47.755


597
CA
TYR
77
57.246
97.657
48.132


598
C
TYR
77
57.092
98.308
49.506


599
O
TYR
77
55.985
98.629
49.953


600
CB
TYR
77
57.540
98.696
47.050


601
CG
TYR
77
56.466
99.752
46.792


602
CD1
TYR
77
56.528
100.969
47.459


603
CD2
TYR
77
55.453
99.518
45.869


604
CE1
TYR
77
55.564
101.938
47.223


605
CE2
TYR
77
54.486
100.486
45.634


606
CZ
TYR
77
54.544
101.694
46.314


607
OH
TYR
77
53.585
102.659
46.087


608
N
ARG
78
58.224
98.493
50.163


609
CA
ARG
78
58.248
99.056
51.521


610
C
ARG
78
59.364
100.091
51.695


611
O
ARG
78
60.507
99.738
52.016


612
CB
ARG
78
58.454
97.898
52.490


613
CG
ARG
78
58.340
98.331
53.946


614
CD
ARG
78
58.445
97.119
54.862


615
NE
ARG
78
57.442
96.111
54.478


616
CZ
ARG
78
56.493
95.657
55.300


617
NH1
ARG
78
56.437
96.090
56.561


618
NH2
ARG
78
55.610
94.756
54.864


619
N
PRO
79
59.017
101.352
51.485


620
CA
PRO
79
59.975
102.462
51.598


621
C
PRO
79
60.339
102.782
53.049


622
O
PRO
79
59.596
102.436
53.975


623
CB
PRO
79
59.281
103.627
50.966


624
CG
PRO
79
57.825
103.271
50.720


625
CD
PRO
79
57.676
101.814
51.120


626
N
GLY
80
61.484
103.417
53.231


627
CA
GLY
80
61.917
103.840
54.564


628
C
GLY
80
62.381
105.298
54.604


629
O
GLY
80
61.566
106.228
54.670


630
N
ILE
81
63.691
105.485
54.607


631
CA
ILE
81
64.273
106.821
54.820


632
C
ILE
81
64.578
107.566
53.516


633
O
ILE
81
65.360
107.097
52.681


634
CB
ILE
81
65.549
106.636
55.642


635
CG1
ILE
81
65.217
106.003
56.989


636
CG2
ILE
81
66.286
107.957
55.845


637
CD1
ILE
81
66.462
105.812
57.846


638
N
ILE
82
63.971
108.734
53.365


639
CA
ILE
82
64.244
109.595
52.203


640
C
ILE
82
65.538
110.400
52.375


641
O
ILE
82
65.596
111.370
53.148


642
CB
ILE
82
63.091
110.576
52.051


643
CG1
ILE
82
61.750
109.860
52.094


644
CG2
ILE
82
63.227
111.348
50.743


645
CD1
ILE
82
60.602
110.861
52.062


646
N
ILE
83
66.535
110.045
51.581


647
CA
ILE
83
67.826
110.744
51.626


648
C
ILE
83
67.888
111.892
50.622


649
O
ILE
83
66.967
112.094
49.822


650
CB
ILE
83
68.950
109.750
51.357


651
CG1
ILE
83
68.701
108.973
50.069


652
CG2
ILE
83
69.126
108.808
52.542


653
CD1
ILE
83
69.789
107.936
49.817


654
N
GLY
84
68.904
112.720
50.787


655
CA
GLY
84
69.129
113.837
49.869


656
C
GLY
84
70.437
113.658
49.101


657
O
GLY
84
70.861
112.531
48.818


658
N
GLY
85
71.037
114.775
48.728


659
CA
GLY
85
72.307
114.756
47.994


660
C
GLY
85
72.124
115.135
46.525


661
O
GLY
85
71.619
116.217
46.200


662
N
ASP
86
72.586
114.255
45.652


663
CA
ASP
86
72.462
114.470
44.203


664
C
ASP
86
72.536
113.157
43.433


665
O
ASP
86
73.587
112.509
43.373


666
CB
ASP
86
73.566
115.399
43.704


667
CG
ASP
86
73.489
115.525
42.180


668
OD1
ASP
86
74.296
114.886
41.519


669
OD2
ASP
86
72.658
116.290
41.708


670
N
LEU
87
71.419
112.781
42.839


671
CA
LEU
87
71.399
111.586
41.991


672
C
LEU
87
71.212
111.954
40.524


673
O
LEU
87
70.855
113.089
40.187


674
CB
LEU
87
70.313
110.626
42.462


675
CG
LEU
87
70.707
109.976
43.786


676
CD1
LEU
87
69.610
109.056
44.306


677
CD2
LEU
87
72.017
109.209
43.644


678
N
THR
88
71.591
111.029
39.663


679
CA
THR
88
71.443
111.226
38.218


680
C
THR
88
70.949
109.936
37.571


681
O
THR
88
71.633
108.907
37.593


682
CB
THR
88
72.787
111.652
37.636


683
OG1
THR
88
73.117
112.909
38.214


684
CG2
THR
88
72.727
111.832
36.122


685
N
HIS
89
69.754
110.006
37.015


686
CA
HIS
89
69.110
108.810
36.464


687
C
HIS
89
68.883
108.922
34.960


688
O
HIS
89
68.242
109.859
34.467


689
CB
HIS
89
67.788
108.605
37.189


690
CG
HIS
89
67.946
108.417
38.686


691
ND1
HIS
89
67.381
109.167
39.651


692
CD2
HIS
89
68.693
107.450
39.318


693
CE1
HIS
89
67.751
108.695
40.858


694
NE2
HIS
89
68.562
107.634
40.652


695
N
ASN
90
69.403
107.939
34.245


696
CA
ASN
90
69.283
107.913
32.786


697
C
ASN
90
68.237
106.900
32.325


698
O
ASN
90
68.384
105.688
32.524


699
CB
ASN
90
70.645
107.560
32.194


700
CG
ASN
90
70.575
107.462
30.670


701
OD1
ASN
90
69.932
108.280
30.002


702
ND2
ASN
90
71.210
106.431
30.141


703
N
CYS
91
67.181
107.426
31.732


704
CA
CYS
91
66.137
106.609
31.104


705
C
CYS
91
65.888
107.152
29.696


706
O
CYS
91
64.867
107.799
29.435


707
CB
CYS
91
64.864
106.679
31.942


708
SG
CYS
91
64.992
106.020
33.621


709
N
PRO
92
66.689
106.653
28.767


710
CA
PRO
92
67.101
107.453
27.605


711
C
PRO
92
66.016
107.677
26.551


712
O
PRO
92
65.079
108.463
26.743


713
CB
PRO
92
68.264
106.716
27.008


714
CG
PRO
92
68.522
105.442
27.795


715
CD
PRO
92
67.526
105.457
28.939


716
N
ASP
93
66.118
106.889
25.490


717
CA
ASP
93
65.404
107.132
24.226


718
C
ASP
93
63.911
107.413
24.372


719
O
ASP
93
63.509
108.574
24.303


720
CB
ASP
93
65.609
105.939
23.300


721
CG
ASP
93
65.566
106.402
21.845


722
OD1
ASP
93
65.195
105.599
21.002


723
OD2
ASP
93
66.105
107.474
21.600


724
N
THR
94
63.119
106.406
24.691


725
CA
THR
94
61.662
106.604
24.668


726
C
THR
94
61.009
106.853
26.030


727
O
THR
94
59.778
106.779
26.115


728
CB
THR
94
61.001
105.392
24.023


729
OG1
THR
94
61.236
104.263
24.851


730
CG2
THR
94
61.567
105.110
22.636


731
N
LYS
95
61.774
107.115
27.078


732
CA
LYS
95
61.130
107.234
28.395


733
C
LYS
95
60.870
108.697
28.752


734
O
LYS
95
61.800
109.509
28.829


735
CB
LYS
95
62.008
106.579
29.455


736
CG
LYS
95
62.423
105.134
29.148


737
CD
LYS
95
61.321
104.081
29.315


738
CE
LYS
95
60.422
103.930
28.090


739
NZ
LYS
95
59.477
102.815
28.246


740
N
SER
96
59.603
109.021
28.961


741
CA
SER
96
59.211
110.401
29.289


742
C
SER
96
59.604
110.798
30.708


743
O
SER
96
59.013
110.321
31.686


744
CB
SER
96
57.702
110.537
29.133


745
OG
SER
96
57.345
111.850
29.546


746
N
ILE
97
60.383
111.866
30.785


747
CA
ILE
97
60.937
112.343
32.058


748
C
ILE
97
59.916
113.114
32.889


749
O
ILE
97
59.978
113.053
34.124


750
CB
ILE
97
62.150
113.215
31.734


751
CG1
ILE
97
63.289
112.352
31.217


752
CG2
ILE
97
62.616
114.033
32.928


753
CD1
ILE
97
63.703
111.305
32.245


754
N
GLY
98
58.829
113.523
32.252


755
CA
GLY
98
57.725
114.195
32.948


756
C
GLY
98
57.073
113.298
34.002


757
O
GLY
98
56.921
113.719
35.158


758
N
TYR
99
56.918
112.022
33.672


759
CA
TYR
99
56.264
111.072
34.583


760
C
TYR
99
57.202
110.551
35.676


761
O
TYR
99
56.733
109.996
36.675


762
CB
TYR
99
55.738
109.905
33.760


763
CG
TYR
99
54.749
110.301
32.667


764
CD1
TYR
99
54.883
109.761
31.395


765
CD2
TYR
99
53.715
111.190
32.941


766
CE1
TYR
99
53.995
110.121
30.390


767
CE2
TYR
99
52.826
111.552
31.937


768
CZ
TYR
99
52.973
111.020
30.662


769
OH
TYR
99
52.154
111.447
29.639


770
N
PHE
100
58.491
110.821
35.545


771
CA
PHE
100
59.424
110.521
36.629


772
C
PHE
100
59.469
111.709
37.578


773
O
PHE
100
59.210
111.558
38.779


774
CB
PHE
100
60.829
110.310
36.072


775
CG
PHE
100
61.034
109.078
35.196


776
CD1
PHE
100
61.183
109.214
33.823


777
CD2
PHE
100
61.105
107.820
35.779


778
CE1
PHE
100
61.388
108.092
33.031


779
CE2
PHE
100
61.310
106.697
34.988


780
CZ
PHE
100
61.452
106.834
33.614


781
N
ILE
101
59.505
112.891
36.980


782
CA
ILE
101
59.642
114.150
37.725


783
C
ILE
101
58.486
114.410
38.682


784
O
ILE
101
58.742
114.686
39.860


785
CB
ILE
101
59.728
115.296
36.716


786
CG1
ILE
101
61.079
115.332
36.022


787
CG2
ILE
101
59.437
116.650
37.350


788
CD1
ILE
101
61.181
116.537
35.098


789
N
GLU
102
57.277
114.065
38.268


790
CA
GLU
102
56.103
114.303
39.118


791
C
GLU
102
56.218
113.594
40.484


792
O
GLU
102
56.476
114.311
41.460


793
CB
GLU
102
54.851
113.956
38.316


794
CG
GLU
102
54.772
114.843
37.076


795
CD
GLU
102
53.877
114.219
36.010


796
OE1
GLU
102
53.431
114.960
35.146


797
OE2
GLU
102
53.782
113.000
35.993


798
N
PRO
103
56.176
112.268
40.591


799
CA
PRO
103
56.306
111.669
41.927


800
C
PRO
103
57.716
111.703
42.538


801
O
PRO
103
57.815
111.771
43.771


802
CB
PRO
103
55.841
110.253
41.761


803
CG
PRO
103
55.586
109.974
40.288


804
CD
PRO
103
55.963
111.247
39.553


805
N
MET
104
58.755
111.875
41.731


806
CA
MET
104
60.120
111.872
42.271


807
C
MET
104
60.548
113.203
42.877


808
O
MET
104
61.597
113.250
43.527


809
CB
MET
104
61.120
111.507
41.183


810
CG
MET
104
61.029
110.044
40.779


811
SD
MET
104
61.526
108.842
42.031


812
CE
MET
104
63.248
109.357
42.225


813
N
LEU
105
59.768
114.256
42.696


814
CA
LEU
105
60.104
115.512
43.365


815
C
LEU
105
59.279
115.720
44.628


816
O
LEU
105
59.655
116.552
45.461


817
CB
LEU
105
59.892
116.695
42.430


818
CG
LEU
105
60.738
116.595
41.165


819
CD1
LEU
105
60.605
117.877
40.359


820
CD2
LEU
105
62.206
116.315
41.475


821
N
MET
106
58.285
114.870
44.848


822
CA
MET
106
57.400
115.031
46.012


823
C
MET
106
58.141
114.759
47.317


824
O
MET
106
58.439
115.674
48.096


825
CB
MET
106
56.274
114.016
45.883


826
CG
MET
106
55.473
114.191
44.598


827
SD
MET
106
54.496
115.706
44.480


828
CE
MET
106
53.651
115.397
42.910


829
N
PHE
107
58.581
113.523
47.457


830
CA
PHE
107
59.270
113.098
48.680


831
C
PHE
107
60.652
113.726
49.000


832
O
PHE
107
60.874
113.885
50.205


833
CB
PHE
107
59.348
111.568
48.695


834
CG
PHE
107
60.175
110.905
47.596


835
CD1
PHE
107
61.483
110.527
47.861


836
CD2
PHE
107
59.620
110.649
46.348


837
CE1
PHE
107
62.248
109.928
46.872


838
CE2
PHE
107
60.388
110.052
45.359


839
CZ
PHE
107
61.703
109.693
45.619


840
N
PRO
108
61.515
114.177
48.081


841
CA
PRO
108
62.710
114.931
48.520


842
C
PRO
108
62.446
116.326
49.116


843
O
PRO
108
63.385
116.920
49.659


844
CB
PRO
108
63.571
115.073
47.317


845
CG
PRO
108
62.830
114.571
46.096


846
CD
PRO
108
61.512
114.037
46.617


847
N
LEU
109
61.211
116.804
49.123


848
CA
LEU
109
60.910
118.047
49.829


849
C
LEU
109
60.592
117.766
51.303


850
O
LEU
109
60.599
118.677
52.141


851
CB
LEU
109
59.735
118.700
49.107


852
CG
LEU
109
60.103
118.962
47.650


853
CD1
LEU
109
58.883
119.266
46.790


854
CD2
LEU
109
61.144
120.064
47.514


855
N
PHE
110
60.422
116.485
51.605


856
CA
PHE
110
60.271
115.968
52.974


857
C
PHE
110
61.502
115.170
53.415


858
O
PHE
110
61.485
114.527
54.470


859
CB
PHE
110
59.060
115.044
53.029


860
CG
PHE
110
57.707
115.744
53.050


861
CD1
PHE
110
57.060
116.073
51.866


862
CD2
PHE
110
57.118
116.045
54.271


863
CE1
PHE
110
55.824
116.703
51.905


864
CE2
PHE
110
55.882
116.675
54.310


865
CZ
PHE
110
55.235
117.003
53.127


866
N
SER
111
62.527
115.178
52.578


867
CA
SER
111
63.773
114.440
52.827


868
C
SER
111
64.506
114.966
54.059


869
O
SER
111
64.210
116.056
54.560


870
CB
SER
111
64.645
114.618
51.584


871
OG
SER
111
65.905
113.982
51.761


872
N
LYS
112
65.437
114.174
54.566


873
CA
LYS
112
66.316
114.652
55.636


874
C
LYS
112
67.439
115.553
55.107


875
O
LYS
112
68.149
116.177
55.904


876
CB
LYS
112
66.930
113.453
56.345


877
CG
LYS
112
65.868
112.611
57.040


878
CD
LYS
112
66.503
111.462
57.815


879
CE
LYS
112
65.455
110.644
58.561


880
NZ
LYS
112
66.083
109.548
59.315


881
N
LYS
113
67.589
115.643
53.794


882
CA
LYS
113
68.602
116.529
53.214


883
C
LYS
113
68.068
117.092
51.897


884
O
LYS
113
67.241
116.437
51.256


885
CB
LYS
113
69.852
115.677
52.982


886
CG
LYS
113
71.059
116.471
52.492


887
CD
LYS
113
72.232
115.553
52.171


888
CE
LYS
113
73.400
116.338
51.587


889
NZ
LYS
113
74.505
115.438
51.223


890
N
LYS
114
68.465
118.305
51.536


891
CA
LYS
114
68.129
118.853
50.211


892
C
LYS
114
68.589
117.881
49.127


893
O
LYS
114
69.586
117.171
49.314


894
CB
LYS
114
68.845
