# Patent

## Title
Method for making insulin precursors and insulin precursor analogues having improved fermentation yield in yeast

## Abstract

Novel insulin precursors and insulin precursor analogs comprising a connecting peptide (mini C-peptide) of preferably up to 15 amino acid residues and comprising at least one Gly are provided. The precursors can be converted into human insulin or a human insulin analog. The precursors will typically have a distance between B27 (atom CG2) and A1 (atom CA) of less than 5 Å.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. application Ser. No. 09/894,711 filed on Jun. 28, 2001 now U.S. Pat. No. 6,777,207, and claims priority under 35 U.S.C. of 119 of Danish application no. PA 1999 01869 filed on Dec. 29, 1999, Danish application no. PA 2000 00443 filed on Mar. 17, 2000, U.S. provisional application No. 60/181,450 filed on Feb. 10, 2000, U.S. provisional application No. 60/211,081 filed on Jun. 13, 2000, and U.S. provisional application No. 09/740,359 filed on Dec. 19, 2000, the contents of which are fully incorporated herein by reference.


BACKGROUND
Yeast organisms produce a number of proteins that have a function outside the cell. Such proteins are referred to as secreted proteins. These secreted proteins are expressed initially inside the cell in a precursor or a pre-form containing a pre-peptide sequence ensuring effective direction (translocation) of the expressed product across the membrane of the endoplasmic reticulum (ER). The pre-peptide, normally named a signal peptide, is generally cleaved off from the desired product during translocation. Once entered in the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi, the protein can follow different routes that lead to compartments such as the cell vacuole or the cell membrane, or it can be routed out of the cell to be secreted to the external medium (Pfeffer et al. (1987) Ann. Rev. Biochem. 56:829-852).
Insulin is a polypeptide hormone secreted by β-cells of the pancreas and consists of two polypeptide chains, A and B, which are linked by two inter-chain disulphide bridges. Furthermore, the A-chain features one intra-chain disulphide bridge.
The hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acid followed by proinsulin containing 86 amino acids, in the configuration: prepeptide -B-Arg Arg-C-Lys Arg-A, in which C is a connecting peptide of 31 amino acids. Arg-Arg and Lys-Arg are cleavage sites for cleavage of the connecting peptide from the A and B chains.
Three major methods have been used for the production of human insulin in microorganisms. Two involve Escherichia coli, with either the expression of a large fusion protein in the cytoplasm (Frank et al. (1981) in Peptides: Proceedings of the 7th American Peptide Chemistry Symposium (Rich & Gross, eds.), Pierce Chemical Co., Rockford, Ill. pp 729-739), or use a signal peptide to enable secretion into the periplasmic space (Chan et al. (1981) PNAS 78:5401-5404). A third method utilizes Saccharomyces cerevisiae to secrete an insulin precursor into the medium (Thim et al. (1986) PNAS 83:6766-6770). The prior art discloses a limited number of insulin precursors which are expressed in either E. coli or Saccharomyces cerevisiae, vide U.S. Pat. No. 5,962,267, WO 95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741188.
SUMMARY OF THE INVENTION
The present invention features novel connecting peptides (mini C-peptides) which confer an increased production yield in insulin precursor molecules and insulin precursor analog molecules when expressed in a transformed microorganism, in particular in yeast. Such insulin precursors or insulin precursor analogs can then be converted into insulin or insulin analogs by one or more suitable, well known conversion steps.
The connecting peptides of the present invention contain at least one Gly and will generally be shorter than the natural human C peptide which, including the flanking dibasic cleavage sites, consists of 35 amino acids. Thus the novel connecting peptides will in general not be of more than 15 amino acid residues in length and preferably not more than 10 amino acid residues. Typically the novel connecting peptides will be of up to 9, 8, 7 or 5 amino acid residues and will preferably not be of more than 4 amino acid residues in length.
As in the natural human insulin molecule, the connecting peptide will contain a cleavage site at its C and N termini enabling in vitro cleavage of the connecting peptide from the A and B chains. Such cleavage sites may be any convenient cleavage sites known in the art, e.g. a Met cleavable by cyanogen bromide; a single basic amino acid residue or a pair of basic amino acid residues (Lys or Arg) cleavable by trypsin or trypsin like proteases; Acromobactor lyticus protease or by a carboxypeptidase protease. The cleavage site enabling cleavage of the connecting peptide from the A-chain is preferably a single basic amino acid residue Lys or Arg, preferably Lys.
Alternatively cleavage of the connecting peptide from the B chain may be enabled by cleavage at the natural LysB29 amino acid residue in the B chain giving rise to a desB30 insulin precursor or desB30 insulin precursor analog. The desired B30 amino acid residue may then be added by well known in vitro, enzymatic procedures.
In one embodiment the connecting peptide will not contain two adjacent basic amino acid residues (Lys,Arg). In this embodiment, cleavage from the A-chain may be accomplished at a single Lys or Arg located at the N-terminal end of the A-chain and the natural Lys in position B29 in the B-chain.
The connecting peptide may comprise more than one Gly but preferably not more than 5. The connecting peptide will preferably not comprise more than 3 Gly and most preferred it will only comprise a single Gly. The Gly may be immediately N-terminal to the cleavage site adjacent to the A chain.
Furthermore, the B27 (atom CG2) will typically have a proximity to the A1 (atom CA) of less than 5 Å.
Accordingly, in one aspect the invention is related to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) being cleavable from the A and B chains said connecting peptide comprising at least one Gly, wherein the B27 (atom CG2) has a proximity to the A1 (atom CA) of less than 5 Å.
In another aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) being cleavable from the A and B chains said connecting peptide comprising at least one Gly and a cleavage site enabling cleavage of the peptide bond between the A-chain and the connecting peptide, wherein one Gly is immediately N-terminal to said cleavage site.
In another aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) being cleavable from the A and B chains said connecting peptide comprising at least one Gly, wherein the connecting peptide is of up to 6 amino acid residues in length.
In a further aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising a sequence of formula:

B(1-27)-X3-X2-X1-Y-A(1-21),

wherein

X1 comprises 1-5 amino acid residues in length comprising at least one Gly,
X2 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain,
X3 is one of Pro, Asp, Lys, or Ile at position 28 of the B chain, and
Y is Lys or Arg.
In one embodiment X1 is 1-4, 1-3 or 1-2 amino acid residues in length.
In a further aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising a sequence of formula:

B(1-27)-X3-X2-X1-Y-A(1-21),

wherein X1 comprises a Gly immediately N-terminal to Y,

X2 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain,
X3 is one of Pro, Asp, Lys, or lie at position 28 of the B chain, and
Y is Lys or Arg.
In one embodiment, X1 is 1-15, 1-10, 1-8, 1-5 or 1-3 amino acid residues in length.
In a further aspect, the present invention is related to insulin precursors or insuin precursor analogs comprising a sequence of formula:

B(1-27)-X3-X2-X1-Y-A(1-21),

wherein X1 comprises at least one Gly,

X2 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain,
X3 is one of Pro, Asp, Lys, or lie at position 28 of the B chain, and
Y is Lys or Arg,
and wherein the B27 (atom CG2) has a proximity to the A1 (atom CA) of less than 5 Å.
In this embodiment X1 is typically 1-15, 1-10, 1-9, 1-8, 1-5, 1-4 or 1-3 amino residues in length.
In the above formula X1 will typically contain 1-5Gly, preferably 1-3 and more preferred only one Gly molecule.
The amino acid residues in X1 can be any codable amino acid residue and may be the same or different with the only proviso that at least one amino acid residue in X1 is Gly.
In one embodiment, X3 is Asp and X2 is Lys. This embodiment encompasses the insulin precursor analogs containing an Asp in position B28 of the B chain (termed hereinafter “AspB28IP”). In another embodiment X2 is Lys and X3 is Pro. In a further embodiment the sequence X1-Y is selected from the group of: (a) Glu-Glu-Gly-Lys(SEQ ID NO:1, (b) Glu-Gly-Lys, (c) Ser-Gly-Lys, (d) Asn-Gly-Lys, (e) Thr-Gly-Lys, (f) Asp-Gly-Lys, (g) Met-Gly-Lys, (h) Ala-Gly-Lys, (i) His-Gly-Lys and (j) Gly-Lys.
In still further specific embodiments, X1 is 1-3 amino acid residues; X3 is Lys and X2 is Pro. In a further embodiment, X1 is 1-3 amino acid residues, X3 is Asp and X2 is Lys. In another embodiment X2 is Pro, X3 is Lys and X, is 1-2 amino acid residues of which one is Trp or Phe.
In another embodiment X3 is Lys, X2 is Pro-Thr and X1 consists of up to 15 amino acid residues of which one is Gly. In this embodiment X, will contain a cleavage site at the C-terminal end, e.g a mono basic or dibasic (Lys, Arg) cleavage site.
In a specific embodiment, the mini C-peptide comprises the sequence Glu-Gly-Lys, Asn-Gly-Lys, or Asp-Gly-Lys.
In a still further aspect, the present invention is related to insulin precursors comprising a sequence of formula:

B(1-29)-X1-Y-A(1-21),

wherein X1 is up to 5 amino acid residues in length and Y is a cleavage site.

X1 may be in a further embodiment be of 1-4, 1-3 or 1-2 amino acid residues in length. In another embodiment Y is Lys or Arg. In a further embodiment X, is GluGly; GluGluGly; SerGly; AsnGly, ThrGly, AspGly; MetGly; AlaGly or HisGly. Thus the sequence X1-Y can be (a) Glu-Glu-Gly-Lys(SEQ ID NO:1, (b) Glu-Gly-Lys, (c) Ser-Gly-Lys, (d) Asn-Gly-Lys, (e) Thr-Gly-Lys, (f) Asp-Gly-Lys, (g) Met-Gly-Lys, (h) Ala-Gly-Lys, or (i) His-Gly-Lys.
The present invention is also related to polynucleotide sequences which code for the claimed insulin precursors or insulin precursor analogs. In a further aspect the present invention is related to vectors containing such polynucleotide sequences and to host cells containing such polynucleotide sequences or vectors.
In another aspect, the invention relates to a process for producing the insulin precursors or insulin precursor analogs in a host cell, said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding the insulin precursors or insulin precursor analogs of the invention under suitable conditions for expression of said precursor or precursor analog; and (ii) isolating the precursor or precursor analog from the culture medium.
In still a further aspect, the invention relates to a process for producing insulin or insulin analogs in a host cell, said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding an insulin precursor or insulin precursor analogs of the invention; (ii) isolating the precursor or precursor analog from the culture medium and (iii) converting the precursor or precursor analog into insulin or an insulin analog by in vitro enzymatic conversion.
In one embodiment of the present invention the host cell is a yeast host cell and in a further embodiment the yeast host cell is selected from to the genus Saccharomyces. In a further embodiment the yeast host cell is selected from the species Saccharomyces cerevisiae. 
In a related aspect, the invention features a mini C-peptide in an insulin precursor or insulin precursor analog wherein the amino acid residues of the C-peptide exhibit sufficient flexibility to allow several geometric arrangements of the C-peptide to accommodate an atomic distance between B27 CG2 and A1 CA less than 5 Å.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 represents the pAK721 S. cerevisiae expression plasmid expressing the LA19 leader-EEAEAEAEPK(SEQ ID NO:2)-IP(AlaAlaLys) fusion protein.
FIG. 2 is the DNA sequence and inferred amino acid sequence of the encoded fusion protein (α-factor-leader-EEAEAEAPK(SEQ ID NO:3)-AspB28IP portion of pAK1150 used as PCR template (SEQ ID NO:4 and 5).
FIG. 3 is the DNA sequence encoding a leader-AspB28IP fusion protein with a synthetic mini C-peptide (DGK or AspGIyLys) generated by randomized optimization (SEQ ID NO:6 and 7). The mini C-peptide (DGK) is indicated by underlining.
FIG. 4 shows the solution structures of AspB28IP(AspGlyLys) as backbone lines of ensemble of 20 converged structures.
FIG. 5 shows a ribbon presentation of AspB28IP(AspGlyLys). The figure is produced using MOLSCRIPT (Kraulis (1991) J. Appl. Crystallog. 24:946-950). Amino acid residue annotation is derived as follows: B1-B29 (Bechain) lare numbered, 1-29, residues C1-C3 (C chain) are numbered 30-32, and residues-A1-A21 (A chain) are numbered 33-53.
FIG. 6 is the ID proton NMR spectrum for AspB28IP(Asp Gly Lys) recorded at 27° C. at 1.0 mM concentration in 10%/90% D2O/H2O with 10 mM phosphate buffer at pH 8.0.
FIG. 7 is DNA and inferred amino acid sequence of the expression cassette expressing the YAP3-TA39-GluGluGlyGluProLys(SEQ ID NO:8)-AspB28IP fusion protein with a synthetic mini C-peptide (DGK or AspGlyLys) (SEQ ID NO:9 and 10).
FIG. 8 is DNA and inferred amino acids sequences of the expression cassette expressing the YAP3-TA57-GluGluGlyGluProLys(SEQ ID NO:8)-AspB28IP fusion protein with a synthetic mini C-peptide (DGK or AspGlyLys) (SEQ ID NOS: 11 and 12).
FIG. 9 represents the pAK855 S. cerevisiae expression plasmid expressing the TA57 leader-GluGluGlyGluProLys SEQ ID NO:8)-B(1-29)-AlaAlaLys-A(1-21) precursor and
FIG. 10 represents the nucleotide sequence of the expression cassette of the pAK855 yeast expression plasmid and the inferred amino acid sequence (SEQ ID NO: 17 and 18).