120.185
50.029


895
CG
LYS
114
68.468
121.175
51.124


896
CD
LYS
114
69.147
122.520
50.899


897
CE
LYS
114
68.739
123.530
51.965


898
NZ
LYS
114
69.387
124.831
51.731


899
N
PHE
115
67.879
117.843
48.014


900
CA
PHE
115
68.153
116.801
47.024


901
C
PHE
115
68.066
117.292
45.582


902
O
PHE
115
66.997
117.641
45.065


903
CB
PHE
115
67.162
115.662
47.234


904
CG
PHE
115
67.344
114.486
46.278


905
CD1
PHE
115
66.340
114.162
45.376


906
CD2
PHE
115
68.514
113.741
46.307


907
CE1
PHE
115
66.504
113.091
44.508


908
CE2
PHE
115
68.681
112.672
45.439


909
CZ
PHE
115
67.676
112.346
44.539


910
N
SER
116
69.211
117.250
44.927


911
CA
SER
116
69.282
117.520
43.493


912
C
SER
116
69.198
116.208
42.710


913
O
SER
116
69.612
115.149
43.205


914
CB
SER
116
70.595
118.234
43.207


915
OG
SER
116
70.662
118.478
41.811


916
N
ILE
117
68.597
116.267
41.535


917
CA
ILE
117
68.461
115.062
40.711


918
C
ILE
117
68.370
115.377
39.213


919
O
ILE
117
67.452
116.054
38.736


920
CB
ILE
117
67.258
114.263
41.212


921
CG1
ILE
117
67.042
112.992
40.401


922
CG2
ILE
117
65.986
115.105
41.252


923
CD1
ILE
117
65.897
112.177
40.986


924
N
ILE
118
69.363
114.895
38.487


925
CA
ILE
118
69.406
115.089
37.035


926
C
ILE
118
68.716
113.929
36.319


927
O
ILE
118
69.091
112.764
36.495


928
CB
ILE
118
70.869
115.157
36.612


929
CG1
ILE
118
71.622
116.160
37.476


930
CG2
ILE
118
70.995
115.527
35.137


931
CD1
ILE
118
73.098
116.228
37.101


932
N
PHE
119
67.682
114.249
35.562


933
CA
PHE
119
66.981
113.235
34.764


934
C
PHE
119
67.338
113.304
33.281


935
O
PHE
119
67.298
114.374
32.663


936
CB
PHE
119
65.482
113.432
34.927


937
CG
PHE
119
64.876
112.760
36.153


938
CD1
PHE
119
63.950
113.438
36.934


939
CD2
PHE
119
65.244
111.462
36.480


940
CE1
PHE
119
63.388
112.817
38.041


941
CE2
PHE
119
64.681
110.841
37.587


942
CZ
PHE
119
63.752
111.517
38.365


943
N
LYS
120
67.702
112.155
32.736


944
CA
LYS
120
67.998
112.030
31.301


945
C
LYS
120
66.945
111.199
30.570


946
O
LYS
120
66.607
110.095
31.009


947
CB
LYS
120
69.349
111.349
31.147


948
CG
LYS
120
70.487
112.230
31.645


949
CD
LYS
120
71.822
111.492
31.596


950
CE
LYS
120
72.133
110.953
30.201


951
NZ
LYS
120
72.253
112.033
29.207


952
N
GLY
121
66.461
111.713
29.452


953
CA
GLY
121
65.476
110.978
28.640


954
C
GLY
121
64.675
111.883
27.700


955
O
GLY
121
65.256
112.643
26.910


956
N
LEU
122
63.369
111.677
27.673


957
CA
LEU
122
62.488
112.540
26.873


958
C
LEU
122
61.962
113.723
27.667


959
O
LEU
122
61.158
113.562
28.594


960
CB
LEU
122
61.283
111.761
26.369


961
CG
LEU
122
61.656
110.785
25.271


962
CD1
LEU
122
60.411
110.093
24.733


963
CD2
LEU
122
62.388
111.506
24.149


964
N
THR
123
62.259
114.908
27.165


965
CA
THR
123
61.762
116.122
27.813


966
C
THR
123
60.539
116.637
27.067


967
O
THR
123
59.849
117.549
27.544


968
CB
THR
123
62.841
117.196
27.827


969
OG1
THR
123
63.022
117.671
26.504


970
CG2
THR
123
64.171
116.667
28.349


971
N
ASN
124
60.267
116.020
25.927


972
CA
ASN
124
59.111
116.387
25.104


973
C
ASN
124
58.689
115.227
24.188


974
O
ASN
124
59.288
114.977
23.132


975
CB
ASN
124
59.413
117.680
24.324


976
CG
ASN
124
60.698
117.646
23.485


977
OD1
ASN
124
61.810
117.486
24.003


978
ND2
ASN
124
60.556
117.950
22.208


979
N
ILE
125
57.694
114.477
24.637


980
CA
ILE
125
57.207
113.349
23.830


981
C
ILE
125
55.946
113.740
23.049


982
O
ILE
125
54.891
114.066
23.617


983
CB
ILE
125
57.033
112.115
24.722


984
CG1
ILE
125
56.646
110.874
23.929


985
CG2
ILE
125
56.035
112.347
25.842


986
CD1
ILE
125
56.524
109.659
24.845


987
N
ALA
126
56.089
113.661
21.733


988
CA
ALA
126
55.110
114.190
20.768


989
C
ALA
126
53.693
113.665
20.947


990
O
ALA
126
53.412
112.480
20.751


991
CB
ALA
126
55.599
113.850
19.366


992
N
GLY
127
52.817
114.571
21.354


993
CA
GLY
127
51.399
114.265
21.585


994
C
GLY
127
51.092
113.275
22.717


995
O
GLY
127
49.968
112.759
22.774


996
N
ASN
128
52.054
112.961
23.572


997
CA
ASN
128
51.763
112.009
24.647


998
C
ASN
128
51.704
112.745
25.976


999
O
ASN
128
51.071
112.290
26.936


1000
CB
ASN
128
52.820
110.909
24.674


1001
CG
ASN
128
52.833
110.111
23.368


1002
OD1
ASN
128
53.896
109.894
22.775


1003
ND2
ASN
128
51.663
109.664
22.945


1004
N
ASP
129
52.379
113.882
26.019


1005
CA
ASP
129
52.209
114.822
27.136


1006
C
ASP
129
52.738
116.200
26.782


1007
O
ASP
129
53.377
116.403
25.741


1008
CB
ASP
129
52.883
114.339
28.432


1009
CG
ASP
129
54.405
114.131
28.370


1010
OD1
ASP
129
54.864
113.264
29.101


1011
OD2
ASP
129
55.065
114.720
27.521


1012
N
THR
130
52.377
117.154
27.620


1013
CA
THR
130
53.101
118.420
27.630


1014
C
THR
130
54.480
118.100
28.189


1015
O
THR
130
54.612
117.216
29.046


1016
CB
THR
130
52.398
119.421
28.535


1017
OG1
THR
130
52.494
118.953
29.873


1018
CG2
THR
130
50.926
119.578
28.172


1019
N
GLY
131
55.485
118.799
27.704


1020
CA
GLY
131
56.857
118.485
28.091


1021
C
GLY
131
57.176
118.945
29.507


1022
O
GLY
131
56.325
119.489
30.227


1023
N
VAL
132
58.463
118.910
29.809


1024
CA
VAL
132
58.965
119.309
31.131


1025
C
VAL
132
58.959
120.832
31.321


1026
O
VAL
132
58.815
121.322
32.451


1027
CB
VAL
132
60.376
118.743
31.250


1028
CG1
VAL
132
61.049
119.155
32.548


1029
CG2
VAL
132
60.356
117.224
31.126


1030
N
ASP
133
58.744
121.531
30.216


1031
CA
ASP
133
58.621
122.988
30.224


1032
C
ASP
133
57.268
123.440
30.791


1033
O
ASP
133
57.206
124.508
31.414


1034
CB
ASP
133
58.749
123.443
28.772


1035
CG
ASP
133
58.998
124.944
28.682


1036
OD1
ASP
133
59.982
125.369
29.272


1037
OD2
ASP
133
58.364
125.579
27.853


1038
N
ALA
134
56.299
122.532
30.839


1039
CA
ALA
134
54.989
122.861
31.412


1040
C
ALA
134
54.969
122.678
32.925


1041
O
ALA
134
54.200
123.350
33.622


1042
CB
ALA
134
53.935
121.954
30.794


1043
N
ILE
135
55.933
121.936
33.441


1044
CA
ILE
135
56.042
121.804
34.889


1045
C
ILE
135
56.772
123.029
35.420


1046
O
ILE
135
56.287
123.677
36.359


1047
CB
ILE
135
56.820
120.532
35.204


1048
CG1
ILE
135
56.178
119.336
34.508


1049
CG2
ILE
135
56.881
120.304
36.711


1050
CD1
ILE
135
56.970
118.054
34.739


1051
N
LYS
136
57.687
123.514
34.596


1052
CA
LYS
136
58.445
124.721
34.919


1053
C
LYS
136
57.546
125.955
34.934


1054
O
LYS
136
57.222
126.451
36.019


1055
CB
LYS
136
59.549
124.875
33.880


1056
CG
LYS
136
60.518
125.998
34.228


1057
CD
LYS
136
61.655
126.057
33.216


1058
CE
LYS
136
62.642
127.172
33.542


1059
NZ
LYS
136
63.743
127.192
32.565


1060
N
TRP
137
56.930
126.256
33.803


1061
CA
TRP
137
56.137
127.488
33.682


1062
C
TRP
137
54.656
127.338
34.043


1063
O
TRP
137
53.877
128.262
33.789


1064
CB
TRP
137
56.246
127.994
32.248


1065
CG
TRP
137
57.655
128.291
31.780


1066
CD1
TRP
137
58.423
127.511
30.944


1067
CD2
TRP
137
58.453
129.450
32.103


1068
NE1
TRP
137
59.622
128.120
30.764


1069
CE2
TRP
137
59.681
129.283
31.440


1070
CE3
TRP
137
58.225
130.575
32.881


1071
CZ2
TRP
137
60.671
130.246
31.568


1072
CZ3
TRP
137
59.222
131.535
33.004


1073
CH2
TRP
137
60.438
131.371
32.351


1074
N
GLY
138
54.254
126.209
34.600


1075
CA
GLY
138
52.834
126.015
34.903


1076
C
GLY
138
52.584
125.462
36.302


1077
O
GLY
138
51.491
125.658
36.849


1078
N
LEU
139
53.533
124.713
36.839


1079
CA
LEU
139
53.339
124.143
38.179


1080
C
LEU
139
54.231
124.818
39.217


1081
O
LEU
139
53.730
125.228
40.277


1082
CB
LEU
139
53.653
122.654
38.133


1083
CG
LEU
139
53.387
121.979
39.472


1084
CD1
LEU
139
51.916
122.079
39.860


1085
CD2
LEU
139
53.833
120.522
39.442


1086
N
LEU
140
55.473
125.087
38.841


1087
CA
LEU
140
56.425
125.731
39.766


1088
C
LEU
140
55.923
127.038
40.399


1089
O
LEU
140
55.908
127.076
41.636


1090
CB
LEU
140
57.770
125.999
39.097


1091
CG
LEU
140
58.505
124.742
38.647


1092
CD1
LEU
140
59.890
125.125
38.144


1093
CD2
LEU
140
58.634
123.727
39.774


1094
N
PRO
141
55.425
128.034
39.664


1095
CA
PRO
141
55.025
129.269
40.352


1096
C
PRO
141
53.886
129.091
41.359


1097
O
PRO
141
54.065
129.537
42.495


1098
CB
PRO
141
54.629
130.229
39.270


1099
CG
PRO
141
54.739
129.544
37.915


1100
CD
PRO
141
55.277
128.158
38.200


1101
N
VAL
142
52.945
128.193
41.104


1102
CA
VAL
142
51.797
128.048
42.009


1103
C
VAL
142
52.190
127.308
43.290


1104
O
VAL
142
51.875
127.775
44.400


1105
CB
VAL
142
50.729
127.254
41.271


1106
CG1
VAL
142
49.467
127.141
42.112


1107
CG2
VAL
142
50.417
127.891
39.915


1108
N
MET
143
53.141
126.399
43.124


1109
CA
MET
143
53.684
125.618
44.235


1110
C
MET
143
54.684
126.409
45.081


1111
O
MET
143
54.846
126.098
46.266


1112
CB
MET
143
54.345
124.381
43.620


1113
CG
MET
143
55.234
123.630
44.597


1114
SD
MET
143
56.012
122.131
43.945


1115
CE
MET
143
56.272
122.688
42.242


1116
N
GLU
144
55.183
127.515
44.558


1117
CA
GLU
144
56.073
128.379
45.340


1118
C
GLU
144
55.319
129.572
45.937


1119
O
GLU
144
55.773
130.151
46.931


1120
CB
GLU
144
57.173
128.854
44.399


1121
CG
GLU
144
58.252
129.658
45.116


1122
CD
GLU
144
59.300
130.100
44.103


1123
OE1
GLU
144
60.340
129.461
44.031


1124
OE2
GLU
144
59.013
131.036
43.369


1125
N
LYS
145
54.151
129.877
45.392


1126
CA
LYS
145
53.326
130.969
45.921


1127
C
LYS
145
52.568
130.539
47.167


1128
O
LYS
145
52.956
130.861
48.296


1129
CB
LYS
145
52.307
131.393
44.869


1130
CG
LYS
145
52.953
132.071
43.672


1131
CD
LYS
145
51.955
132.252
42.532


1132
CE
LYS
145
52.630
132.845
41.298


1133
NZ
LYS
145
51.695
132.922
40.164


1134
N
PHE
146
51.467
129.841
46.943


1135
CA
PHE
146
50.608
129.430
48.059


1136
C
PHE
146
50.836
127.978
48.467


1137
O
PHE
146
50.202
127.485
49.407


1138
CB
PHE
146
49.147
129.677
47.711


1139
CG
PHE
146
48.752
131.149
47.795


1140
CD1
PHE
146
49.278
131.941
48.807


1141
CD2
PHE
146
47.872
131.701
46.871


1142
CE1
PHE
146
48.935
133.282
48.895


1143
CE2
PHE
146
47.528
133.044
46.958


1144
CZ
PHE
146
48.060
133.836
47.969


1145
N
GLY
147
51.710
127.299
47.745


1146
CA
GLY
147
52.134
125.967
48.170


1147
C
GLY
147
53.120
126.089
49.329


1148
O
GLY
147
52.717
126.321
50.479


1149
N
VAL
148
54.369
125.750
49.037


1150
CA
VAL
148
55.500
125.911
49.968


1151
C
VAL
148
56.848
125.617
49.316


1152
O
VAL
148
57.890
126.104
49.772


1153
CB
VAL
148
55.428
124.955
51.165


1154
CG1
VAL
148
55.201
125.681
52.482


1155
CG2
VAL
148
54.514
123.746
50.984


1156
N
ARG
149
56.823
124.840
48.248


1157
CA
ARG
149
58.018
124.063
47.903


1158
C
ARG
149
59.010
124.680
46.925


1159
O
ARG
149
58.675
125.251
45.878


1160
CB
ARG
149
57.534
122.690
47.466


1161
CG
ARG
149
56.866
122.072
48.688


1162
CD
ARG
149
56.251
120.699
48.479


1163
NE
ARG
149
55.797
120.216
49.791


1164
CZ
ARG
149
54.518
120.200
50.168


1165
NH1
ARG
149
53.549
120.390
49.271


1166
NH2
ARG
149
54.207
119.858
51.418


1167
N
GLU
150
60.264
124.513
47.319


1168
CA
GLU
150
61.422
124.944
46.528


1169
C
GLU
150
61.924
123.824
45.622


1170
O
GLU
150
62.583
122.881
46.081


1171
CB
GLU
150
62.544
125.330
47.488


1172
CG
GLU
150
63.803
125.761
46.740


1173
CD
GLU
150
64.955
125.945
47.725


1174
OE1
GLU
150
66.096
125.879
47.292


1175
OE2
GLU
150
64.669
126.147
48.897


1176
N
VAL
151
61.631
123.964
44.341


1177
CA
VAL
151
62.118
123.020
43.329


1178
C
VAL
151
62.362
123.731
41.996


1179
O
VAL
151
61.430
124.117
41.282


1180
CB
VAL
151
61.118
121.875
43.187


1181
CG1
VAL
151
59.679
122.371
43.224


1182
CG2
VAL
151
61.390
121.031
41.948


1183
N
SER
152
63.629
123.964
41.706


1184
CA
SER
152
63.991
124.634
40.452


1185
C
SER
152
64.182
123.627
39.323


1186
O
SER
152
64.934
122.654
39.453


1187
CB
SER
152
65.269
125.431
40.659


1188
OG
SER
152
65.527
126.121
39.445


1189
N
LEU
153
63.494
123.874
38.222


1190
CA
LEU
153
63.551
122.975
37.063


1191
C
LEU
153
64.433
123.569
35.963


1192
O
LEU
153
64.096
124.589
35.352


1193
CB
LEU
153
62.107
122.766
36.604


1194
CG
LEU
153
61.916
121.676
35.554


1195
CD1
LEU
153
60.544
121.029
35.698


1196
CD2
LEU
153
62.116
122.194
34.136


1197
N
HIS
154
65.551
122.910
35.710


1198
CA
HIS
154
66.501
123.392
34.700


1199
C
HIS
154
66.540
122.491
33.472


1200
O
HIS
154
67.121
121.400
33.515


1201
CB
HIS
154
67.900
123.405
35.307


1202
CG
HIS
154
68.074
124.257
36.548


1203
ND1
HIS
154
68.014
123.836
37.827


1204
CD2
HIS
154
68.339
125.605
36.583


1205
CE1
HIS
154
68.235
124.879
38.651


1206
NE2
HIS
154
68.433
125.973
37.880


1207
N
ILE
155
65.992
122.965
32.369


1208
CA
ILE
155
66.090
122.192
31.125


1209
C
ILE
155
67.403
122.526
30.427


1210
O
ILE
155
67.520
123.548
29.741


1211
CB
ILE
155
64.917
122.528
30.214


1212
CG1
ILE
155
63.603
122.334
30.955


1213
CG2
ILE
155
64.955
121.644
28.972


1214
CD1
ILE
155
62.416
122.796
30.121


1215
N
LEU
156
68.368
121.636
30.584


1216
CA
LEU
156
69.712
121.858
30.050


1217
C
LEU
156
69.778
121.391
28.605


1218
O
LEU
156
70.508
121.954
27.781


1219
CB
LEU
156
70.684
120.995
30.850


1220
CG
LEU
156
70.543
121.167
32.359


1221
CD1
LEU
156
71.266
120.046
33.098


1222
CD2
LEU
156
71.042
122.530
32.824


1223
N
LYS
157
68.949
120.407
28.301


1224
CA
LYS
157
68.947
119.817
26.963


1225
C
LYS
157
67.572
119.283
26.574


1226
O
LYS
157
66.973
118.479
27.298


1227
CB
LYS
157
69.980
118.691
26.965


1228
CG
LYS
157
70.025
117.926
25.649


1229
CD
LYS
157
71.132
116.881
25.647


1230
CE
LYS
157
71.199
116.154
24.309


1231
NZ
LYS
157
72.308
115.188
24.291


1232
N
ARG
158
67.070
119.777
25.455


1233
CA
ARG
158
65.844
119.246
24.844


1234
C
ARG
158
66.032
117.782
24.413


1235
O
ARG
158
67.122
117.397
23.978


1236
CB
ARG
158
65.572
120.114
23.613


1237
CG
ARG
158
64.231
119.866
22.920


1238
CD
ARG
158
63.097
120.730
23.475


1239
NE
ARG
158
62.673
120.336
24.827


1240
CZ
ARG
158
62.526
121.191
25.840


1241
NH1
ARG
158
62.820
122.481
25.671


1242
NH2
ARG
158
62.109
120.750
27.028


1243
N
GLY
159
65.036
116.950
24.673


1244
CA
GLY
159
65.060
115.558
24.195


1245
C
GLY
159
63.760
115.212
23.471


1246
O
GLY
159
62.785
114.770
24.103


1247
N
SER
160
63.795
115.364
22.155


1248
CA
SER
160
62.596
115.223
21.318


1249
C
SER
160
62.317
113.788
20.891


1250
O
SER
160
63.030
113.220
20.055


1251
CB
SER
160
62.766
116.083
20.071


1252
OG
SER
160
62.770
117.450
20.467


1253
N
ALA
161
61.161
113.305
21.318


1254
CA
ALA
161
60.722
111.929
21.027


1255
C
ALA
161
60.721
111.593
19.537


1256
O
ALA
161
60.370
112.440
18.709


1257
CB
ALA
161
59.309
111.757
21.559


1258
N
PRO
162
61.101
110.368
19.196


1259
CA
PRO
162
61.537
109.334
20.155


1260
C
PRO
162
63.014
109.398
20.576


1261
O
PRO
162
63.474
108.489
21.280


1262
CB
PRO
162
61.294
108.047
19.428


1263
CG
PRO
162
61.138
108.345
17.943


1264
CD
PRO
162
61.101
109.861
17.822


1265
N
LEU
163
63.752
110.390
20.098


1266
CA
LEU
163
65.180
110.515
20.397


1267
C
LEU
163
65.366
111.149
21.774


1268
O
LEU
163
65.230
112.366
21.962


1269
CB
LEU
163
65.808
111.377
19.299


1270
CG
LEU
163
67.327
111.235
19.189


1271
CD1
LEU
163
68.101
112.085
20.194


1272
CD2
LEU
163
67.756
109.772
19.233


1273
N
GLY
164
65.750
110.307
22.712


1274
CA
GLY
164
65.948
110.743
24.093


1275
C
GLY
164
67.413
111.001
24.399


1276
O
GLY
164
68.322
110.474
23.746


1277
N
GLY
165
67.619
111.817
25.414


1278
CA
GLY
165
68.966
112.204
25.830


1279
C
GLY
165
68.923
113.572
26.497


1280
O
GLY
165
69.929
114.053
27.033


1281
N
GLY
166
67.729
114.139
26.529


1282
CA
GLY
166
67.518
115.455
27.126


1283
C
GLY
166
67.734
115.401
28.629


1284
O
GLY
166
67.408
114.403
29.280


1285
N
GLU
167
68.344
116.448
29.152


1286
CA
GLU
167
68.707
116.478
30.569


1287
C
GLU
167
68.023
117.616
31.313


1288
O
GLU
167
68.103
118.788
30.919


1289
CB
GLU
167
70.217
116.629
30.699


1290
CG
GLU
167
70.954
115.518
29.963


1291
CD
GLU
167
72.434
115.557
30.319


1292
OE1
GLU
167
72.723
115.730
31.496


1293
OE2
GLU
167
73.248
115.403
29.421


1294
N
VAL
168
67.339
117.243
32.379


1295
CA
VAL
168
66.693
118.219
33.263


1296
C
VAL
168
67.249
118.112
34.683


1297
O
VAL
168
67.110
117.081
35.354


1298
CB
VAL
168
65.188
117.968
33.266


1299
CG1
VAL
168
64.467
118.947
34.186


1300
CG2
VAL
168
64.615
118.059
31.856


1301
N
HIS
169
67.886
119.182
35.123


1302
CA
HIS
169
68.437
119.243
36.477


1303
C
HIS
169
67.385
119.766
37.448


1304
O
HIS
169
66.964
120.928
37.371


1305
CB
HIS
169
69.633
120.189
36.471


1306
CG
HIS
169
70.330
120.345
37.811


1307
ND1
HIS
169
71.443
119.706
38.210


1308
CD2
HIS
169
69.959
121.176
38.844


1309
CE1
HIS
169
71.770
120.104
39.455


1310
NE2
HIS
169
70.848
121.013
39.848


1311
N
LEU
170
66.944
118.900
38.339


1312
CA
LEU
170
66.007
119.333
39.376


1313
C
LEU
170
66.769
119.712
40.638


1314
O
LEU
170
67.683
119.003
41.078


1315
CB
LEU
170
64.985
118.244
39.706


1316
CG
LEU
170
63.917
118.013
38.631


1317
CD1
LEU
170
63.421
119.334
38.055


1318
CD2
LEU
170
64.364
117.069
37.516


1319
N
LEU
171
66.429
120.870
41.169


1320
CA
LEU
171
67.034
121.330
42.419


1321
C
LEU
171
65.978
121.460
43.516


1322
O
LEU
171
65.324
122.505
43.648


1323
CB
LEU
171
67.696
122.676
42.155


1324
CG
LEU
171
68.483
123.162
43.366


1325
CD1
LEU
171
69.567
122.159
43.748


1326
CD2
LEU
171
69.090
124.534
43.097


1327
N
CYS
172
65.795
120.379
44.257


1328
CA
CYS
172
64.816
120.355
45.347


1329
C
CYS
172
65.475
120.561
46.700


1330
O
CYS
172
66.650
120.237
46.915


1331
CB
CYS
172
64.118
119.004
45.359


1332
SG
CYS
172
63.261
118.568
43.837


1333
N
SER
173
64.714
121.136
47.608


1334
CA
SER
173
65.204
121.285
48.975


1335
C
SER
173
64.140
120.894
49.984


1336
O
SER
173
63.000
121.358
49.880


1337
CB
SER
173
65.586
122.741
49.195


1338
OG
SER
173
66.562
123.087
48.221


1339
N
SER
174
64.490
119.986
50.881


1340
CA
SER
174
63.633
119.736
52.045


1341
C
SER
174
63.503
121.043
52.822


1342
O
SER
174
64.504
121.663
53.206


1343
CB
SER
174
64.235
118.628
52.900


1344
OG
SER
174
65.558
118.990
53.269


1345
N
LEU
175
62.269
121.486
52.984


1346
CA
LEU
175
62.039
122.886
53.359


1347
C
LEU
175
61.242
123.104
54.642


1348
O
LEU
175
61.056
122.208
55.473


1349
CB
LEU
175
61.317
123.577
52.197


1350
CG
LEU
175
60.000
122.888
51.827


1351
CD1
LEU
175
58.833
123.866
51.894


1352
CD2
LEU
175
60.066
122.243
50.451


1353
N
ILE
176
60.891
124.372
54.808


1354
CA
ILE
176
60.044
124.876
55.902


1355
C
ILE
176
58.804
124.003
56.114


1356
O
ILE
176
58.058
123.720
55.170


1357
CB
ILE
176
59.625
126.288
55.477


1358
CG1
ILE
176
60.859
127.131
55.168


1359
CG2
ILE
176
58.768
126.987
56.531


1360
CD1
ILE
176
60.479
128.533
54.702


1361
N
PRO
177
58.565
123.646
57.368


1362
CA
PRO
177
57.519
122.677
57.749


1363
C
PRO
177
56.066
123.191
57.751


1364
O
PRO
177
55.213
122.531
58.352


1365
CB
PRO
177
57.889
122.247
59.136


1366
CG
PRO
177
58.989
123.151
59.670


1367
CD
PRO
177
59.379
124.059
58.518


1368
N
GLN
178
55.780
124.336
57.150


1369
CA
GLN
178
54.404
124.866
57.185


1370
C
GLN
178
53.783
124.982
55.794


1371
O
GLN
178
53.845
126.065
55.202


1372
CB
GLN
178
54.410
126.267
57.786


1373
CG
GLN
178
54.863
126.308
59.239


1374
CD
GLN
178
54.747
127.745
59.744


1375
OE1
GLN
178
53.685
128.369
59.639


1376
NE2
GLN
178
55.845
128.259
60.270


1377
N
PRO
179
53.128
123.931
55.321


1378
CA
PRO
179
52.407
124.003
54.044


1379
C
PRO
179
51.164
124.874
54.181


1380
O
PRO
179
50.500
124.838
55.222


1381
CB
PRO
179
52.048
122.585
53.730


1382
CG
PRO
179
52.316
121.720
54.954


1383
CD
PRO
179
52.942
122.639
55.989


1384
N
LEU
180
50.890
125.699
53.186


1385
CA
LEU
180
49.709
126.555
53.309


1386
C
LEU
180
48.634
126.285
52.257


1387
O
LEU
180
48.765
125.453
51.348


1388
CB
LEU
180
50.079
128.041
53.376


1389
CG
LEU
180
50.983
128.550
52.257


1390
CD1
LEU
180
50.476
129.890
51.737


1391
CD2
LEU
180
52.438
128.673
52.713


1392
N
THR
181
47.507
126.921
52.521


1393
CA
THR
181
46.278
126.763
51.743


1394
C
THR
181
46.322
127.464
50.387


1395
O
THR
181
46.488
128.687
50.287


1396
CB
THR
181
45.165
127.332
52.619


1397
OG1
THR
181
44.983
126.425
53.697


1398
CG2
THR
181
43.831
127.466
51.899


1399
N
ILE
182
46.138
126.668
49.347


1400
CA
ILE
182
46.101
127.205
47.983


1401
C
ILE
182
44.665
127.531
47.578


1402
O
ILE
182
43.777
126.669
47.588


1403
CB
ILE
182
46.709
126.171
47.038


1404
CG1
ILE
182
48.063
125.706
47.563


1405
CG2
ILE
182
46.871
126.741
45.632


1406
CD1
ILE
182
48.726
124.721
46.608


1407
N
HIS
183
44.431
128.795
47.275


1408
CA
HIS
183
43.100
129.211
46.816


1409
C
HIS
183
43.090
129.591
45.340


1410
O
HIS
183
43.258
130.761
44.968


1411
CB
HIS
183
42.602
130.370
47.665


1412
CG
HIS
183
41.759
129.938
48.844


1413
ND1
HIS
183
40.871
130.700
49.503


1414
CD2
HIS
183
41.736
128.696
49.437


1415
CE1
HIS
183
40.302
129.978
50.490


1416
NE2
HIS
183
40.838
128.737
50.446


1417
N
ALA
184
42.777
128.602
44.523


1418
CA
ALA
184
42.663
128.781
43.070


1419
C
ALA
184
41.279
129.294
42.672


1420
O
ALA
184
40.394
128.517
42.285


1421
CB
ALA
184
42.918
127.436
42.391


1422
N
LEU
185
41.109
130.601
42.794


1423
CA
LEU
185
39.857
131.263
42.404


1424
C
LEU
185
39.880
131.571
40.913


1425
O
LEU
185
40.163
130.696
40.094


1426
CB
LEU
185
39.648
132.565
43.186


1427
CG
LEU
185
38.879
132.399
44.501


1428
CD1
LEU
185
37.586
131.617
44.278


1429
CD2
LEU
185
39.702
131.754
45.610


1430
N
ASP
186
39.515
132.793
40.573


1431
CA
ASP
186
39.534
133.249
39.177


1432
C
ASP
186
40.919
133.762
38.777


1433
O
ASP
186
41.787
133.963
39.633


1434
CB
ASP
186
38.490
134.353
39.008


1435
CG
ASP
186
38.658
135.428
40.083


1436
OD1
ASP
186
39.597
136.201
39.976


1437
OD2
ASP
186
37.872
135.420
41.019


1438
N
ILE
187
41.118
133.946
37.480


1439
CA
ILE
187
42.383
134.517
36.975


1440
C
ILE
187
42.579
136.004
37.355


1441
O
ILE
187
43.716
136.356
37.689


1442
CB
ILE
187
42.462
134.307
35.461


1443
CG1
ILE
187
42.244
132.843
35.104


1444
CG2
ILE
187
43.804
134.785
34.910


1445
CD1
ILE
187
42.333
132.618
33.598


1446
N
PRO
188
41.569
136.875
37.322


1447
CA
PRO
188
40.422
136.840
36.387


1448
C
PRO
188
40.714
137.303
34.948


1449
O
PRO
188
39.951
136.952
34.041


1450
CB
PRO
188
39.432
137.780
37.007


1451
CG
PRO
188
40.171
138.692
37.977


1452
CD
PRO
188
41.590
138.153
38.043


1453
N
LYS
189
41.810
138.015
34.728


1454
CA
LYS
189
42.073
138.614
33.414


1455
C
LYS
189
43.418
138.193
32.831


1456
O
LYS
189
44.367
137.863
33.548


1457
CB
LYS
189
42.059
140.132
33.567


1458
CG
LYS
189
40.726
140.644
34.105


1459
CD
LYS
189
40.760
142.149
34.348


1460
CE
LYS
189
41.017
142.925
33.060


1461
NZ
LYS
189
39.932
142.709
32.089


1462
N
PHE
190
43.469
138.177
31.514


1463
CA
PHE
190
44.729
137.911
30.819


1464
C
PHE
190
45.540
139.197
30.753


1465
O
PHE
190
45.016
140.251
30.378


1466
CB
PHE
190
44.435
137.433
29.400


1467
CG
PHE
190
43.502
136.229
29.317


1468
CD1
PHE
190
42.213