DETAILED DESCRIPTION
Abbreviations and Nomenclature.
By “connecting peptide” or “C-peptide” is meant the connection moiety “C” of the B-C-A polypeptide sequence of a single chain preproinsulin-like molecule. Specifically, in the natural insulin chain, the C-peptide connects position 30 of the B chain and position 1 of the A chain. A “mini C-peptide” or “connecting peptide” such as those described herein, connect B29 or B30 to A1, and differ in sequence and length from that of the natural C-peptide.
By “IP” is meant a single-chain insulin precursor in which a desB30 chain is linked to the A chain of insulin via a connecting peptide. The single-chain insulin precursor will contain correctly positioned disulphide bridges (three) as in human insulin.
With “desB30” or “B(1-29)” is meant a natural insulin B chain lacking the B30 amino acid residue, “A(1-21)” means the natural insulin A chain, “B(1-27)” means the natural B chain lacking the B28, B29, and B30 amino acid residues; “AspB28IP” means a single-chain insulin precursor with aspartic acid at position 28 of the B-chain and no C-peptide (B29 is linked to A1). The mini C-peieptide and its amino acid sequence is indicated: in the three, letter amino acid code in parenthesis following the IP; Thus “AspB28IP(MetTrpLys)” means a single chain insulin precursor with aspartic acid at position 28 of the B-chain and a mini C-peptide with the sequence Met-Trp-Lys connecting B29 to A1.
By “insulin precursor” is meant a single-chain polypeptide which by one or more subsequent chemical and/or enzymatic processes can be converted into human insulin.
By “insulin precursor analog” is meant an insulin precursor molecule having one or more mutations, substitutions, deletions and or additions of the A and/or B amino acid chains relative to the human insulin molecule. The insulin analogs are preferably such wherein one or more of the naturally occurring amino acid residues, preferably one, two, or three of them, have been substituted by another codable amino acid residue. In one embodiment, the instant invention comprises analog molecules having position 28 of the B chain altered relative to the natural human insulin molecule. In this embodiment, position 28 is modified from the natural Pro residue to one of Asp, Lys, or Ile. In a preferred embodiment, the natural Pro residue at position B28 is modified to an Asp residue. In another embodiment Lys at position B29 is modified to Pro; Also, Asn at position A21 may be modified to Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr or Val, in particular to Gly, Ala, Ser, or Thr and preferably to Gly. Furthermore, Asn at position B3 may be modified to Lys. Further examples of insulin precursor analogs are des(B30) human insulin, insulin analogs wherein PheB1 has been deleted; insulin analogs wherein the A-chain and/or the B-chain have an N-terminal extension and insulin analogs wherein the A-chain and/or the B-chain have a C-terminal extension. Thus one or two Arg may be added to position B1.
The term “immediately N-terminal to” is meant to illustrate the situation where an amino acid residue or a peptide sequence is directly linked at its C-terminal end to the N-terminal end of another amino acid residue or amino acid sequence by means of a peptide bond.
In the present context, the term “functional analog of insulin” and the like, is meant to indicate a polypeptide with a similar biological action as the native human insulin protein.
By a distance shorter than 5 Å between two amino acid residues is meant the shortest inter-atomic distance less than 5 Å between any atom in the first amino acid and any atom in the second amino acid. Atomic distances are measured from three-dimensional structures of the molecule determined either by NMR (Wüthrich, K., 1986, NMR of Proteins and Nucleic Acids, Wiley, N.Y.), or by X-ray crystallography (Drenth, J., 1994, Principles of Protein X-ray crystallography, springer Verlag Berlin). A distance from one amino acid to another is measured asthe shortest inter-atomic distance between any atom in the first amino acid andany:atom in the second amino acid if not stated differently.
The present invention features novel mini C-peptides connecting position 29 of the insulin B chain and position 1 of the insulin A chain which significantly increased production yields in a yeast host cell. By the term “significantly increased production,” “increased fermentation yield,” and the like, is meant an increase in secreted amount of the insulin precursor molecule or insulin precursor analog molecule present in the culture supernatant compared to the yield of an insulin precursor or insulin precursor analog with no aromatic amino acid residue in the mini C peptide. An “increased” fermentation yield is an absolute number larger than the control; preferably, the increase is 50% or more larger than the control (AspB28IP) level; even more preferably, the increase is 100% or more larger than control levels.
“POT” is the Schizosaccharomyces pombe triose phosphate isomerase gene, and “TPI1” is the S. cerevisiae triose phosphate isomerase gene.
By a “leader” is meant an amino acid sequence consisting of a pre-peptide (the signal peptide) and a pro-peptide.
The term “signal peptide” is understood to mean a pre-peptide which is present as an N-terminal sequence on the precursor form of a protein. The function of the signal peptide is to allow the heterologous protein to facilitate translocation into the endoplasmic reticulum. The signal peptide is normally cleaved off in the course of this process. The signal peptide may be heterologous or homologous to the yeast organism producing the protein. A number of signal peptides which may be used with the DNA construct of the invention including yeast aspartic protease 3 (YAP3) signal peptide or any functional analog (Egel-Mitani et al. (1990) YEAST 6:127-137 and U.S. Pat. No. 5,726,038) and the α-factor signal of the MFα1 gene (Thorner (1981) in The Molecular Biology of the Yeast Saccharomyces cerevisiae Strathern et al., eds., pp 143-180, Cold Spring Harbor Laboratory, NY and U.S. Pat. No. 4,870,00.
The term “pro-peptide” means a polypeptide sequence whose function is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e. exportation of the polypeptide across the cell wall or at least through the cellular membrane into the periplasmic space of the yeast cell). The pro-peptide may be the yeast α-factor pro-peptide, vide U.S. Pat. Nos. 4,546,082 and 4,870,008. Alternatively, the pro-peptide may be a synthetic pro-peptide, which is to say a pro-peptide not found in nature. Suitable synthetic pro-peptides are those disclosed in U.S. Pat. Nos. 5,395,922; 5,795,746; 5,162,498 and WO 98/32867. The pro-peptide will preferably contain an endopeptidase processing site at the C-terminal end, such as a Lys-Arg sequence or any functional analog thereof.
The polynucleotide sequence of the invention may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage et al. (1981) Tetrahedron Letters 22:1859-1869, or the method described by Matthes et al. (1984) EMBO Journal 3:801-805. According to the phosphoamidite method, oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, purified, duplexed and ligated to form the synthetic DNA construct. A currently preferred way of preparing the DNA construct is by polymerase chain reaction (PCR).
The polynucleotide sequence of the invention may also be of mixed genomic, cDNA, and synthetic origin. For example, a genomic or cDNA sequence encoding a leader peptide may be joined to a genomic or cDNA sequence encoding the A and B chains, after which the DNA sequence may be modified at a site by inserting synthetic oligonucleotides encoding the desired amino acid sequence for homologous recombination in accordance with well-known procedures or preferably generating the desired sequence by PCR using suitable oligonucleotides.
The invention encompasses a vector which is capable of replicating in the selected microorganism or cell line and which carries a polynucleotide sequence encoding the insulin precursors or insulin precursor analogs of the invention. The recombinant vector may be an autonomously replicating vector, i.e., a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used. The vectors may be linear or closed circular plasmids and will preferably contain an element(s) that permits stable.integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
In a preferred embodiment, the recombinant expression vector is capable of replicating in yeast organisms. Examples of sequences which enable the vector to replicate in yeast are the yeast plasmid 2 μm replication genes REP 1-3 and origin of replication.
The vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Selectable markers for use in a filamentous fungal host cell include amdS (acetamidase), argB (ornithine carbamoyltransferase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase) and trpC (anthranilate synthase. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. A preferred selectable marker for yeast is the Schizosaccharomyces pompe TPI gene (Russell (1985) Gene 40:125-130).
In the vector, the polynucleotide sequence is operably connected to a suitable promoter sequence. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing the transcription in a bacterial host cell, are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and Bacillus licheniformis penicillinase gene (penP). Examples of suitable promoters for directing the transcription in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, and Aspergillus niger acid stable alpha-amylase. In a yeast host, useful promoters are the Saccharomyces cerevisiae Ma1, TPI, ADH or PGK promoters.
The polynucleotide construct of the invention will also typically be operably connected to a suitable terminator. In yeast a suitable terminator is the TPI terminator (Alber et al. (1982) J. Mol. Appl. Genet. 1:419-434).
The procedures used to ligate the polynucleotide sequence of the invention, the promoter and the terminator, respectively, and to insert them into suitable yeast vectors containing the information necessary for yeast replication, are well known to persons skilled in the art. It will be understood that the vector may be constructed either by first preparing a DNA construct containing the entire DNA sequence of the invention, and subsequently inserting this fragment into a suitable expression vector, or by sequentially inserting DNA fragments containing genetic information for the individual elements (such as the signal, pro-peptide, mini C-peptide, A and B chains) followed by ligation.
The present invention also relates to recombinant host cells, comprising a polynucleotide sequence encoding the insulin precursors or the insulin precursor analogs of the invention. A vector comprising such polynucleotide sequence is introduced into the host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, Streptomyces cell, or gram negative bacteria such as E. coli and Pseudomonas sp. Eukaryote cells may be mammalian, insect, plant, or fungal cells. In a preferred embodiment, the host cell is a yeast cell. The yeast organism used in the process of the invention may be any suitable yeast organism which, on cultivation, produces large amounts of the insulin precursor and insulin precursor analogs of the invention. Examples of suitable yeast organisms are strains selected from the yeast species Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Sacchoromyces uvarum, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp., and Geotrichum fermentans. 
The transformation of the yeast cells may for instance be effected by protoplast formation followed by transformation in a manner known per se. The medium used to cultivate the cells may be any conventional medium suitable for growing yeast organisms. The secreted insulin precursor or insulin precursor analogs of the invention, a significant proportion of which will be present in the medium in correctly processed form, may be recovered from the medium by conventional procedures including separating the yeast cells from the medium by centrifugation, filtration or catching the insulin precursor or insulin precursor analog by an ion exchange matrix or by a reverse phase absorption matrix, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like.
The insulin precursors and insulin precursor analogs of the invention may be expressed with an N-terminal amino acid residue extension, as described in U.S. Pat. No. 5,395,922, and European Patent No. 765,395A, both of which patents are herein specifically incorporated by reference. The extension is found to be stably attached to the insulin precursor or insulin precursor analogs of the invention during fermentation, protecting the N-terminal end of the insulin precursor or insulin precursor analog against the proteolytic activity of yeast proteases such as DPAP. The presence of an N-terminal extension on the insulin precursor or insulin precursor analog may also serve as a protection of the N-terminal amino group during chemical processing of the protein, i.e. it may serve as a substitute for a BOC (t-butyl-oxycarbonyl) or similar protecting group.
The N-terminal extension may be removed from the recovered insulin precursor or insulin precursor analog by means of a proteolytic enzyme which is specific for a basic amino acid (e.g., Lys) so that the terminal extension is cleaved off at the Lys residue. Examples of such proteolytic enzymes are trypsin or Achromobacter lyticus protease .
After secretion to the culture medium and recovery, the insulin precursor or insulin precursor analogs of the invention will be subjected to various in vitro procedures to remove the possible N-terminal extension sequence and the mini C-peptide to give insulin or the desired insulin analog. Such methods include enzymatic conversion by means of trypsin or an Achromobacter lyticus protease in the presence of an L-threonine ester followed by conversion of the threonine ester of the insulin or insulin analog into insulin or the insulin analog by basic or acid hydrolysis as described in U.S. Pat. No. 4,343,898 or 4,916,212 or Research Disclosure, September 1994/487 the disclosures of which are incorporated by reference hereinto. Cleavage of the connecting peptide from the B chain is preferentially enabled by cleavage at the natural LysB29 amino acid residue in the B chain giving rise to a desB30 insulin precursor or desB30 insulin precursor analogue. If the insulin precursor is to be converted into human insulin, the B30 Thr amino acid residue (Thr) can be added by in vitro, enzymatic procedures such methods include enzymatic conversion by means of trypsin or an Achromobacter lyticus protease (see above). The desB30 insulin may also be converted into an acylated insulin as disclosed in U.S. Pat. No. 5,750,497 and U.S. Pat. No. 5,905,140 the disclosures of which are incorporated by reference hereinto.
As described below, IPs with synthetic C-peptides were constructed featuring a Gly residue (Examples 1 and 3). A Saccharomyces cerevisiae expression plasmid containing a DNA sequences of formula I was constructed by PCR and used to transform a S. cerevisiae host cell. The amount of insulin analog produced was measured as a percentage of the control level AspB28IP lacking mini C-peptide (Table 1 and 5). The novel C-peptides of the invention containing a Gly in the sequence X1 of the mini C-peptide increased yields by up to 4-fold levels. In Example 4 production of human insulin precursors with a Gly in the C-peptide is described. The increase in yield is up to 2-fold (Table 6).
As described below in Example 2 for AspB28IP(Asp Gly Lys), the mini C-peptides of the invention result in a region of flexibility between B27 and A1 which all allow a proximity of A1 to B27 measured as the atomic distance between A1 (atom CA) and B27 (atom CG2) (e.g., less than 5 Å). Accordingly, the invention encompasses mini C-peptide constructs which induce the structural effects shown in Example 2 below.
The present invention is described in further detail in the following examples which are not in any way intended to limit the scope of the invention as claimed. The attached Figures are meant to be considered as integral parts of the specification and description of the invention. All references cited are herein specifically incorporated by reference for all that is described therein.