136.384
28.825


1469
CD2
PHE
190
43.945
134.976
29.723


1470
CE1
PHE
190
41.360
135.291
28.754


1471
CE2
PHE
190
43.091
133.883
29.651


1472
CZ
PHE
190
41.798
134.040
29.169


1473
N
SER
191
46.786
139.129
31.181


1474
CA
SER
191
47.639
140.313
31.087


1475
C
SER
191
48.741
140.081
30.063


1476
O
SER
191
49.246
141.023
29.441


1477
CB
SER
191
48.234
140.597
32.458


1478
OG
SER
191
47.148
140.737
33.364


1479
N
ALA
192
49.078
138.818
29.874


1480
CA
ALA
192
50.089
138.461
28.876


1481
C
ALA
192
49.671
137.266
28.028


1482
O
ALA
192
49.009
136.335
28.506


1483
CB
ALA
192
51.403
138.160
29.586


1484
N
ILE
193
50.057
137.331
26.764


1485
CA
ILE
193
49.837
136.232
25.815


1486
C
ILE
193
51.168
135.922
25.138


1487
O
ILE
193
51.621
136.677
24.269


1488
CB
ILE
193
48.814
136.644
24.755


1489
CG1
ILE
193
47.476
137.033
25.374


1490
CG2
ILE
193
48.606
135.522
23.743


1491
CD1
ILE
193
46.459
137.404
24.300


1492
N
ARG
194
51.846
134.893
25.608


1493
CA
ARG
194
53.152
134.556
25.027


1494
C
ARG
194
53.076
133.343
24.108


1495
O
ARG
194
52.325
132.401
24.367


1496
CB
ARG
194
54.137
134.319
26.158


1497
CG
ARG
194
54.424
135.623
26.894


1498
CD
ARG
194
55.306
135.389
28.112


1499
NE
ARG
194
56.515
134.634
27.754


1500
CZ
ARG
194
56.953
133.592
28.464


1501
NH1
ARG
194
58.026
132.911
28.055


1502
NH2
ARG
194
56.290
133.200
29.555


1503
N
GLY
195
53.859
133.373
23.046


1504
CA
GLY
195
53.848
132.275
22.073


1505
C
GLY
195
55.264
131.899
21.662


1506
O
GLY
195
55.803
132.435
20.687


1507
N
VAL
196
55.838
130.952
22.384


1508
CA
VAL
196
57.223
130.539
22.127


1509
C
VAL
196
57.311
129.094
21.628


1510
O
VAL
196
56.910
128.138
22.304


1511
CB
VAL
196
58.042
130.772
23.398


1512
CG1
VAL
196
57.240
130.518
24.667


1513
CG2
VAL
196
59.366
130.014
23.402


1514
N
ALA
197
57.783
128.966
20.400


1515
CA
ALA
197
57.925
127.654
19.765


1516
C
ALA
197
59.366
127.149
19.790


1517
O
ALA
197
60.279
127.814
19.284


1518
CB
ALA
197
57.457
127.773
18.320


1519
N
TYR
198
59.546
125.969
20.358


1520
CA
TYR
198
60.849
125.293
20.357


1521
C
TYR
198
61.149
124.716
18.981


1522
O
TYR
198
60.228
124.393
18.215


1523
CB
TYR
198
60.860
124.198
21.415


1524
CG
TYR
198
60.890
124.749
22.835


1525
CD1
TYR
198
59.890
124.416
23.741


1526
CD2
TYR
198
61.922
125.597
23.216


1527
CE1
TYR
198
59.927
124.930
25.031


1528
CE2
TYR
198
61.960
126.110
24.505


1529
CZ
TYR
198
60.962
125.775
25.409


1530
OH
TYR
198
61.006
126.278
26.691


1531
N
CYS
199
62.428
124.517
18.718


1532
CA
CYS
199
62.885
124.202
17.364


1533
C
CYS
199
64.254
123.521
17.342


1534
O
CYS
199
65.302
124.180
17.396


1535
CB
CYS
199
62.961
125.528
16.612


1536
SG
CYS
199
63.649
125.477
14.943


1537
N
THR
200
64.242
122.202
17.302


1538
CA
THR
200
65.487
121.462
17.068


1539
C
THR
200
65.469
120.868
15.658


1540
O
THR
200
64.741
119.908
15.369


1541
CB
THR
200
65.689
120.402
18.148


1542
OG1
THR
200
64.577
119.518
18.184


1543
CG2
THR
200
65.833
121.025
19.529


1544
N
ARG
201
66.317
121.442
14.814


1545
CA
ARG
201
66.341
121.175
13.362


1546
C
ARG
201
64.952
121.196
12.745


1547
O
ARG
201
64.401
120.157
12.360


1548
CB
ARG
201
67.006
119.847
13.034


1549
CG
ARG
201
68.526
119.946
13.037


1550
CD
ARG
201
69.154
118.617
12.625


1551
NE
ARG
201
68.676
118.185
11.302


1552
CZ
ARG
201
68.038
117.030
11.091


1553
NH1
ARG
201
67.752
116.227
12.118


1554
NH2
ARG
201
67.640
116.705
9.859


1555
N
VAL
202
64.363
122.377
12.754


1556
CA
VAL
202
63.076
122.608
12.100


1557
C
VAL
202
63.213
123.885
11.279


1558
O
VAL
202
63.796
124.859
11.767


1559
CB
VAL
202
61.988
122.761
13.169


1560
CG1
VAL
202
60.626
123.078
12.561


1561
CG2
VAL
202
61.888
121.521
14.050


1562
N
SER
203
62.779
123.847
10.029


1563
CA
SER
203
62.826
125.046
9.181


1564
C
SER
203
62.186
126.236
9.881


1565
O
SER
203
61.087
126.119
10.440


1566
CB
SER
203
62.062
124.771
7.888


1567
OG
SER
203
61.853
126.016
7.227


1568
N
PRO
204
62.807
127.399
9.737


1569
CA
PRO
204
62.288
128.620
10.358


1570
C
PRO
204
60.926
129.065
9.810


1571
O
PRO
204
60.137
129.613
10.585


1572
CB
PRO
204
63.331
129.663
10.098


1573
CG
PRO
204
64.434
129.074
9.232


1574
CD
PRO
204
64.060
127.620
9.007


1575
N
SER
205
60.526
128.564
8.649


1576
CA
SER
205
59.221
128.938
8.101


1577
C
SER
205
58.116
128.036
8.649


1578
O
SER
205
56.959
128.461
8.718


1579
CB
SER
205
59.265
128.819
6.582


1580
OG
SER
205
59.469
127.451
6.251


1581
N
ILE
206
58.509
126.923
9.249


1582
CA
ILE
206
57.546
125.983
9.816


1583
C
ILE
206
57.205
126.380
11.246


1584
O
ILE
206
56.023
126.443
11.606


1585
CB
ILE
206
58.175
124.594
9.777


1586
CG1
ILE
206
58.342
124.128
8.336


1587
CG2
ILE
206
57.363
123.586
10.579


1588
CD1
ILE
206
58.942
122.729
8.273


1589
N
VAL
207
58.193
126.911
11.947


1590
CA
VAL
207
57.920
127.414
13.293


1591
C
VAL
207
57.348
128.830
13.243


1592
O
VAL
207
56.467
129.150
14.052


1593
CB
VAL
207
59.184
127.331
14.142


1594
CG1
VAL
207
59.381
125.923
14.688


1595
CG2
VAL
207
60.416
127.786
13.373


1596
N
ASN
208
57.580
129.515
12.132


1597
CA
ASN
208
56.945
130.811
11.908


1598
C
ASN
208
55.495
130.629
11.473


1599
O
ASN
208
54.641
131.413
11.900


1600
CB
ASN
208
57.708
131.561
10.822


1601
CG
ASN
208
57.091
132.940
10.631


1602
OD1
ASN
208
57.034
133.736
11.576


1603
ND2
ASN
208
56.595
133.190
9.432


1604
N
ARG
209
55.179
129.469
10.917


1605
CA
ARG
209
53.794
129.163
10.559


1606
C
ARG
209
52.950
128.889
11.802


1607
O
ARG
209
51.829
129.406
11.883


1608
CB
ARG
209
53.796
127.932
9.664


1609
CG
ARG
209
52.390
127.527
9.242


1610
CD
ARG
209
51.735
128.590
8.370


1611
NE
ARG
209
50.422
128.127
7.896


1612
CZ
ARG
209
50.220
127.643
6.668


1613
NH1
ARG
209
51.229
127.592
5.795


1614
NH2
ARG
209
49.004
127.233
6.305


1615
N
MET
210
53.572
128.372
12.852


1616
CA
MET
210
52.844
128.200
14.114


1617
C
MET
210
52.700
129.527
14.855


1618
O
MET
210
51.623
129.801
15.400


1619
CB
MET
210
53.585
127.224
15.015


1620
CG
MET
210
53.706
125.843
14.392


1621
SD
MET
210
54.360
124.584
15.506


1622
CE
MET
210
55.898
125.403
15.982


1623
N
ILE
211
53.640
130.433
14.636


1624
CA
ILE
211
53.536
131.773
15.227


1625
C
ILE
211
52.505
132.625
14.481


1626
O
ILE
211
51.701
133.312
15.122


1627
CB
ILE
211
54.913
132.429
15.167


1628
CG1
ILE
211
55.920
131.649
16.005


1629
CG2
ILE
211
54.849
133.878
15.636


1630
CD1
ILE
211
55.540
131.641
17.483


1631
N
ASP
212
52.326
132.330
13.202


1632
CA
ASP
212
51.282
132.973
12.399


1633
C
ASP
212
49.900
132.427
12.747


1634
O
ASP
212
48.939
133.202
12.808


1635
CB
ASP
212
51.545
132.694
10.920


1636
CG
ASP
212
52.871
133.283
10.447


1637
OD1
ASP
212
53.170
134.403
10.838


1638
OD2
ASP
212
53.448
132.697
9.537


1639
N
SER
213
49.849
131.187
13.210


1640
CA
SER
213
48.579
130.599
13.644


1641
C
SER
213
48.172
131.144
15.010


1642
O
SER
213
47.012
131.539
15.185


1643
CB
SER
213
48.738
129.085
13.734


1644
OG
SER
213
49.112
128.596
12.452


1645
N
ALA
214
49.163
131.453
15.833


1646
CA
ALA
214
48.890
132.051
17.143


1647
C
ALA
214
48.468
133.512
17.011


1648
O
ALA
214
47.482
133.901
17.649


1649
CB
ALA
214
50.149
131.955
17.997


1650
N
ARG
215
48.973
134.167
15.975


1651
CA
ARG
215
48.630
135.561
15.653


1652
C
ARG
215
47.288
135.679
14.913


1653
O
ARG
215
46.698
136.763
14.837


1654
CB
ARG
215
49.757
136.077
14.757


1655
CG
ARG
215
49.590
137.533
14.331


1656
CD
ARG
215
49.714
138.489
15.511


1657
NE
ARG
215
51.064
138.424
16.092


1658
CZ
ARG
215
51.502
139.288
17.009


1659
NH1
ARG
215
50.700
140.262
17.443


1660
NH2
ARG
215
52.738
139.168
17.500


1661
N
ALA
216
46.780
134.558
14.429


1662
CA
ALA
216
45.480
134.549
13.761


1663
C
ALA
216
44.360
134.126
14.707


1664
O
ALA
216
43.176
134.269
14.373


1665
CB
ALA
216
45.545
133.587
12.579


1666
N
VAL
217
44.727
133.608
15.867


1667
CA
VAL
217
43.718
133.228
16.857


1668
C
VAL
217
43.643
134.282
17.954


1669
O
VAL
217
42.552
134.710
18.350


1670
CB
VAL
217
44.093
131.870
17.445


1671
CG1
VAL
217
43.155
131.472
18.581


1672
CG2
VAL
217
44.103
130.798
16.362


1673
N
LEU
218
44.806
134.723
18.400


1674
CA
LEU
218
44.900
135.788
19.401


1675
C
LEU
218
45.994
136.764
19.007


1676
O
LEU
218
46.592
136.656
17.933


1677
CB
LEU
218
45.223
135.202
20.773


1678
CG
LEU
218
44.018
134.517
21.410


1679
CD1
LEU
218
44.399
133.867
22.734


1680
CD2
LEU
218
42.876
135.508
21.614


1681
N
LYS
219
46.215
137.741
19.867


1682
CA
LYS
219
47.306
138.695
19.650


1683
C
LYS
219
48.416
138.476
20.675


1684
O
LYS
219
48.311
138.954
21.811


1685
CB
LYS
219
46.740
140.101
19.807


1686
CG
LYS
219
45.524
140.298
18.910


1687
CD
LYS
219
44.872
141.658
19.129


1688
CE
LYS
219
43.593
141.783
18.308


1689
NZ
LYS
219
42.641
140.719
18.667


1690
N
PRO
220
49.418
137.691
20.308


1691
CA
PRO
220
50.568
137.493
21.186


1692
C
PRO
220
51.311
138.797
21.445


1693
O
PRO
220
51.572
139.599
20.543


1694
CB
PRO
220
51.440
136.491
20.494


1695
CG
PRO
220
50.827
136.142
19.148


1696
CD
PRO
220
49.551
136.961
19.047


1697
N
THR
221
51.670
138.970
22.701


1698
CA
THR
221
52.432
140.136
23.142


1699
C
THR
221
53.918
139.795
23.179


1700
O
THR
221
54.771
140.680
23.306


1701
CB
THR
221
51.954
140.543
24.535


1702
OG1
THR
221
52.232
139.485
25.445


1703
CG2
THR
221
50.452
140.802
24.558


1704
N
GLY
222
54.213
138.511
23.050


1705
CA
GLY
222
55.606
138.057
22.988


1706
C
GLY
222
55.751
136.777
22.168


1707
O
GLY
222
55.650
135.665
22.706


1708
N
CYS
223
55.933
136.944
20.869


1709
CA
CYS
223
56.148
135.798
19.974


1710
C
CYS
223
57.635
135.446
19.880


1711
O
CYS
223
58.499
136.328
19.815


1712
CB
CYS
223
55.588
136.134
18.596


1713
SG
CYS
223
56.303
137.581
17.780


1714
N
GLU
224
57.922
134.158
19.977


1715
CA
GLU
224
59.306
133.672
19.950


1716
C
GLU
224
59.495
132.406
19.113


1717
O
GLU
224
58.704
131.456
19.177


1718
CB
GLU
224
59.751
133.353
21.370


1719
CG
GLU
224
60.016
134.578
22.233


1720
CD
GLU
224
60.633
134.127
23.552


1721
OE1
GLU
224
61.854
134.024
23.608


1722
OE2
GLU
224
59.878
133.905
24.490


1723
N
VAL
225
60.574
132.411
18.350


1724
CA
VAL
225
61.018
131.221
17.612


1725
C
VAL
225
62.380
130.803
18.162


1726
O
VAL
225
63.415
131.395
17.827


1727
CB
VAL
225
61.121
131.557
16.126


1728
CG1
VAL
225
61.759
130.417
15.341


1729
CG2
VAL
225
59.759
131.916
15.541


1730
N
ASN
226
62.375
129.782
18.998


1731
CA
ASN
226
63.584
129.447
19.748


1732
C
ASN
226
64.244
128.156
19.296


1733
O
ASN
226
63.802
127.054
19.647


1734
CB
ASN
226
63.236
129.335
21.229


1735
CG
ASN
226
62.920
130.711
21.808


1736
OD1
ASN
226
61.859
131.291
21.545


1737
ND2
ASN
226
63.855
131.226
22.588


1738
N
ILE
227
65.324
128.323
18.548


1739
CA
ILE
227
66.233
127.208
18.258


1740
C
ILE
227
66.727
126.709
19.611


1741
O
ILE
227
67.151
127.504
20.462


1742
CB
ILE
227
67.377
127.733
17.398


1743
CG1
ILE
227
66.819
128.524
16.219


1744
CG2
ILE
227
68.247
126.588
16.889


1745
CD1
ILE
227
67.934
129.110
15.359


1746
N
THR
228
66.562
125.424
19.858


1747
CA
THR
228
66.580
124.988
21.252


1748
C
THR
228
67.921
124.479
21.772


1749
O
THR
228
68.161
123.277
21.925


1750
CB
THR
228
65.480
123.960
21.454


1751
OG1
THR
228
64.304
124.484
20.851


1752
CG2
THR
228
65.206
123.741
22.938


1753
N
ALA
229
68.695
125.449
22.232


1754
CA
ALA
229
69.884
125.190
23.045


1755