EXAMPLES
General Procedures
All expressions plasmids are of the C-POT type, similar to those described in EP 171 142, which are characterized by containing the Schizosaccharomyces pombe triose phosphate isomerase gene (POT) for the purpose of plasmid selection and stabilization in S. cerevisiae. The plasmids furthermore contain the S. cerevisiae triose phosphate isomerase promoter and terminator. These sequences are similar to the corresponding sequences in plasmid pKFN1003 (described in WO 90/100075) as are all sequences except the sequence of the EcoRI-XbaI fragment encoding the fusion of the leader and insulin precursor. In order to express different fusion proteins, the EcoRI-XbaI fragment of pKFN1003 is simply replaced by an EcoRI-XbaI fragment encoding the leader insulin precursor or leader insulin precursor analog of interest. Such EcoRI-XbaI fragments may be synthesized using synthetic oligonucleotides and PCR according to standard techniques.
Yeast transformants were prepared by transformation of the host strain: S. cerevisiae strain MT663 (MATa/MATα pep4-31pep4-3 HIS4/his4 tpi::LEU2/tpi::LEU2 Cir+). yeast strain MT663 was deposited in the Deutsche Sammlung von Mikroorganismen und Zelikulturen in connection with filing WO 92/11378 and was given the deposit number DSM 6278.
MT663 was grown on YPGaL (1% Bacto yeast extract, 2% Bacto peptone, 2% galactose, 1% lactate) to an O.D. at 600 nm of 0.6. 100 ml of culture was harvested by centrifugation, washed with 10 ml of water, recentrifuged and resuspended in 10 ml of a solution containing 1.2 M sorbitol, 25 mM Na2EDTA pH=8.0 and 6.7 mg/ml dithiotreitol. The suspension was incubated at 30° C. for 15 minutes, centrifuged and the cells resuspended in 10 ml of a solution containing 1.2 M sorbitol, 10 mM Na2EDTA, 0.1 M sodium citrate, pH 0 5.8, and 2 mg NOVOZYM®234. The suspension was incubated at 30° C. for 30 minutes, the cells collected by centrifugation, washed in 10 ml of 1.2 M sorbitol and 10 ml of CAS (1.2 M sorbitol, 10 mM CaCl2, 10 mM Tris HCl (Tris=Tris(hydroxymethyl)aminomethane) pH=7.5) and resuspended in 2 ml of CAS. For transformation, 1 ml of CAS-suspended cells was mixed with approx. 0.1 mg of plasmid DNA and left at room temperature for 15 minutes. 1 ml of (20% polyethylene glycol 4000, 10 mM CaCl2, 10 mM Tris HCl, pH=7.5) was added and the mixture left for a further 30 minutes at room temperature. The mixture was centrifuged and the pellet resuspended in 0.1 ml of SOS (1.2 M sorbitol, 33% v/v YPD, 6.7 mM CaCl2) and incubated at 30° C. for 2 hours. The suspension was then centrifuged and the pellet resuspended in 0.5 ml of 1.2 M sorbitol. Then, 6 ml of top agar (the SC medium of Sherman et al. (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratory) containing 1.2 M sorbitol plus 2.5% agar) at 52° C. was added and the suspension poured on top of plates containing the same agar-solidified, sorbitol containing medium.
S. cerevisiae stain MT663 transformed with expression plasmids were grown in YPD for 72 h at 30° C. Quantitation of the insulin-precursor yield in the culture supernatants was performed by reverse-phase HPLC analysis with human insulin as an external standard (Snel & Damgaard (1988) Proinsulin heterogenity in pigs. Horm. Metabol. Res. 20:476-488).
Example 1
Construction of Insulin Analog Precursors Comprising Synthetic C-eptides with a Glycine Residue
Synthetic genes encoding fusion proteins consisting of AspB28IP associated with a leader sequence consisting of a pre-peptide (signal peptide) and a pro-peptide, were constructed using PCR under standard conditions (Sambrook et al. (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press) and E.H.F. polymerase (Boehringer Mannheim GmbH, Sandhoefer Strasse 116, Mannheim, Germany). The resulting DNA fragments were isolated and digested with endonucleases and purified using the Gene Clean kit (Bio101 Inc., La Jolla, Calif., USA). Standard methods were used for DNA ligation and transformation of E. coli cells were performed by the CaCl2 method (Sambrook et al. (1989) supra). Plasmids were purified from transformed E. coli cells using QIAGEN columns (QIAGEN, Hilden, Germany). Nucleotide sequences were determined using the ALF Pharmacia Biotech DNA sequencing system with purified double-stranded plasmid DNA as template. Oligonucleotide primers for PCR were obtained from DNA technology (Århus, Denmark).
Secretory expression of the AspB28IP in S. cerevisiae was performed using S. cerevisiae strain MT663 and the 2 μm based yeast expression vector CPOT (see FIG. 1) as described in Thim, L. et al. (1986) Proc. NatL. Acad. Sci. USA 83:6766-6770. The yeast expression vector contains the Schizosaccharomyces pombe triose phosphate isomerase gene (POT) for plasmid selection and stabilization in S. cerevisiae. Furthermore, the S. cerevisiae triose phosphate isomerase gene (TPI1) promoter and terminator are used for transcription initiation and termination of the recombinant gene encoding the leader-AspB28IP fusion protein. Secretion of the AspB28IP was facilitated by the α-factor leader, although a variety of known yeast leader sequences may be used.
As shown in FIG. 1, the pAK721 S. cerevisiae expression plasmid expressing the LA19 leader-EEAEAEAEPK(SEQ ID NO:2)-IP fusion protein was constructed based on the S. cerevisiae-E. coli shuttle POT plasmid (U.S. Pat. No. 5,871,957). L-IP indicates the fusion protein expression cassette encoding the leader-IP fusion protein, TPI-PROMOTER is the S. cerevisiae TPI1 promoter and TPI-TERMINATOR is the S. cerevisiae TPI1 terminator; TPI-POMBE indicates the S. pombe POT gene used for selection in S. cerevisiae; ORIGIN indicates a S. cerevisiae origin of replication derived from the 2 μm plasmid; AMP-R indicates the β-lactamase gene conferring resistance toward ampicillin, facilitating selection in E. coli and ORIGIN-PBR322 indicates an E. coli origin of replication.
DNA encoding a number of fusion proteins of leader sequences and AspB28IP with different mini-C-peptides was generated by PCR using appropriate oligonucleotides as primers, as described below. Standard methods were used to subclone DNA fragments encoding the leader-AspB28IP fusion proteins into the CPOT expression vector in the following configuration: leader-Lys-Arg-spacer-AspB28IP, where Lys Arg is a potential dibasic endoprotease processing site. To optimize processing of the fusion protein by the S. cerevisiae Kex2 endoprotease, DNA encoding a spacer peptide, e.g. EEAEAEAPK (SEQ ID NO:3), was inserted between the DNA encoding the leader and the AspB28IP (Kjeldsen et al. (1996) Gene 170, 107-112.). However, the present of the spacer peptide is not mandatory. The mature AspB28IP was secreted as a single-chain N-terminally extended insulin precursor analogue with a synthetic mini C-peptide connecting LySB29 and GlyA1. After purification of the AspB28IP and proteolytic removal of the N-terminal extension and the synthetic mini C-peptide, a threonine amino acid residue (ThrB30) may be added to LysB29 by enzyme-mediated transpeptidation, to generate AspB28 human insulin (Markussen, et al. (1987) in “Peptides 1986” (Theodoropoulos, D., Ed.), pp. 189-194, Walter de Gruyter & Co., Berlin.).
Development of synthetic mini C-peptides was performed by randomization of one or more codon(s) encoding the amino acids in the mini C-peptide. All synthetic mini C-peptides feature an enzymatic processing site (Lys) at the C-terminus which allows enzymatic removal of the synthetic mini C-peptide (U.S. Pat. No. 4,916,212, herein specifically incorporated by reference). Randomization was performed using doped oligonucleotides which introduced codon(s) variations at one or more positions of the synthetic mini C-peptides. Typically one of the two primers (oligonucleotides) used for PCR was doped. An example of an oligonucleotides pair used for PCR generation of leader-AspB28IP with randomized synthetic mini C-peptides used to generated synthetic mini C-peptides with the general formula: Xaa-Gly-Lys (XGK) are as follows:








Primer A:









(SEQ ID NO:13)









5′TAAATCTATAACTACAAAAAACACATA-3′



and


 


Primer B:








(SEQ ID NO:14)









3′-CCAAAGAAGATGTGACTGTTCNNMCCCTTCCCATAGCAACTTGTTA-



 


CAACATGAAGATAGACAAGAAACATGGTTAACCTTTTGATGACATTGAT-


 


CAGATCTTTGATTC 5′,


where N is A, C, G, or T and M is C or A.






PCR was typically performed as indicated below: 5 μl Primer A (20 pmol), 5 μl Primer B (20 pmol), 10 μl 10× PCR buffer, 8 μl dNTP mix, 0.75 μl E.H.F. enzyme, 1 μl pAK1150 plasmid as template (approximately 0.2 μg DNA) (SEQ ID NO:3), and 70.25 μl distilled water.
Typically between 10 and 15 cycles were performed, one cycle typically was 94° C. for 45 sec.; 55° C. for 1 min; 72° C. for 1.5 min. The PCR mixture was subsequently loaded onto an 2% agarose gel and electrophoresis was performed using standard techniques. The resulting DNA fragment was cut out of the agarose gel and isolated by the Gene Clean kit.
FIG. 2 shows the sequence of pAK1150 DNA used as template for PCR and inferred amino acids of the encoded fusion protein (α-factor-leader-EEAEAEAPK(SEQ ID NO:3)-AspB28IP of pAK1150 (SEQ ID NO:4 and 5). The pAK1150 plasmid is similar to pAK721 shown in FIG. 1. The α-factor-leader's C-terminus was modified to introduce a Nco I restriction endonuclease site, which changes the inferred amino acid sequences linked to LysArg from SerLeuAsp to SerMetAla. Moreover, the encoded AspB28IP does not feature a mini C-peptide but LysB29 is directly connected to GlyA1.
The purified PCR DNA fragment was dissolved in water and restriction endonucleases buffer and digested with suitable restriction endonucleases (e.g. BgI II and Xba I) according to standard techniques. The BgIII-XbaI DNA fragments were subjected to agarose electrophoresis and purified using The Gene Clean Kit.
The expression plasmid pAK1150 or a similar plasmid of the CPOT type (see FIG. 1) was digested with the restriction endonucleases Bgl II and Xba I and the vector fragment of 10765 nucleotide basepairs isolated using The Gene Clean Kit.
The two digested and isolated DNA fragments (the vector fragment and the PCR fragment) were ligated together using T4 DNA ligase and standard conditions. The ligation mix was subsequently transformed into a competent E. coli strain (R−, M+) followed by selection with ampicillin resistance. Plasmids from the resulting E. coli's were isolated using QIAGEN columns.
The plasmids were subsequently used for transformation of a suitable S. cerevisiae strainMT663 (MATa/MATα pep4-3/pep4-3 HIS4/his4 tpi::LEU2/tpi::LEU2 Cir+). Individual transformed S. cerevisiae clones were grown in liquid culture, and the quantity of AspB28IP secreted to the culture supernatants were determined by RP-HPLC. The DNA sequence encoding the synthetic mini C-peptide of the expression plasmids from S. cerevisiae clones secreting increased quantity of the AspB28IP were then determined. Subsequently, the identified synthetic mini C-peptide sequence might be subjected to another round of randomization optimization.
An example on a DNA sequence encoding a leader-AspB28IP(AspGyLys) fusion protein featuring a synthetic mini C-peptide (AspGlyLys) resulting from the randomized optimization process described are shown in FIG. 3 (SEQ ID NO:6 and 7).
Table 1 shows the insulin analogue precursors generated by the above method and production yield expressed as a percent of the control. Fermentation was at 30° C. for 72 h in 5 ml YPD. Yield of the insulin precursor analogs was determined by RP-HPLC of the culture supernatant, and is expressed relative to the yield of insulin precursor of a control strain. In the table, “α*” indicates an α-factor leader in which the C-terminus up to the LysArg has been modified from SLD (SerLeuAsp) to SMA (SerMetAla) and “ex4” is an N-terminal extension peptide with the amino acid sequence EEAEAEAPK (SEQ ID NO:3).












TABLE 1





Leader-N-terminal







extension
Precursor
C-peptide
Yield*
SEQ ID







α*-ex4
AspB28IP
—
100




 


α*-ex4
AspB28IP
GluGluGlyLys
245
SEQ ID NO:1


 


α*-ex4
AspB28IP
GluGlyLys
350


 


α*-ex4
AspB28IP
SerGlyLys
294


 


α*-ex4
AspB28IP
AsnGlyLys
341


 


α*-ex4
AspB28IP
ThrGlyLys
258


 


α*-ex4
AspB28IP
AspGlyLys
428


 


α*-ex4
AspB28IP
MetGlyLys
225


 


α*-ex4
AspB28IP
AlaGlyLys
243


 


α*-ex4
AspB28IP
HisGlyLys
225


 


α*-ex4
AspB28IP
TyrGlyLys
214









Example 2
Structure Determination of AspB28IP (AspGlyLys) in Aqueous Solution by NMR Spectroscopy
NMR spectroscopy. Samples for NMR were prepared by dissolving the lyophilized protein powder in 10/90 D2O/H2O with a 10 mM phosphate buffer and adjusting the pH as desired by addition of small volumes of 1 M DCl or NaOD. All pH meter readings are without correction for isotope effects. Samples of AspB28IP(AspGlyLys) for NMR were prepared at concentrations ranging from 25 μM to 1 mM at pH 8.0. Two-dimensional 1H-1H NMR spectra of 1 mM samples, DQF-COSY (Piantini et al. (1982) J. Am. Chem. Soc. 104:6800-6801, Rance et al. (1983) Biochem. Biophys. Res. Commun. 117:479-485), TOCSY (Braunschweiler et al. (1983) J. Magn. Reson. 53:521-528, Bax et al. (1985) J. Magn. Reson. 65:355-360) and NOESY (Jeener et al. (1979) J. Chem. Phys. 71:4546-4553) were recorded at 600 MHz on a Varian Unity Inova NMR spectrometer equipped with a 1H/13C/15N triple resonance probe with a self-shielded triple-axis gradient coil using standard pulse sequences from the Varian user library. The operating temperature was set to 27° C. For each phase sensitive two-dimensional NMR spectrum 512 t1 increments were acquired each with 2048 or 4096 real data points according to the TPPI-States method (Marion et al. (1989) J. Magn. Reson. 85:393-399). Spectral widths of 6983 Hz in both dimensions were used, with the carrier placed exactly on the water resonance which was attenuated by using either saturation between scans for 1.5 seconds or selective excitation by a gradient-tailored excitation pulse sequence (WATERGATE, Piotto et al. (1992) J. Biomol. NMR 2:661-665). DQFCOSY spectra were recorded using a gradient enhanced version applying magic-angle gradients (Mattiello et al. (1996) J. Am. Chem. Soc. 118:3253-3261). For TOCSY spectra mixing times between 30 and 80 ms were used and for NOESY mixing times between 50 and 200 ms.
The processing of the two-dimensional NMR spectra was performed using the software package Xwinnmr (version 2.5, NMR processing software from Bruker Analytische Messtechnik GmbH, D-76275 Ettlingen, Germany). Each dimension was processed with shifted sine-bell apodization and zero-filling performed once in each dimension. Baseline corrections were applied if necessary using Xwinnmr standard procedures. The spectral assignment, cross peak integration, sequence specific assignment, stereo specific assignment, and all other bookkeeping were performed using the program PRONTO (PRONTO Software Development and Distribution, Copenhagen Denmark) (Kjaer et al. (1991) NATO ASI Series (Hoch, J. C., Redfield C., & Poulsen, F. M., Eds.) Plenum, New York). Chemical shifts are measured in ppm and the water resonance set to 4.75 ppm.
Structure calculations. Distance restraints for the subsequent structure calculation were obtained from integrated NOESY cross peaks classified as either weak, medium or strong corresponding to upper distance restraints of 5.5, 3.3, and 2.7 Å, respectively. For distance restraints involving methyl groups, an additional 0.5 Å was added to the upper limit (Wagner et al. (1985) J. Mol. Biol. 196:611-639). Structure calculations were performed using the hybrid method combining distance geometry (Crippen et al. (1988) Distance Geometry and Molecular Conformation, Research Studies Press, Taunton, Somerset, England; Kuszewski et al. (1992) J. Biomol NMR 2:33-56) and simulated annealing based on the ideas of Nilges et al. (1988) FEBS Lett. 229:317-324 using X-PLOR 3.0 (Bronger (1992) X-PLOR Version 3.1: A System for X-ray Crystallography and NMR, Yale University Press, New Haven) according to the examples given by the X-PLOR manual (dg_sub_embed.inp, dgsa.inp, refine.inp). Residue numbers are derived from standard insulin residue numbering, residues in the B-chain are numbered B1-29, residues in the C-peptide (e. g. AspGlyLys) are numbered C1-C3 and residues in the A-chain are numbered A1-A21.
Spectral assignment of the NMR spectra followed for most resonances the standard sequential assignment procedure described by Wuthrich (1986 NMR of Proteins and Nucleic Acids, Wiley, New York). The standard assignment procedure fails when the amid proton of a particular amino acid residue exchanges to rapidly with protons in the water. At pH 8.0 this occurs for several amino acid residues, however, comparison with earlier mutant insulin NMR spectral assignments and identification of neighboring (in space) amino acid residues through NOEs allow an almost total spectral assignment. Analysis of the NOESY spectra showed that several amino acid residues had a NOE network to the surrounding residues similar to what has previously been determined for other insulin molecules, i.e., human insulin HisB16 mutant (Ludvigsen et al. (1994) Biochemistry 33:7998-8006) and these similar connections are found for residues B1-B10, B13-B14, B17-B24 and A4-A21. Additionally the dihedral angle restraints for the above listed residues were adopted from those used previously (Ludvigsen et al. (1994) supra).
Several amino acids in particular B27-B29, C1-C3, A1-A3 have cross peaks patterns which are consistent with peptide chains that are less well ordered than commonly well-defined secondary structural elements. Thus additional NOEs were converted into distance restraints without any further classification than upper limits of 5.5 Å or 6.0 Å if a methyl group were included. An ensemble of 20 converged structures (FIG. 4) was calculated and the relevant parameters listed in Table 2 for the converged structures. Each NOE here identical to a distance restraint is only counted once even though it might occur several times in the NOESY spectrum. Ramachandran plot quality assessment is standard quality parameters to evaluate local geometry quality. In general the described quality parameters are comparable to 2.5 Å resolution of X-ray based protein structures (Laskowski et al. (1996) J. Biomol. NMR 8:477-486).