C
ALA
229
69.561
125.487
24.511


1756
O
ALA
229
70.435
125.423
25.386


1757
CB
ALA
229
71.028
126.078
22.569


1758
N
ASP
230
68.278
125.710
24.772


1759
CA
ASP
230
67.807
126.182
26.081


1760
C
ASP
230
68.536
127.471
26.441


1761
O
ASP
230
68.783
128.301
25.559


1762
CB
ASP
230
68.005
125.093
27.136


1763
CG
ASP
230
67.055
123.926
26.864


1764
OD1
ASP
230
65.861
124.186
26.809


1765
OD2
ASP
230
67.536
122.841
26.564


1766
N
VAL
231
68.826
127.669
27.716


1767
CA
VAL
231
69.587
128.857
28.140


1768
C
VAL
231
71.103
128.589
28.100


1769
O
VAL
231
71.920
129.489
28.327


1770
CB
VAL
231
69.136
129.211
29.559


1771
CG1
VAL
231
69.703
130.548
30.031


1772
CG2
VAL
231
67.614
129.248
29.642


1773
N
TRP
232
71.459
127.374
27.720


1774
CA
TRP
232
72.844
126.927
27.771


1775
C
TRP
232
73.398
126.652
26.380


1776
O
TRP
232
72.897
127.150
25.365


1777
CB
TRP
232
72.903
125.662
28.625


1778
CG
TRP
232
72.629
125.910
30.100


1779
CD1
TRP
232
73.569
126.240
31.050


1780
CD2
TRP
232
71.359
125.847
30.791


1781
NE1
TRP
232
72.942
126.406
32.241


1782
CE2
TRP
232
71.618
126.195
32.129


1783
CE3
TRP
232
70.064
125.567
30.385


1784
CZ2
TRP
232
70.573
126.275
33.039


1785
CZ3
TRP
232
69.024
125.647
31.303


1786
CH2
TRP
232
69.275
126.000
32.624


1787
N
ARG
233
74.566
126.037
26.383


1788
CA
ARG
233
75.216
125.610
25.145


1789
C
ARG
233
74.946
124.126
24.911


1790
O
ARG
233
75.135
123.304
25.816


1791
CB
ARG
233
76.709
125.882
25.292


1792
CG
ARG
233
76.960
127.381
25.406


1793
CD
ARG
233
78.090
127.704
26.381


1794
NE
ARG
233
77.779
127.190
27.729


1795
CZ
ARG
233
77.025
127.825
28.634


1796
NH1
ARG
233
76.555
129.050
28.386


1797
NH2
ARG
233
76.787
127.252
29.815


1798
N
GLY
234
74.510
123.794
23.706


1799
CA
GLY
234
74.180
122.397
23.401


1800
C
GLY
234
74.000
122.131
21.909


1801
O
GLY
234
74.051
123.039
21.068


1802
N
GLU
235
73.819
120.860
21.592


1803
CA
GLU
235
73.640
120.467
20.192


1804
C
GLU
235
72.186
120.576
19.755


1805
O
GLU
235
71.429
119.607
19.875


1806
CB
GLU
235
74.096
119.023
20.009


1807
CG
GLU
235
75.588
118.856
20.266


1808
CD
GLU
235
76.006
117.419
19.974


1809
OE1
GLU
235
75.372
116.813
19.123


1810
OE2
GLU
235
76.940
116.960
20.615


1811
N
ASN
236
71.891
121.629
19.012


1812
CA
ASN
236
70.547
121.832
18.446


1813
C
ASN
236
70.199
120.734
17.434


1814
O
ASN
236
69.129
120.117
17.514


1815
CB
ASN
236
70.537
123.184
17.732


1816
CG
ASN
236
70.729
124.349
18.708


1817
OD1
ASN
236
69.762
124.785
19.342


1818
ND2
ASN
236
71.908
124.951
18.688


1819
N
SER
237
71.228
120.299
16.722


1820
CA
SER
237
71.119
119.202
15.753


1821
C
SER
237
71.275
117.803
16.369


1822
O
SER
237
71.343
116.819
15.628


1823
CB
SER
237
72.188
119.404
14.687


1824
OG
SER
237
71.933
120.654
14.059


1825
N
GLY
238
71.317
117.716
17.690


1826
CA
GLY
238
71.426
116.430
18.381


1827
C
GLY
238
70.212
116.211
19.282


1828
O
GLY
238
69.977
115.102
19.774


1829
N
LYS
239
69.511
117.302
19.561


1830
CA
LYS
239
68.228
117.248
20.283


1831
C
LYS
239
67.075
117.149
19.285


1832
O
LYS
239
65.899
117.022
19.653


1833
CB
LYS
239
68.088
118.545
21.066


1834
CG
LYS
239
69.318
118.808
21.922


1835
CD
LYS
239
69.293
120.205
22.529


1836
CE
LYS
239
70.593
120.492
23.268


1837
NZ
LYS
239
70.565
121.812
23.912


1838
N
SER
240
67.449
117.354
18.035


1839
CA
SER
240
66.577
117.240
16.864


1840
C
SER
240
66.207
115.797
16.536


1841
O
SER
240
66.826
114.867
17.062


1842
CB
SER
240
67.401
117.776
15.709


1843
OG
SER
240
68.461
116.857
15.484


1844
N
PRO
241
65.248
115.612
15.638


1845
CA
PRO
241
64.312
116.660
15.198


1846
C
PRO
241
63.159
116.848
16.186


1847
O
PRO
241
62.725
115.887
16.832


1848
CB
PRO
241
63.795
116.147
13.891


1849
CG
PRO
241
64.069
114.649
13.820


1850
CD
PRO
241
64.894
114.318
15.055


1851
N
GLY
242
62.629
118.057
16.262


1852
CA
GLY
242
61.484
118.287
17.154


1853
C
GLY
242
61.102
119.752
17.356


1854
O
GLY
242
61.896
120.574
17.837


1855
N
PHE
243
59.859
120.048
17.018


1856
CA
PHE
243
59.283
121.380
17.260


1857
C
PHE
243
58.117
121.301
18.245


1858
O
PHE
243
57.487
120.248
18.405


1859
CB
PHE
243
58.864
122.059
15.948


1860
CG
PHE
243
57.708
121.500
15.099


1861
CD1
PHE
243
56.975
122.394
14.328


1862
CD2
PHE
243
57.398
120.147
15.055


1863
CE1
PHE
243
55.927
121.939
13.538


1864
CE2
PHE
243
56.351
119.690
14.263


1865
CZ
PHE
243
55.613
120.587
13.506


1866
N
GLY
244
57.872
122.393
18.946


1867
CA
GLY
244
56.792
122.394
19.946


1868
C
GLY
244
56.437
123.790
20.444


1869
O
GLY
244
57.248
124.447
21.110


1870
N
ILE
245
55.202
124.196
20.203


1871
CA
ILE
245
54.790
125.561
20.556


1872
C
ILE
245
54.087
125.615
21.917


1873
O
ILE
245
53.304
124.724
22.277


1874
CB
ILE
245
53.912
126.107
19.425


1875
CG1
ILE
245
53.384
127.510
19.715


1876
CG2
ILE
245
52.767
125.157
19.105


1877
CD1
ILE
245
52.483
128.012
18.594


1878
N
THR
246
54.528
126.561
22.731


1879
CA
THR
246
53.890
126.820
24.026


1880
C
THR
246
53.205
128.196
24.040


1881
O
THR
246
53.831
129.239
23.800


1882
CB
THR
246
54.956
126.726
25.119


1883
OG1
THR
246
55.854
127.815
24.998


1884
CG2
THR
246
55.778
125.448
25.000


1885
N
LEU
247
51.905
128.176
24.286


1886
CA
LEU
247
51.121
129.416
24.376


1887
C
LEU
247
50.700
129.690
25.823


1888
O
LEU
247
49.806
129.046
26.384


1889
CB
LEU
247
49.901
129.293
23.470


1890
CG
LEU
247
49.715
130.535
22.599


1891
CD1
LEU
247
48.697
130.286
21.492


1892
CD2
LEU
247
49.337
131.766
23.416


1893
N
VAL
248
51.313
130.716
26.381


1894
CA
VAL
248
51.135
131.060
27.792


1895
C
VAL
248
50.056
132.121
28.008


1896
O
VAL
248
50.246
133.294
27.658


1897
CB
VAL
248
52.468
131.605
28.296


1898
CG1
VAL
248
52.440
131.829
29.802


1899
CG2
VAL
248
53.618
130.678
27.918


1900
N
ALA
249
48.943
131.694
28.584


1901
CA
ALA
249
47.918
132.632
29.071


1902
C
ALA
249
48.334
133.051
30.476


1903
O
ALA
249
48.318
132.235
31.406


1904
CB
ALA
249
46.570
131.921
29.115


1905
N
GLU
250
48.724
134.303
30.629


1906
CA
GLU
250
49.469
134.666
31.837


1907
C
GLU
250
48.967
135.875
32.636


1908
O
GLU
250
48.535
136.888
32.064


1909
CB
GLU
250
50.877
134.941
31.315


1910
CG
GLU
250
51.908
135.234
32.392


1911
CD
GLU
250
53.232
135.569
31.721


1912
OE1
GLU
250
53.463
135.035
30.647


1913
OE2
GLU
250
53.964
136.381
32.269


1914
N
LEU
251
48.860
135.641
33.942


1915
CA
LEU
251
48.929
136.677
35.001


1916
C
LEU
251
47.644
137.042
35.736


1917
O
LEU
251
46.972
136.205
36.359


1918
CB
LEU
251
49.621
137.956
34.544


1919
CG
LEU
251
51.138
137.825
34.619


1920
CD1
LEU
251
51.819
139.091
34.115


1921
CD2
LEU
251
51.587
137.494
36.040


1922
N
LYS
252
47.486
138.356
35.808


1923
CA
LYS
252
46.547
139.066
36.688


1924
C
LYS
252
46.782
138.704
38.152


1925
O
LYS
252
47.785
139.132
38.732


1926
CB
LYS
252
45.107
138.799
36.284


1927
CG
LYS
252
44.248
140.007
36.637


1928
CD
LYS
252
44.786
141.248
35.942


1929
CE
LYS
252
43.990
142.486
36.331


1930
NZ
LYS
252
44.521
143.675
35.647


1931
N
ARG
253
45.899
137.904
38.731


1932
CA
ARG
253
46.022
137.530
40.144


1933
C
ARG
253
46.887
136.292
40.360


1934
O
ARG
253
47.139
135.919
41.512


1935
CB
ARG
253
44.633
137.323
40.737


1936
CG
ARG
253
43.932
138.667
40.906


1937
CD
ARG
253
44.741
139.573
41.830


1938
NE
ARG
253
44.143
140.913
41.925


1939
CZ
ARG
253
44.349
141.729
42.960


1940
NH1
ARG
253
45.089
141.322
43.994


1941
NH2
ARG
253
43.786
142.939
42.978


1942
N
GLY
254
47.348
135.676
39.286


1943
CA
GLY
254
48.354
134.628
39.440


1944
C
GLY
254
48.062
133.351
38.666


1945
O
GLY
254
48.444
132.259
39.108


1946
N
TRP
255
47.429
133.467
37.514


1947
CA
TRP
255
47.210
132.252
36.730


1948
C
TRP
255
47.996
132.220
35.435


1949
O
TRP
255
47.821
133.042
34.529


1950
CB
TRP
255
45.727
132.001
36.523


1951
CG
TRP
255
45.133
131.400
37.776


1952
CD1
TRP
255
44.065
131.871
38.508


1953
CD2
TRP
255
45.599
130.210
38.448


1954
NE1
TRP
255
43.891
131.060
39.585


1955
CE2
TRP
255
44.803
130.069
39.598


1956
CE3
TRP
255
46.624
129.316
38.193


1957
CZ2
TRP
255
45.076
129.052
40.498


1958
CZ3
TRP
255
46.877
128.286
39.091


1959
CH2
TRP
255
46.105
128.159
40.243


1960
N
ARG
256
48.944
131.300
35.427


1961
CA
ARG
256
49.773
131.063
34.249


1962
C
ARG
256
49.480
129.677
33.673


1963
O
ARG
256
49.763
128.640
34.289


1964
CB
ARG
256
51.234
131.186
34.656


1965
CG
ARG
256
52.152
131.167
33.442


1966
CD
ARG
256
53.601
131.387
33.856


1967
NE
ARG
256
53.719
132.611
34.661


1968
CZ
ARG
256
54.648
133.546
34.449


1969
NH1
ARG
256
55.530
133.399
33.457


1970
NH2
ARG
256
54.685
134.634
35.223


1971
N
ILE
257
48.826
129.684
32.526


1972
CA
ILE
257
48.447
128.440
31.854


1973
C
ILE
257
49.251
128.251
30.571


1974
O
ILE
257
49.005
128.910
29.551


1975
CB
ILE
257
46.956
128.511
31.542


1976
CG1
ILE
257
46.165
128.826
32.809


1977
CG2
ILE
257
46.471
127.201
30.931


1978
CD1
ILE
257
44.683
129.020
32.512


1979
N
VAL
258
50.226
127.361
30.640


1980
CA
VAL
258
51.072
127.081
29.476


1981
C
VAL
258
50.495
125.981
28.584


1982
O
VAL
258
50.701
124.779
28.799


1983
CB
VAL
258
52.470
126.723
29.970


1984
CG1
VAL
258
53.221
127.980
30.385


1985
CG2
VAL
258
52.425
125.720
31.118


1986
N
THR
259
49.848
126.426
27.523


1987
CA
THR
259
49.233
125.521
26.548


1988
C
THR
259
50.271
125.017
25.549


1989
O
THR
259
50.579
125.686
24.557


1990
CB
THR
259
48.159
126.310
25.811


1991
OG1
THR
259
47.248
126.826
26.770


1992
CG2
THR
259
47.389
125.435
24.837


1993
N
GLU
260
50.837
123.859
25.834


1994
CA
GLU
260
51.879
123.307
24.963


1995
C
GLU
260
51.353
122.229
24.026


1996
O
GLU
260
50.212
121.766
24.164


1997
CB
GLU
260
52.958
122.660
25.820


1998
CG
GLU
260
53.497
123.579
26.906


1999
CD
GLU
260
54.736
122.926
27.509


2000
OE1
GLU
260
55.630
123.649
27.921


2001
OE2
GLU
260
54.839
121.709
27.403


2002
N
ASN
261
52.129
121.986
22.981


2003
CA
ASN
261
52.034
120.717
22.244


2004
C
ASN
261
53.325
120.421
21.477


2005
O
ASN
261
53.928
121.292
20.834


2006
CB
ASN
261
50.799
120.628
21.340


2007
CG
ASN
261
50.806
121.498
20.083


2008
OD1
ASN
261
51.733
122.264
19.789


2009
ND2
ASN
261
49.735
121.336
19.330


2010
N
VAL
262
53.818
119.214
21.692


2011
CA
VAL
262
54.988
118.742
20.951


2012
C
VAL
262
54.524
118.135
19.630


2013
O
VAL
262
53.765
117.157
19.622


2014
CB
VAL
262
55.703
117.693
21.793


2015
CG1
VAL
262
57.037
117.306
21.162


2016
CG2
VAL
262
55.916
118.202
23.214


2017
N
GLY
263
55.011
118.696
18.538


2018
CA
GLY
263
54.576
118.276
17.206


2019
C
GLY
263
55.420
117.136
16.648


2020
O
GLY
263
56.652
117.214
16.563


2021
N
SER
264
54.728
116.071
16.280


2022
CA
SER
264
55.378
114.919
15.646


2023
C
SER
264
55.832
115.287
14.241


2024
O
SER
264
55.270
116.200
13.624


2025
CB
SER
264
54.385
113.770
15.532


2026
OG
SER
264
53.481
114.098
14.486


2027
N
ALA
265
56.639
114.410
13.664


2028
CA
ALA
265
57.214
114.618
12.323


2029
C
ALA
265
56.256
114.392
11.142


2030
O
ALA
265
56.695
114.404
9.988


2031
CB
ALA
265
58.419
113.696
12.172


2032
N
GLY
266
54.979
114.181
11.418


2033
CA
GLY
266
53.991
114.030
10.354


2034
C
GLY
266
52.956
115.148
10.448


2035
O
GLY
266
52.029
115.222
9.632


2036
N
SER
267
53.116
116.003
11.444


2037
CA
SER
267
52.166
117.098
11.644


2038
C
SER
267
52.596
118.342
10.879


2039
O
SER
267
53.711
118.843
11.055


2040
CB
SER
267
52.110
117.431
13.131


2041
OG
SER
267
51.766
116.246
13.833


2042
N
LEU
268
51.687
118.857
10.069


2043
CA
LEU
268
51.922
120.137
9.394


2044
C
LEU
268
51.970
121.245
10.442


2045
O
LEU
268
51.316
121.129
11.488


2046
CB
LEU
268
50.777
120.393
8.416


2047
CG
LEU
268
50.705
119.322
7.335


2048
CD1
LEU
268
49.448
119.485
6.489


2049
CD2
LEU
268
51.954
119.338
6.459


2050
N
PRO
269
52.662
122.335
10.144


2051
CA
PRO
269
52.839
123.402
11.140


2052
C
PRO
269
51.524
124.077
11.544


2053
O
PRO
269
51.290
124.262
12.746


2054
CB
PRO
269
53.756
124.387
10.490


2055
CG
PRO
269
54.071
123.939
9.071


2056
CD
PRO
269
53.389
122.594
8.896


2057
N
GLU
270
50.571
124.119
10.623


2058
CA
GLU
270
49.252
124.679
10.931


2059
C
GLU
270
48.416
123.742
11.807


2060
O
GLU
270
47.663
124.240
12.648


2061
CB
GLU
270
48.508
124.931
9.622


2062
CG
GLU
270
47.112
125.502
9.864


2063
CD
GLU
270
46.375
125.667
8.540


2064
OE1
GLU
270
46.754
124.979
7.602


2065
OE2
GLU
270
45.506
126.524
8.470


2066
N
ASP
271
48.762
122.463
11.841


2067
CA
ASP
271
48.011
121.523
12.674


2068
C
ASP
271
48.472
121.654
14.117


2069
O
ASP
271
47.629
121.799
15.011


2070
CB
ASP
271
48.257
120.084
12.225


2071
CG
ASP
271
47.899
119.854
10.761


2072
OD1
ASP
271
48.538
118.994
10.166


2073
OD2
ASP
271
47.067
120.581
10.236


2074
N
SER
272
49.759
121.905
14.299


2075
CA
SER
272
50.282
122.062
15.655


2076
C
SER
272
49.857
123.403
16.238


2077
O
SER
272
49.289
123.422
17.339


2078
CB
SER
272
51.799
121.969
15.631


2079
OG
SER
272
52.249
122.188
16.961


2080
N
GLY
273
49.831
124.419
15.388


2081
CA
GLY
273
49.357
125.748
15.788


2082
C
GLY
273
47.878
125.724
16.175


2083
O
GLY
273
47.527
126.090
17.305


2084
N
GLU
274
47.067
125.094
15.341


2085
CA
GLU
274
45.619
125.045
15.561


2086
C
GLU
274
45.207
124.129
16.715


2087
O
GLU
274
44.235
124.446
17.410


2088
CB
GLU
274
44.991
124.538
14.269


2089
CG
GLU
274
43.473
124.476
14.341


2090
CD
GLU
274
42.938
123.899
13.037


2091
OE1
GLU
274
41.839
124.272
12.652


2092
OE2
GLU
274
43.673
123.144
12.413


2093
N
LEU
275
46.021
123.142
17.053


2094
CA
LEU
275
45.706
122.311
18.220


2095
C
LEU
275
46.132
122.991
19.517


2096
O
LEU
275
45.407
122.898
20.516


2097
CB
LEU
275
46.399
120.962
18.085


2098
CG
LEU
275
45.834
120.161
16.919


2099
CD1
LEU
275
46.656
118.904
16.666


2100
CD2
LEU
275
44.365
119.820
17.142


2101
N
THR
276
47.099
123.891
19.421


2102
CA
THR
276
47.499
124.677
20.590


2103
C
THR
276
46.428
125.721
20.869


2104
O
THR
276
45.928
125.803
22.000


2105
CB
THR
276
48.818
125.383
20.297


2106
OG1
THR
276
49.797
124.397
20.029


2107
CG2
THR
276
49.297
126.203
21.488


2108
N
ALA
277
45.861
126.233
19.788


2109
CA
ALA
277
44.775
127.207
19.883


2110
C
ALA
277
43.467
126.577
20.353


2111
O
ALA
277
42.796
127.172
21.204


2112
CB
ALA
277
44.568
127.826
18.507


2113
N
TYR
278
43.239
125.314
20.021


2114
CA
TYR
278
42.037
124.625
20.505


2115
C
TYR
278
42.098
124.369
22.004


2116
O
TYR
278
41.175
124.786
22.714


2117
CB
TYR
278
41.875
123.291
19.785


2118
CG
TYR
278
41.321
123.377
18.367


2119
CD1
TYR
278
41.561
122.341
17.473


2120
CD2
TYR
278
40.555
124.469
17.979


2121
CE1
TYR
278
41.044
122.401
16.186


2122
CE2
TYR
278
40.040
124.532
16.691


2123
CZ
TYR
278
40.285
123.497
15.799


2124
OH
TYR
278
39.757
123.548
14.527


2125
N
GLN
279
43.272
124.003
22.494


2126
CA
GLN
279
43.434
123.736
23.927


2127
C
GLN
279
43.370
125.025
24.745


2128
O
GLN
279
42.666
125.070
25.764


2129
CB
GLN
279
44.790
123.069
24.127


2130
CG
GLN
279
44.890
121.773
23.332


2131
CD
GLN
279
46.321
121.238
23.342


2132
OE1
GLN
279
46.540
120.025
23.238


2133
NE2
GLN
279
47.276
122.152
23.372


2134
N
LEU
280
43.839
126.111
24.151


2135
CA
LEU
280
43.805
127.410
24.821


2136
C
LEU
280
42.382
127.968
24.871


2137
O
LEU
280
41.902
128.312
25.961


2138
CB
LEU
280
44.711
128.352
24.035


2139
CG
LEU
280
44.842
129.714
24.703


2140
CD1
LEU
280
45.413
129.584
26.110


2141
CD2
LEU
280
45.711
130.638
23.860


2142
N
LEU
281
41.631
127.758
23.801


2143
CA
LEU
281
40.245
128.237
23.742


2144
C
LEU
281
39.271
127.339
24.503


2145
O
LEU
281
38.180
127.796
24.853


2146
CB
LEU
281
39.810
128.328
22.285


2147
CG
LEU
281
40.605
129.381
21.521


2148
CD1
LEU
281
40.284
129.328
20.032


2149
CD2
LEU
281
40.364
130.779
22.082


2150
N
GLU
282
39.714
126.165
24.919


2151
CA
GLU
282
38.875
125.319
25.770


2152
C
GLU
282
38.956
125.741
27.234


2153
O
GLU
282
37.990
125.530
27.980


2154
CB
GLU
282
39.312
123.869
25.613


2155
CG
GLU
282
38.959
123.346
24.226


2156
CD
GLU
282
39.552
121.959
24.009


2157
OE1
GLU
282
38.873
120.994
24.330


2158
OE2
GLU
282
40.651
121.885
23.473


2159
N
GLU
283
39.995
126.481
27.592


2160
CA
GLU
283
40.061
127.057
28.938


2161
C
GLU
283
39.304
128.380
28.940


2162
O
GLU
283
38.369
128.552
29.729


2163
CB
GLU
283
41.518
127.327
29.300


2164
CG
GLU
283
41.699
127.654
30.782


2165
CD
GLU
283
41.888
126.369
31.587


2166
OE1
GLU
283
42.344
126.489
32.715


2167
OE2
GLU
283
42.004
125.348
30.919


2168
N
ILE
284
39.470
129.103
27.839


2169
CA
ILE
284
38.855
130.433
27.654


2170
C
ILE
284
37.353
130.370
27.312


2171
O
ILE
284
36.654
131.390
27.339


2172
CB
ILE
284
39.641
131.128
26.536


2173
CG1
ILE
284
41.124
131.188
26.878


2174
CG2
ILE
284
39.139
132.541
26.257


2175
CD1
ILE
284
41.913
131.892
25.780


2176
N
SER
285
36.839
129.167
27.115


2177
CA
SER
285
35.409
128.979
26.857


2178
C
SER
285
34.616
128.797
28.150


2179
O
SER
285
33.388
128.664
28.103


2180
CB
SER
285
35.214
127.744
25.986


2181
OG
SER
285
35.642
126.611
26.729


2182
N
ASN
286
35.306
128.720
29.276


2183
CA
ASN
286
34.623
128.613
30.567


2184
C
ASN
286
35.338
129.426
31.640


2185
O
ASN
286
36.011
130.420
31.348


2186
CB
ASN
286
34.489
127.146
30.971


2187
CG
ASN
286
35.798
126.385
30.811


2188
OD1
ASN
286
36.767
126.632
31.539


2189
ND2
ASN
286
35.741
125.354
29.987


2190
N
SER
287
35.095
129.055
32.884


2191
CA
SER
287
35.691
129.780
34.007


2192
C
SER
287
36.600
128.891
34.855


2193
O
SER
287
36.827
129.189
36.035


2194
CB
SER
287
34.581
130.415
34.835


2195
OG
SER
287
33.577
129.436
35.050


2196
N
GLY
288
37.035
127.778
34.287


2197
CA
GLY
288
38.014
126.919
34.954


2198
C
GLY
288
39.405
127.499
34.735


2199
O
GLY
288
39.862
127.653
33.595


2200
N
VAL
289
40.075
127.792
35.835


2201
CA
VAL
289
41.344
128.525
35.779


2202
C
VAL
289
42.576
127.621
35.752


2203
O
VAL
289
43.701
128.111
35.596


2204
CB
VAL
289
41.386
129.439
36.994


2205
CG1
VAL
289
40.172
130.362
36.973


2206
CG2
VAL
289
41.427
128.628
38.287


2207
N
VAL
290
42.371
126.329
35.953


2208
CA
VAL
290
43.477
125.375
35.846


2209
C
VAL
290
43.186
124.295
34.812


2210
O
VAL
290
42.105
123.690
34.790


2211
CB
VAL
290
43.762
124.756
37.210


2212
CG1
VAL
290
44.732
125.607
38.017


2213
CG2
VAL
290
42.481
124.496
37.992


2214
N
GLY
291
44.188
124.043
33.988


2215
CA
GLY
291
44.094
123.004
32.963


2216
C
GLY
291
44.186
121.615
33.583


2217
O
GLY
291
44.699
121.440
34.697


2218
N
ARG
292
43.897
120.620
32.762


2219
CA
ARG
292
43.802
119.231
33.237


2220
C
ARG
292
45.149
118.554
33.510


2221
O
ARG
292
45.181
117.538
34.208


2222
CB
ARG
292
43.031
118.397
32.214


2223
CG
ARG
292
41.509
118.474
32.359


2224
CD
ARG
292
40.857
119.729
31.792


2225
NE
ARG
292
39.389
119.643
31.881


2226
CZ
ARG
292
38.611
119.442
30.812


2227
NH1
ARG
292
39.162
119.263
29.609


2228
NH2
ARG
292
37.285
119.406
30.946


2229
N
TYR
293
46.249
119.159
33.087


2230
CA
TYR
293
47.567
118.611
33.427


2231
C
TYR
293
48.109
119.265
34.701


2232
O
TYR
293
48.928
118.669
35.411


2233
CB
TYR
293
48.519
118.857
32.259


2234
CG
TYR
293
49.870
118.147
32.364


2235
CD1
TYR
293
49.985
116.826
31.945


2236
CD2
TYR
293
50.978
118.812
32.874


2237
CE1
TYR
293
51.209
116.175
32.027


2238
CE2
TYR
293
52.203
118.161
32.959


2239
CZ
TYR
293
52.315
116.845
32.530


2240
OH
TYR
293
53.548
116.229
32.523


2241
N
GLN
294
47.534
120.402
35.063


2242
CA
GLN
294
47.941
121.084
36.291


2243
C
GLN
294
47.114
120.533
37.440


2244
O
GLN
294
47.651
120.266
38.523


2245
CB
GLN
294
47.680
122.582
36.139


2246
CG
GLN
294
48.530
123.199
35.032


2247
CD
GLN
294
48.090
124.635
34.753


2248
OE1
GLN
294
47.073
124.854
34.078


2249
NE2
GLN
294
48.825
125.588
35.301


2250
N
LEU
295
45.906
120.122
37.087


2251
CA
LEU
295
44.943
119.510
38.013


2252
C
LEU
295
45.523
118.421
38.942


2253
O
LEU
295
45.606
118.719
40.142


2254
CB
LEU
295
43.771
119.011
37.164


2255
CG
LEU
295
42.723
118.239
37.954


2256
CD1
LEU
295
42.285
118.994
39.204


2257
CD2
LEU
295
41.533
117.893
37.064


2258
N
PRO
296
46.056
117.294
38.470


2259
CA
PRO
296
46.402
116.235
39.427


2260
C
PRO
296
47.643
116.548
40.269


2261
O
PRO
296
47.660
116.218
41.461


2262
CB
PRO
296
46.632
115.012
38.592


2263
CG
PRO
296
46.602
115.389
37.120


2264
CD
PRO
296
46.266
116.866
37.077


2265
N
LEU
297
48.515
117.404
39.759


2266
CA
LEU
297
49.759
117.706
40.462


2267
C
LEU
297
49.519
118.792
41.498


2268
O
LEU
297
49.912
118.640
42.663


2269
CB
LEU
297
50.769
118.188
39.427


2270
CG
LEU
297
50.972
117.150
38.328


2271
CD1
LEU
297
51.825
117.707
37.194


2272
CD2
LEU
297
51.581
115.867
38.886


2273
N
ALA
298
48.618
119.699
41.157


2274
CA
ALA
298
48.266
120.781
42.067


2275
C
ALA
298
47.349
120.286
43.171


2276
O
ALA
298
47.530
120.717
44.313


2277
CB
ALA
298
47.574
121.892
41.288


2278
N
LEU
299
46.629
119.201
42.927


2279
CA
LEU
299
45.784
118.642
43.984


2280
C
LEU
299
46.622
117.888
45.019


2281
O
LEU
299
46.287
117.969
46.204


2282
CB
LEU
299
44.735
117.720
43.364


2283
CG
LEU
299
43.355
117.882
44.012


2284
CD1
LEU
299
42.285
117.129
43.232


2285
CD2
LEU
299
43.319
117.467
45.480


2286
N
VAL
300
47.821
117.457
44.659


2287
CA
VAL
300
48.696
116.845
45.662


2288
C
VAL
300
49.277
117.908
46.596


2289
O
VAL
300
49.229
117.736
47.821


2290
CB
VAL
300
49.829
116.118
44.945


2291
CG1
VAL
300
50.812
115.520
45.944


2292
CG2
VAL
300
49.293
115.038
44.012


2293
N
TYR
301
49.494
119.096
46.056


2294
CA
TYR
301
50.025
120.191
46.869


2295
C
TYR
301
48.927
120.903
47.653


2296
O
TYR
301
49.171
121.334
48.785


2297
CB
TYR
301
50.738
121.160
45.939


2298
CG
TYR
301
51.921
120.513
45.205


2299
CD1
TYR
301
52.882
119.831
45.927


2300
CD2
TYR
301
52.013
120.608
43.823


2301
CE1
TYR
301
53.956
119.241
45.269


2302
CE2
TYR
301
53.071
120.004
43.162


2303
CZ
TYR
301
54.050
119.322
43.886


2304
OH
TYR
301
55.121
118.761
43.242


2305
N
MET
302
47.695
120.781
47.186


2306
CA
MET
302
46.532
121.295
47.918


2307
C
MET
302
46.122
120.355
49.047


2308
O
MET
302
45.704
120.808
50.123


2309
CB
MET
302
45.389
121.394
46.923


2310
CG
MET
302
45.632
122.480
45.886


2311
SD
MET
302
44.368
122.617
44.601


2312
CE
MET
302
45.061
123.995
43.660


2313
N
THR
303
46.447
119.087
48.864


2314
CA
THR
303
46.198
118.070
49.878


2315
C
THR
303
47.129
118.247
51.062


2316
O
THR
303
46.637
118.404
52.183


2317
CB
THR
303
46.422
116.704
49.245


2318
OG1
THR
303
45.315
116.442
48.393


2319
CG2
THR
303
46.472
115.602
50.292


2320
N
ILE
304
48.398
118.519
50.794


2321
CA
ILE
304
49.331
118.722
51.906


2322
C
ILE
304
49.297
120.170
52.404


2323
O
ILE
304
49.766
120.457
53.510


2324
CB
ILE
304
50.739
118.332
51.476


2325
CG1
ILE
304
50.712
117.094
50.590


2326
CG2
ILE
304
51.578
118.036
52.715


2327
CD1
ILE
304
52.113
116.714
50.126


2328
N
GLY
305
48.658
121.044
51.639


2329
CA
GLY
305
48.333
122.403
52.096


2330
C
GLY
305
47.222
122.371
53.149


2331
O
GLY
305
47.021
123.356
53.873


2332
N
LYS
306
46.388
121.340
53.038


2333
CA
LYS
306
45.443
120.881
54.077


2334
C
LYS
306
44.129
121.644
54.035


2335
O
LYS
306
43.298
121.496
54.943


2336
CB
LYS
306
46.046
121.027
55.474


2337
CG
LYS
306
47.338
120.241
55.668


2338
CD
LYS
306
47.948
120.563
57.025


2339
CE
LYS
306
49.345
119.978
57.174


2340
NZ
LYS
306
49.857
120.204
58.534


2341
N
GLU
307
43.860
122.215
52.873


2342
CA
GLU
307
42.732
123.133
52.678


2343
C
GLU
307
42.937
123.857
51.353


2344
O
GLU
307
43.987
124.474
51.135


2345
CB
GLU
307
42.672
124.145
53.823


2346
CG
GLU
307
41.457
125.067
53.734


2347
CD
GLU
307
40.161
124.259
53.752


2348
OE1
GLU
307
39.687
123.933
52.669


2349
OE2
GLU
307
39.632
124.047
54.833


2350
N
ASP
308
41.970
123.750
50.458


2351
CA
ASP
308
42.130
124.421
49.165


2352
C
ASP
308
40.830
124.683
48.424


2353
O
ASP
308
39.834
123.962
48.555


2354
CB
ASP
308
43.008
123.569
48.255


2355
CG
ASP
308
42.270
122.315
47.787


2356
OD1
ASP
308
41.672
122.344
46.721


2357
OD2
ASP
308
42.279
121.339
48.524


2358
N
ILE
309
40.904
125.725
47.618


2359
CA
ILE
309
39.920
125.988
46.566


2360
C
ILE
309
40.612
125.712
45.232


2361
O
ILE
309
41.801
126.021
45.091


2362
CB
ILE
309
39.438
127.435
46.698


2363
CG1
ILE
309
38.407
127.546
47.812


2364
CG2
ILE
309
38.875
128.011
45.403


2365
CD1
ILE
309
37.824
128.951
47.874


2366
N
GLY
310
39.939
125.019
44.332


2367
CA
GLY
310
40.523
124.728
43.017


2368
C
GLY
310
39.461
124.714
41.922


2369
O
GLY
310
38.816
123.683
41.694


2370
N
ARG
311
39.290
125.846
41.260


2371
CA
ARG
311
38.293
125.971
40.188


2372
C
ARG
311
38.727
125.351
38.852


2373
O
ARG
311
39.494
125.928
38.067


2374
CB
ARG
311
37.997
127.455
40.039


2375
CG
ARG
311
37.229
127.946
41.260


2376
CD
ARG
311
36.976
129.445
41.212


2377
NE
ARG
311
36.332
129.836
39.952


2378
CZ
ARG
311
35.842
131.058
39.734


2379
NH1
ARG
311
35.269
131.346
38.565


2380
NH2
ARG
311
35.926
131.990
40.686


2381
N
LEU
312
38.177
124.175
38.607


2382
CA
LEU
312
38.447
123.378
37.401


2383
C
LEU
312
37.406
123.605
36.316


2384
O
LEU
312
36.288
124.057
36.582


2385
CB
LEU
312
38.390
121.890
37.754


2386
CG
LEU
312
39.736
121.282
38.130


2387
CD1
LEU
312
40.766
121.566
37.044


2388
CD2
LEU
312
40.234
121.750
39.496


2389
N
LYS
313
37.751
123.220
35.102


2390
CA
LYS
313
36.746
123.213
34.041


2391
C
LYS
313
35.769
122.067
34.303


2392
O
LYS
313
34.631
122.310
34.732


2393
CB
LYS
313
37.435
123.048
32.692


2394
CG
LYS
313
38.471
124.148
32.489


2395
CD
LYS
313
38.990
124.192
31.057


2396
CE
LYS
313
39.727
122.918
30.681


2397
NZ
LYS
313
40.173
122.943
29.281


2398
N
LEU
314
36.259
120.839
34.191


2399
CA
LEU
314
35.425
119.662
34.493


2400
C
LEU
314
36.274
118.404
34.714


2401
O
LEU
314
36.939
117.910
33.797


2402
CB
LEU
314
34.459
119.434
33.330


2403
CG
LEU
314
33.376
118.419
33.679


2404
CD1
LEU
314
32.574
118.887
34.889


2405
CD2
LEU
314
32.448
118.174
32.493


2406
N
GLN
315
36.209
117.863
35.920


2407
CA
GLN
315
36.957
116.638
36.235


2408
C
GLN
315
36.107
115.374
36.022


2409
O
GLN
315
35.774
115.072
34.864


2410
CB
GLN
315
37.549
116.769
37.640


2411
CG
GLN
315
36.584
117.321
38.693


2412
CD
GLN
315
36.287
116.216
39.707


2413
OE1
GLN
315
37.065
115.264
39.816


2414
NE2
GLN
315
35.073
116.212
40.221


2415
N
LYS
316
35.996
114.544
37.049


2416
CA
LYS
316
35.071
113.401
37.090


2417
C
LYS
316
35.551
112.212
36.286


2418
O
LYS
316
36.173
112.383
35.236


2419
CB
LYS
316
33.692
113.789
36.559


2420
CG
LYS
316
32.932
114.742
37.468


2421
CD
LYS
316
31.545
114.998
36.893


2422
CE
LYS
316
30.710
115.897
37.794


2423
NZ
LYS
316
31.341
117.212
37.953


2424
N
SER
317
34.917
111.086
36.568


2425
CA
SER
317
35.173
109.838
35.833


2426
C
SER
317
34.554
109.823
34.428


2427
O
SER
317
34.672
108.817
33.725


2428
CB
SER
317
34.591
108.676
36.625


2429
OG
SER
317
33.176
108.796
36.591


2430
N
GLU
318
33.909
110.910
34.029


2431
CA
GLU
318
33.371
111.039
32.680


2432
C
GLU
318
34.353
111.778
31.771


2433
O
GLU
318
34.303
111.607
30.548


2434
CB
GLU
318
32.082
111.846
32.763


2435
CG
GLU
318
31.053
111.168
33.657


2436
CD
GLU
318
29.913
112.141
33.928


2437
OE1
GLU
318
30.228
113.292
34.200


2438
OE2
GLU
318
28.792
111.677
34.069


2439
N
ILE
319
35.228
112.586
32.355


2440
CA
ILE
319
36.217
113.333
31.562


2441
C
ILE
319
37.608
113.048
32.123


2442
O
ILE
319
38.374
112.233
31.589


2443
CB
ILE
319
35.928
114.838
31.654


2444
CG1
ILE
319
34.485
115.173
31.292


2445
CG2
ILE
319
36.862
115.627
30.737


2446
CD1
ILE
319
34.201
114.933
29.812


2447
N
ASP
320
37.864
113.618
33.287


2448
CA
ASP
320
39.141
113.415
33.975


2449
C
ASP
320
39.077
112.249
34.950


2450
O
ASP
320
39.151
112.424
36.176


2451
CB
ASP
320
39.549
114.681
34.710


2452
CG
ASP
320
40.332
115.586
33.773


2453
OD1
ASP
320
39.917
115.743
32.635


2454
OD2
ASP
320
41.445
115.919
34.157


2455
N
GLU
321
39.223
111.068
34.372


2456
CA
GLU
321
39.204
109.811
35.127


2457
C
GLU
321
40.531
109.588
35.854


2458
O
GLU
321
40.565
108.989
36.938


2459
CB
GLU
321
38.985
108.706
34.105


2460
CG
GLU
321
37.794
109.045
33.215


2461
CD
GLU
321
37.759
108.108
32.015


2462
OE1
GLU
321
38.832
107.646
31.651


2463
OE2
GLU
321
36.695
107.928
31.441


2464
N
ASN
322
41.536
110.323
35.404


2465
CA
ASN
322
42.838
110.334
36.062


2466
C
ASN
322
42.821
111.161
37.348


2467
O
ASN
322
43.491
110.774
38.313


2468
CB
ASN
322
43.855
110.933
35.098


2469
CG
ASN
322
45.205
111.003
35.796


2470
OD1
ASN
322
45.548
110.103
36.570


2471
ND2
ASN
322
45.921
112.091
35.570


2472
N
LEU
323
41.870
112.074
37.480


2473
CA
LEU
323
41.810
112.835
38.726


2474
C
LEU
323
41.126
112.010
39.805


2475
O
LEU
323
41.545
112.063
40.965


2476
CB
LEU
323
41.027
114.127
38.555


2477
CG
LEU
323
41.105
114.899
39.868


2478
CD1
LEU
323
42.550
115.282
40.170


2479
CD2
LEU
323
40.201
116.122
39.883


2480
N
VAL
324
40.308
111.060
39.386


2481
CA
VAL
324
39.658
110.189
40.358


2482
C
VAL
324
40.624
109.079
40.767


2483
O
VAL
324
40.686
108.734
41.955


2484
CB
VAL
324
38.401
109.621
39.719


2485
CG1
VAL
324
37.453
109.114
40.797


2486
CG2
VAL
324
37.705
110.702
38.906


2487
N
SER
325
41.539
108.757
39.863


2488
CA
SER
325
42.654
107.853
40.175


2489
C
SER
325
43.513
108.460
41.276


2490
O
SER
325
43.507
107.961
42.412


2491
CB
SER
325
43.540
107.699
38.942


2492
OG
SER
325
42.784
107.155
37.872


2493
N
VAL
326
43.977
109.674
41.015


2494
CA
VAL
326
44.840
110.403
41.953


2495
C
VAL
326
44.153
110.712
43.285


2496
O
VAL
326
44.743
110.426
44.337


2497
CB
VAL
326
45.259
111.703
41.266


2498
CG1
VAL
326
45.946
112.675
42.221


2499
CG2
VAL
326
46.142
111.415
40.057


2500
N
LEU
327
42.859
110.985
43.246


2501
CA
LEU
327
42.109
111.275
44.468


2502
C
LEU
327
41.956
110.050
45.358


2503
O
LEU
327
42.303
110.136
46.541


2504
CB
LEU
327
40.714
111.765
44.107


2505
CG
LEU
327
39.924
112.065
45.375


2506
CD1
LEU
327
40.512
113.270
46.104


2507
CD2
LEU
327
38.450
112.285
45.069


2508
N
ARG
328
41.686
108.889
44.782


2509
CA
ARG
328
41.531
107.695
45.617


2510
C
ARG
328
42.873
107.103
46.022


2511
O
ARG
328
42.982
106.577
47.137


2512
CB
ARG
328
40.699
106.661
44.882


2513
CG
ARG
328
39.252
107.120
44.781


2514
CD
ARG
328
38.453
106.128
43.957


2515
NE
ARG
328
39.091
105.998
42.644


2516
CZ
ARG
328
38.427
105.742
41.520


2517
NH1
ARG
328
39.059
105.834
40.352


2518
NH2
ARG
328
37.107
105.543
41.553


2519
N
ASP
329
43.918
107.431
45.278


2520
CA
ASP
329
45.272
107.089
45.714


2521
C
ASP
329
45.579
107.857
46.999


2522
O
ASP
329
45.780
107.236
48.054


2523
CB
ASP
329
46.282
107.490
44.636


2524
CG
ASP
329
46.078
106.738
43.319


2525
OD1
ASP
329
46.327
107.336
42.280


2526
OD2
ASP
329
45.683
105.580
43.367


2527
N
ILE
330
45.299
109.153
46.966


2528
CA
ILE
330
45.572
110.023
48.116


2529
C
ILE
330
44.669
109.722
49.306


2530
O
ILE
330
45.195
109.509
50.404


2531
CB
ILE
330
45.356
111.473
47.692


2532
CG1
ILE
330
46.371
111.896
46.640


2533
CG2
ILE
330
45.423
112.407
48.894


2534
CD1
ILE
330
46.177
113.351
46.227


2535
N
GLN
331
43.420
109.383
49.034


2536
CA
GLN
331
42.432
109.130
50.086


2537
C
GLN
331
42.588
107.758
50.757


2538
O
GLN
331
42.023
107.535
51.836


2539
CB
GLN
331
41.062
109.231
49.416


2540
CG
GLN
331
39.891
109.207
50.393


2541
CD
GLN
331
39.864
110.470
51.251


2542
OE1
GLN
331
39.593
111.572
50.753


2543
NE2
GLN
331
40.215
110.304
52.514


2544
N
GLU
332
43.396
106.882
50.177


2545
CA
GLU
332
43.664
105.581
50.797


2546
C
GLU
332
44.935
105.582
51.647


2547
O
GLU
332
45.181
104.605
52.364


2548
CB
GLU
332
43.793
104.528
49.700


2549
CG
GLU
332
42.458
104.265
49.012


2550
CD
GLU
332
42.661
103.419
47.757


2551
OE1
GLU
332
41.973
103.681
46.777


2552
OE2
GLU
332
43.519
102.548
47.781


2553
N
VAL
333
45.733
106.636
51.572


2554
CA
VAL
333
46.958
106.671
52.379


2555
C
VAL
333
46.950
107.863
53.336


2556
O
VAL
333
47.444
107.776
54.467


2557
CB
VAL
333
48.163
106.750
51.443


2558
CG1
VAL
333
49.469
106.791
52.228


2559
CG2
VAL
333
48.182
105.580
50.464


2560
N
PHE
334
46.376
108.960
52.877


2561
CA
PHE
334
46.261
110.173
53.689


2562
C
PHE
334
44.855
110.343
54.244


2563
O
PHE
334
43.859
110.024
53.580


2564
CB
PHE
334
46.576
111.398
52.837


2565
CG
PHE
334
48.046
111.667
52.534


2566
CD1
PHE
334
48.384
112.531
51.500


2567
CD2
PHE
334
49.044
111.088
53.307


2568
CE1
PHE
334
49.718
112.795
51.222


2569
CE2
PHE
334
50.379
111.351
53.027


2570
CZ
PHE
334
50.716
112.202
51.983


2571
N
GLY
335
44.789
111.004
55.389


2572
CA
GLY
335
43.508
111.340
56.024


2573
C
GLY
335
42.982
112.676
55.497


2574
O
GLY
335
43.079
113.716
56.165


2575
N
THR
336
42.549
112.630
54.249


2576
CA
THR
336
42.004
113.793
53.546


2577
C
THR
336
40.494
113.697
53.403


2578
O
THR
336
39.847
112.810
53.977


2579
CB
THR
336
42.605
113.860
52.147


2580
OG1
THR
336
42.238
112.687
51.433


2581
CG2
THR
336
44.119
113.931
52.192


2582
N
GLU
337
39.962
114.650
52.656


2583
CA
GLU
337
38.539
114.690
52.302


2584
C
GLU
337
38.297
115.823
51.311


2585
O
GLU
337
38.263
117.001
51.696


2586
CB
GLU
337
37.681
114.883
53.548


2587
CG
GLU
337
36.201
114.771
53.199


2588
CD
GLU
337
35.363
114.614
54.464


2589
OE1
GLU
337
34.982
113.487
54.750


2590
OE2
GLU
337
35.034
115.629
55.060


2591
N
ALA
338
38.163
115.459
50.045


2592
CA
ALA
338
37.987
116.453
48.977


2593
C
ALA
338
36.565
116.522
48.426


2594
O
ALA
338
36.043
115.545
47.879


2595
CB
ALA
338
38.939
116.112
47.837


2596
N
PHE
339
35.970
117.694
48.549


2597
CA
PHE
339
34.655
117.951
47.950


2598
C
PHE
339
34.813
118.540
46.550


2599
O
PHE
339
35.838
119.160
46.240


2600
CB
PHE
339
33.879
118.908
48.845


2601
CG
PHE
339
33.652
118.373
50.255


2602
CD1
PHE
339
32.786
117.307
50.461


2603
CD2
PHE
339
34.320
118.945
51.332


2604
CE1
PHE
339
32.584
116.815
51.744


2605
CE2
PHE
339
34.118
118.452
52.614


2606
CZ
PHE
339
33.249
117.388
52.820


2607
N
PHE
340
33.839
118.269
45.698


2608
CA
PHE
340
33.878
118.747
44.307


2609
C
PHE
340
32.528
119.294
43.846


2610
O
PHE
340
31.656
118.531
43.414


2611
CB
PHE
340
34.252
117.578
43.407


2612
CG
PHE
340
35.675
117.055
43.569


2613
CD1
PHE
340
36.754
117.925
43.479


2614
CD2
PHE
340
35.892
115.701
43.784


2615
CE1
PHE
340
38.049
117.445
43.619


2616
CE2
PHE
340
37.187
115.221
43.922


2617
CZ
PHE
340
38.266
116.092
43.842


2618
N
LYS
341
32.418
120.609
43.808


2619
CA
LYS
341
31.137
121.238
43.451


2620
C
LYS
341
31.156
121.984
42.110


2621
O
LYS
341
31.710
123.083
41.980


2622
CB
LYS
341
30.744
122.167
44.596


2623
CG
LYS
341
31.888
123.093
44.996


2624
CD
LYS
341
31.537
123.916
46.227


2625
CE
LYS
341
31.280
123.014
47.428


2626
NZ
LYS
341
30.948
123.810
48.620


2627
N
ASP
342
30.552
121.365
41.108


2628
CA
ASP
342
30.406
122.026
39.802


2629
C
ASP
342
29.117
122.832
39.763


2630
O
ASP
342
28.033
122.293
40.014


2631
CB
ASP
342
30.385
120.991
38.677


2632
CG
ASP
342
30.263
121.672
37.310


2633
OD1
ASP
342
29.143
121.900
36.877


2634
OD2
ASP
342
31.294
121.991
36.729


2635
N
ASP
343
29.229
124.107
39.438


2636
CA
ASP
343
28.019
124.923
39.322


2637
C
ASP
343
27.608
125.130
37.861


2638
O
ASP
343
26.407
125.054
37.569


2639
CB
ASP
343
28.173
126.256
40.070


2640
CG
ASP
343
29.155
127.259
39.447


2641
OD1
ASP
343
28.810
128.432
39.438


2642
OD2
ASP
343
30.223
126.852
39.010


2643
N
ALA
344
28.584
125.196
36.961


2644
CA
ALA
344
28.332
125.407
35.518


2645
C
ALA
344
29.630
125.741
34.789


2646
O
ALA
344
30.019
126.915
34.759


2647
CB
ALA
344
27.384
126.587
35.309


2648
N
GLU
345
30.222
124.745
34.137


2649
CA
GLU
345
31.533
124.906
33.472


2650
C
GLU
345
32.539
125.479
34.457


2651
O
GLU
345
33.230
126.469
34.161


2652
CB
GLU
345
31.412
125.836
32.266


2653
CG
GLU
345
30.776
125.160
31.058


2654
CD
GLU
345
31.669
124.021
30.579


2655
OE1
GLU
345
31.335
122.881
30.876


2656
OE2
GLU
345
32.687
124.304
29.955


2657
N
GLU
346
32.665
124.768
35.565


2658
CA
GLU
346
33.356
125.273
36.745


2659
C
GLU
346
33.070
124.351
37.920


2660
O
GLU
346
31.949
124.327
38.450


2661
CB
GLU
346
32.840
126.659
37.118


2662
CG
GLU
346
33.983
127.626
37.405


2663
CD
GLU
346
35.064
126.968
38.244


2664
OE1
GLU
346
36.115
126.687
37.689


2665
OE2
GLU
346
34.768
126.609
39.379


2666
N
LEU
347
34.073
123.579
38.281


2667
CA
LEU
347
33.997
122.747
39.475


2668
C
LEU
347
35.009
123.203
40.517


2669
O
LEU
347
36.221
123.064
40.323


2670
CB
LEU
347
34.258
121.299
39.085


2671
CG
LEU
347
34.200
120.383
40.304


2672
CD1
LEU
347
33.337
119.165
40.030


2673
CD2
LEU
347
35.589
119.984
40.798


2674
N
ASP
348
34.515
123.651
41.657


2675
CA
ASP
348
35.416
124.039
42.740


2676
C
ASP
348
35.764
122.841
43.626


2677
O
ASP
348
34.889
122.172
44.192


2678
CB
ASP
348
34.765
125.140
43.567


2679
CG
ASP
348
35.809
125.762
44.490


2680
OD1
ASP
348
36.967
125.764
44.093


2681
OD2
ASP
348
35.405
126.416
45.439


2682
N
SER
349
37.045
122.516
43.615


2683
CA
SER
349
37.616
121.477
44.484


2684
C
SER
349
37.920
122.035
45.874


2685
O
SER
349
38.319
123.198
45.992


2686
CB
SER
349
38.918
121.010
43.843


2687
OG
SER
349
38.626
120.600
42.512


2688
N
ASP
350
37.640
121.254
46.906


2689
CA
ASP
350
37.927
121.664
48.298


2690
C
ASP
350
38.315
120.488
49.202


2691
O
ASP
350
37.450
119.831
49.797


2692
CB
ASP
350
36.712
122.380
48.892


2693
CG
ASP
350
36.543
123.803
48.352


2694
OD1
ASP
350
36.817
124.719
49.113


2695
OD2
ASP
350
35.888
123.932
47.322


2696
N
ASP
351
39.613
120.250
49.324


2697
CA
ASP
351
40.139
119.150
50.153


2698
C
ASP
351
40.745
119.641
51.465


2699
O
ASP
351
41.492
120.625
51.478


2700
CB
ASP
351
41.226
118.439
49.344


2701
CG
ASP
351
41.927
117.327
50.128


2702
OD1
ASP
351
43.145
117.399
50.218


2703
OD2
ASP
351
41.281
116.314
50.366


2704
N
LYS
352
40.389
118.971
52.551


2705
CA
LYS
352
41.075
119.167
53.837


2706
C
LYS
352
41.965
117.961
54.127


2707
O
LYS
352
41.877
116.954
53.414


2708
CB
LYS
352
40.057
119.337
54.953


2709
CG
LYS
352
39.130
120.510
54.670


2710
CD
LYS
352
38.175
120.738
55.833


2711
CE
LYS
352
37.131
121.790
55.491


2712
NZ
LYS
352
36.271
121.334
54.385


2713
N
PHE
353
42.835
118.096
55.117


2714
CA
PHE
353
43.780
117.022
55.482


2715
C
PHE
353
44.367
117.219
56.883


2716
O
PHE
353
44.536
118.371
57.298


2717
CB
PHE
353
44.910
117.047
54.447


2718
CG
PHE
353
46.138
116.169
54.714


2719
CD1
PHE
353
47.401
116.745
54.724


2720
CD2
PHE
353
46.002
114.805
54.931


2721
CE1
PHE
353
48.524
115.964
54.958


2722
CE2
PHE
353
47.124
114.023
55.170


2723
CZ
PHE
353
48.386
114.602
55.183


2724
N
MET
354
44.466
116.116
57.623


2725
CA
MET
354
45.300
115.953
58.843


2726
C
MET
354
44.570
115.348
60.030


2727
O
MET
354
43.540
115.834
60.501


2728
CB
MET
354
46.033
117.191
59.329


2729
CG
MET
354
47.425
117.262
58.727


2730
SD
MET
354
48.464
115.826
59.062


2731
CE
MET
354
50.030
116.428
58.389


2732
N
THR
355
45.312
114.461
60.664


2733
CA
THR
355
44.830
113.730
61.834


2734
C
THR
355
45.611
114.209
63.063


2735
O
THR
355
46.122
113.404
63.857


2736
CB
THR
355
45.078
112.243
61.577


2737
OG1
THR
355
44.905
112.007
60.186


2738
CG2
THR
355
44.111
111.354
62.345


2739
N
VAL
356
45.674
115.525
63.226


2740
CA
VAL
356
46.530
116.117
64.262


2741
C
VAL
356
46.063
115.734
65.661


2742
O
VAL
356
44.855
115.676
65.918


2743
CB
VAL
356
46.535
117.636
64.095


2744
CG1
VAL
356
47.129
118.348
65.303


2745
CG2
VAL
356
47.268
118.031
62.819


2746
N
SER
357
47.020
115.141
66.370


2747
CA
SER
357
46.944
114.730
67.793


2748
C
SER
357
46.129
113.459
68.034


2749
O
SER
357
46.257
112.855
69.105


2750
CB
SER
357
46.371
115.851
68.654


2751
OG
SER
357
44.950
115.767
68.637


2752
N
ILE
358
45.497
112.925
66.999


2753
CA
ILE
358
44.728
111.692
67.157


2754
C
ILE
358
45.637
110.503
66.902


2755
O
ILE
358
45.426
109.411
67.442


2756
CB
ILE
358
43.548
111.702
66.191


2757
CG1
ILE
358
42.659
112.908
66.460


2758
CG2
ILE
358
42.737
110.419
66.315


2759
CD1
ILE
358
41.455
112.932
65.524


2760
N
LYS
359
46.801
110.837
66.369


2761
CA
LYS
359
47.897
109.877
66.220


2762
C
LYS
359
48.479
109.484
67.581


2763
O
LYS
359
48.817
108.311
67.777


2764
CB
LYS
359
48.985
110.582
65.425


2765
CG
LYS
359
48.391
111.352
64.253


2766
CD
LYS
359
49.425
112.268
63.607


2767
CE
LYS
359
48.804
113.111
62.500


2768
NZ
LYS
359
49.755
114.100
61.975


2769
N
GLY
360
48.361
110.373
68.559


2770
CA
GLY
360
48.835
110.098
69.921


2771
C
GLY
360
47.899
109.112
70.609


2772
O
GLY
360
48.352
108.128
71.209


2773
N
VAL
361
46.610
109.277
70.350


2774
CA
VAL
361
45.604
108.354
70.879


2775
C
VAL
361
45.742
106.983
70.219


2776
O
VAL
361
45.765
105.967
70.921


2777
CB
VAL
361
44.231
108.935
70.568


2778
CG1
VAL
361
43.122
108.051
71.126


2779
CG2
VAL
361
44.107
110.357
71.105


2780
N
GLY
362
46.085
106.984
68.941


2781
CA
GLY
362
46.393
105.750
68.216


2782
C
GLY
362
47.545
104.987
68.868


2783
O
GLY
362
47.361
103.830
69.271


2784
N
PHE
363
48.633
105.688
69.150


2785
CA
PHE
363
49.813
105.055
69.751


2786
C
PHE
363
49.576
104.579
71.183


2787
O
PHE
363
49.944
103.443
71.499


2788
CB
PHE
363
50.971
106.049
69.746


2789
CG
PHE
363
51.474
106.455
68.363


2790
CD1
PHE
363
51.934
107.748
68.151


2791
CD2
PHE
363
51.484
105.535
67.321


2792
CE1
PHE
363
52.390
108.126
66.895


2793
CE2
PHE
363
51.940
105.913
66.065


2794
CZ
PHE
363
52.392
107.208
65.852


2795
N
THR
364
48.779
105.305
71.951


2796
CA
THR
364
48.508
104.870
73.327


2797
C
THR
364
47.523
103.702
73.379


2798
O
THR
364
47.739
102.772
74.167


2799
CB
THR
364
47.972
106.041
74.146


2800
OG1
THR
364
46.796
106.545
73.524


2801
CG2
THR
364
48.987
107.174
74.237


2802
N
ASN
365
46.659
103.606
72.380


2803
CA
ASN
365
45.732
102.478
72.304


2804
C
ASN
365
46.466
101.214
71.888


2805
O
ASN
365
46.381
100.203
72.599


2806
CB
ASN
365
44.652
102.769
71.269


2807
CG
ASN
365
43.709
103.882
71.715


2808
OD1
ASN
365
43.714
104.325
72.871


2809
ND2
ASN
365
42.856
104.281
70.788


2810
N
VAL
366
47.395
101.349
70.952


2811
CA
VAL
366
48.136
100.172
70.490


2812
C
VAL
366
49.264
99.774
71.443


2813
O
VAL
366
49.670
98.609
71.411


2814
CB
VAL
366
48.676
100.382
69.077


2815
CG1
VAL
366
47.542
100.668
68.100


2816
CG2
VAL
366
49.736
101.473
69.004


2817
N
SER
367
49.542
100.590
72.450


2818
CA
SER
367
50.495
100.178
73.483


2819
C
SER
367
49.882
99.106
74.382


2820
O
SER
367
50.598
98.212
74.846


2821
CB
SER
367
50.877
101.388
74.329


2822
OG
SER
367
51.546
102.320
73.492


2823
N
LYS
368
48.561
99.090
74.476


2824
CA
LYS
368
47.892
98.016
75.207


2825
C
LYS
368
47.389
96.934
74.252


2826
O
LYS
368
47.540
95.739
74.534


2827
CB
LYS
368
46.715
98.621
75.961


2828
CG
LYS
368
47.171
99.771
76.856


2829
CD
LYS
368
46.048
100.389
77.692


2830
CE
LYS
368
45.832
99.703
79.045


2831
NZ
LYS
368
45.232
98.361
78.941


2832
N
LYS
369
47.030
97.347
73.046


2833
CA
LYS
369
46.435
96.424
72.065


2834
C
LYS
369
47.430
95.568
71.273


2835
O
LYS
369
46.998
94.674
70.537


2836
CB
LYS
369
45.582
97.234
71.099


2837
CG
LYS
369
44.395
97.854
71.823


2838
CD
LYS
369
43.577
98.744
70.897


2839
CE
LYS
369
42.417
99.392
71.644


2840
NZ
LYS
369
41.646
100.273
70.753


2841
N
ILE
370
48.725
95.792
71.434


2842
CA
ILE
370
49.711
94.880
70.839


2843
C
ILE
370
50.001
93.695
71.773


2844
O
ILE
370
50.481
92.645
71.325


2845
CB
ILE
370
50.974
95.682
70.504


2846
CG1
ILE
370
50.701
96.630
69.341


2847
CG2
ILE
370
52.167
94.792
70.170


2848
CD1
ILE
370
51.931
97.466
69.005


2849
N
ALA
371
49.548
93.802
73.013


2850
CA
ALA
371
49.715
92.703
73.967


2851
C
ALA
371
48.548
91.720
73.892


2852
O
ALA
371
47.662
91.823
74.727


2853
CB
ALA
371
49.813
93.282
75.375


2854
OXT
ALA
371
48.638
90.804
73.082

## Claims

1. An isolated nucleic acid molecule consisting of a polynucleotide selected from the group consisting of
(a) an isolated polynucleotide encoding a polypeptide consisting of amino acids 1 to 622 of SEQ ID NO:13; and
(b) an isolated polynucleotide encoding a polypeptide consisting of amino acids 2 to 622 of SEQ ID NO:13, wherein said encoded polypeptide has arginyl-tRNA synthetase activity.

2. The isolated nucleic acid molecule of claim 1, wherein said polynucleotide is (a).

3. The isolated nucleic acid molecule of claim 1, wherein said polynucleotide consists of nucleotides 1 to 1866 of SEQ ID NO:2.

4. The isolated nucleic acid molecule of claim 1, wherein said polynucleotide is (b).

5. The isolated nucleic acid molecule of claim 1, wherein said polynucleotide consists of nucleotides 4 to 1866 of SEQ ID NO:2.

6. A recombinant vector comprising the isolated nucleic acid molecule of claim 1, wherein said recombinant vector expresses an isolated polypeptide consisting of amino acids 1 to 622 of SEQ ID NO:13 or an isolated polypeptide consisting of amino acids 2 to 622 of SEQ ID NO:13 when transfected into a host cell under conditions suitable for expression of said polypeptide.

7. An isolated recombinant host cell comprising the recombinant vector of claim 6.

8. A method of making an isolated polypeptide consisting of amino acids 1 to 622 of SEQ ID NO:13 or an isolated polypeptide consisting of amino acids 2 to 622 of SEQ ID NO:13, comprising:
(a) culturing the isolated recombinant host cell of claim 7 under conditions such that said polypeptide is expressed; and
(b) recovering said polypeptide.

9. An isolated polynucleotide consisting of the complete complementary sequence of (a) or (b) of claim 1.