TABLE 2





Structural quality assessment
AspB28IP(AspGlyLys)

















Number of
Total
913


NOEs



Intra
454



short range
297



(within 5 residue positions away



but not intra NOEs)



Long range
162



(more than 5 residue positions



away)








Violations of NOEs >0.4 Å (average for 20
 0


structures)


RMS of NOE violations
 0.020(±0.002) Å


RMS of dihedral angle restraints
 0.32(±0.12)°









Deviations from ideal geometry
Impropers
 0.37(±0.05)°



Angles
 0.43(±0.03)°



Bonds
0.0034(±0.0002) Å









Ramachandran
Favoured regions
76.1%


Plot (Procheck,


Laskowski et


al, 1996)



additional allowed regions
20.8%



generously allowed regions
 2.2%



disallowed regions
 1.0%









Description of the Calculated Structure.

A representative structure resembling the average of the ensemble is displayed in FIG. 5. AspB28IP(AspGlyLys) is structurally similar to the native insulin structure for regions comprising residues B1-B10, B14-B23, A4-A21. The differences are mostly pronounced for regions in the vicinity of the connecting peptide in positions B26-B29, C1-C3, A1-A3 and less pronounced for residues B11-B13. The structure of AspB28IP(AspGlyLys) near the C-peptide is strikingly different from the native like structure in solution (Ludvigsen (1994) supra) and AspB28IP(AlaAlaLys) structure in the crystal phase (Whittingham et al. (1998) Biochemistry 37:11516-11523). The connecting peptide of AspB28IP(AspGlyLys) is poorly determined in terms of accuracy, but a few structural restraints obtained from the NOESY spectra (NOEs between ThrB27 and GlyC2 and between ThrB27 and GlyA1) clearly indicate important structural arrangements of the C-peptide. The relative intense NOEs between ThrB27 (methyl group HG2) and GlyC2 (atom HA) and between ThrB27 (methyl group HG2) and GlyA1 (atom HA) in a flexible region shows that these proton pairs are close in space (<5 Å). The tight arrangement of ThrB27, GlyC2 and GlyA1 defined as the atomic distance between B27 (CG2) and A1 (CA) is less than 5 Å, not seen previously in any single chain insulin molecule shows that the C-peptide accommodates this structural arrangement and in fact the C-peptide can do this in several ways which appears to be a prerequisite for the C-peptide. However, it is clear that the presence of Glycine in the connecting peptide allows more flexibility in the connecting peptide and subsequently less structural constraints are imposed on the neighboring amino acids in their quest to accommodate an optimal packing with the remainder of the insulin molecule. Secondly the arrangement of AspB28, LysB29, AspC1 and LysC3 charged side-chains creates a highly polar surface compared to other connecting peptides.
Under the conditions used for NMR both the spectra AspB28IP(AspGlyLys) are influenced by some degree of self-association but the exchange between monomer and dimer is on the timescale of NMR only observed here as an average between the two states. Below concentrations of 0.2 mM the degree of self-association does not change as seen by NMR at even lower concentrations (at least until 25 μM). Table 3 provides the NMR spectral assignments for AspB28IP(AspGlyLys) and Table 4 provides the atomic coordinates of AspB28IP(AspGlyLys) in PDB format. The structure selected to represent the ensemble (FIG. 5 and Table 4 atomic coordinates) has 84.8% residues in “favored” regions and 15.2% in “additionally allowed” regions of the ramachandran plot as described in Table 2.
Table 3 shows chemical shifts of AspB28IP(AspGlyLys) at 27° Celcius obtained at 600 MHz, pH 8 in 10%/90% D2O/H2O with 10 mM phosphate buffer. Chemical shifts are referenced by setting the residual water signal to 4.75 ppm. N/A means no assignment. AspB28IP(AspGlyLys) assignments (1-29=B1-B29; 30-32=C1-C3 and 33-53=A1-A21):










TABLE 3





Spin





system
HN
HA
Other:







Phe-1

4.52
HB#a: N/A, HB#b: 2.992, HD#: 7.087, HE#:





7.203, HZ: 7.145


Val-2
7.70
3.99
HB: 1.912, HG#a: 0.797, HG#b: N/A


Asn-3

4.50
HB#a: 2.989, HB#b: 2.321, HD2#a: 7.372,





HD2#b: 6.920


Glu-4

4.38
HB#a: 1.999, HB#b: 2.103


His-5

4.31
HB#a: 3.314, HB#b: 2.978, HD2: 6.798, HE1:





7.595


Leu-6

4.44
HB#a: 1.648, HB#b: N/A, HG: 1.503, HD#a:





0.755, HD#b: 0.662


Cys-7
8.28
4.87
HB#a: 2.925, HB#b: 3.143


Gly-8

3.94,




3.76


Ser-9

4.05
HB#: 3.812


His-10
8.55
4.38
HB#a: 3.124, HB#b: 3.282, HD2: 7.081, HE1:





7.708


Leu-11
6.93
3.85
HB#a: 1.749, HB#b: 1.175, HG: 1.222, HD#a:





0.602, HD#b: 0.722


Val-12
7.02
3.17
HB: 2.031, HG#a: 0.970, HG#b: N/A


Glu-13
7.84
3.97
HB#a: 2.025, HG#a: 2.222, HG#b: 2.383


Ala-14
7.55
3.94
HB#: 1.253


Leu-15
7.91
3.59
HB#a: 0.905, HB#b: 0.067, HG: 1.075, HD#a:





0.377, HD#b: −0.051


Tyr-16
8.10
4.30
HB#a: 3.064, HD#: 7.193, HE#: 6.771


Leu-17
7.66
4.02
HB#a: N/A, HB#b: 1.844, HG: 1.712, HD#a:





0.892, HD#b: 0.872


Val-18
8.27
3.68
HB: 1.882, HG#a: 0.945, HG#b: 0.792


Cys-19
8.69
4.76
HB#a: 2.759, HB#b: 3.254


Gly-20
7.78
3.90,




3.76


Arg-22
8.03
4.08
HB#a: 1.801, HB#b: 1.845, HG#a: 2.041, HG#b:





2.088, HD#a: 3.352, HD#b: 3.280


Gly-23
7.23
4.07,




3.67


Phe-24
7.37
5.37
HB#a: 2.857, HB#b: 3.013, HD#: 6.502, HE#:





6.688, HZ: 6.944


Phe-25
8.51
4.87
HB#a: 3.151, HB#b: 3.321, HD#: 7.162, HE#:





7.073


Tyr-26
8.17
4.69
HB#a: 2.920, HB#b: 3.168, HD#: 7.029, HE#:





6.662


Thr-27
7.80
5.06
HB: 3.965, HG2#: 1.198


Asp-28
8.43
4.50
HB#a: 2.764, HB#b: 2.660


Lys-29


Asp-30
8.06
4.76
HB#a: 2.615, HB#b: 2.821


Gly-31
8.15
4.14,




3.58


Lys-32


Gly-33

4.03,




4.87


Ile-34
8.16
3.78
HB: N/A, HG1#a: 0.730, HG1#b: 0.885, HG2#:





0.722, HD#: 0.345


Val-35
8.08
3.54
HB: 1.966, HB: N/A, HG#a: 0.862, HG#b: 0.957


Glu-36
8.23
4.11
HB#a: 2.094, HG#a: 2.249


Gln-37
7.79
4.09
HB#: N/A, HG#: N/A


Cys-38
8.11
5.04
HB#a: 3.273, HB#b: 2.695


Cys-39
8.33
4.86
HB#a: 3.732, HB#b: 3.275


Thr-40

4.06
HB: 4.402, HG2#: 1.188


Ser-41
7.23
4.62
HB#a: 3.732, HB#b: 3.874


Ile-42
7.74
4.18
HB: 1.488, HG1#a: 1.038, HG2#: 0.604, HD#:





0.446


Cys-43
9.61
4.95
HB#a: 3.125


Ser-44
8.52
4.58
HB#a: 4.084, HB#b: 3.930


Leu-45

3.88
HB#a: N/A, HB#b: 1.452, HG: 1.543, HD#a:





0.812, HD#b: 0.750


Tyr-46
7.58
4.28
HB#a: 2.956, HD#: 7.074, HE#: 6.806


Gln-47
7.43
3.95
HB#a: 2.287, HB#b: 1.983, HG#a: 2.382, HG#b:





2.140


Leu-48
7.70
3.99
HB#a: 1.905, HB#b: 1.329, HG: 1.658, HD#a:





0.666, HD#b: 0.614


Glu-49
7.82
4.15
HB#a: N/A, HB#b: 1.946, HG#a: 2.307, HG#b:





2.171


Asn-50
7.27
4.44
HB#a: 2.716, HB#b: 2.592


Tyr-51
7.88
3.94
HB#a: 3.534, HB#b: 2.573, HD#: 7.192, HE#:





6.682


Cys-52
7.03
5.10
HB#a: 2.711, HB#b: 3.231


Asn-53
7.88
4.46
HB#a: 2.475, HB#b: 2.683, HD2#a: 7.480,





HD2#b: 6.549
















TABLE 4





Atomic coordinates of Asp28IP(AspGlyLys) in PDB format
























ATOM
1
CA
PHE
1
5.563
−10.343
1.925
1.00
0.00


ATOM
2
HA
PHE
1
5.191
−11.286
1.550
1.00
0.00


ATOM
3
CB
PHE
1
4.581
−9.224
1.558
1.00
0.00


ATOM
4
HB1
PHE
1
5.131
−8.325
1.323
1.00
0.00


ATOM
5
HB2
PHE
1
3.923
−9.037
2.393
1.00
0.00


ATOM
6
CG
PHE
1
3.765
−9.641
0.357
1.00
0.00


ATOM
7
CD1
PHE
1
3.789
−8.866
−0.809
1.00
0.00


ATOM
8
HD1
PHE
1
4.392
−7.970
−0.851
1.00
0.00


ATOM
9
CD2
PHE
1
2.985
−10.800
0.410
1.00
0.00


ATOM
10
HD2
PHE
1
2.968
−11.397
1.311
1.00
0.00


ATOM
11
CE1
PHE
1
3.033
−9.253
−1.921
1.00
0.00


ATOM
12
HE1
PHE
1
3.052
−8.657
−2.822
1.00
0.00


ATOM
13
CE2
PHE
1
2.229
−11.187
−0.701
1.00
0.00


ATOM
14
HE2
PHE
1
1.627
−12.083
−0.658
1.00
0.00


ATOM
15
CZ
PHE
1
2.252
−10.414
−1.867
1.00
0.00


ATOM
16
HZ
PHE
1
1.670
−10.713
−2.726
1.00
0.00


ATOM
17
C
PHE
1
6.925
−10.049
1.295
1.00
0.00


ATOM
18
O
PHE
1
7.945
−10.088
1.957
1.00
0.00


ATOM
19
N
PHE
1
5.702
−10.416
3.406
1.00
0.00


ATOM
20
HT1
PHE
1
6.005
−9.491
3.772
1.00
0.00


ATOM
21
HT2
PHE
1
6.412
−11.136
3.653
1.00
0.00


ATOM
22
HT3
PHE
1
4.787
−10.672
3.829
1.00
0.00


ATOM
23
N
VAL
2
6.943
−9.757
0.020
1.00
0.00


ATOM
24
HN
VAL
2
6.105
−9.735
−0.486
1.00
0.00


ATOM
25
CA
VAL
2
8.231
−9.458
−0.667
1.00
0.00


ATOM
26
HA
VAL
2
9.024
−10.032
−0.210
1.00
0.00


ATOM
27
CB
VAL
2
8.122
−9.830
−2.148
1.00
0.00


ATOM
28
HB
VAL
2
7.420
−9.167
−2.633
1.00
0.00


ATOM
29
CG1
VAL
2
9.494
−9.691
−2.810
1.00
0.00


ATOM
30
HG11
VAL
2
9.807
−8.658
−2.773
1.00
0.00


ATOM
31
HG12
VAL
2
9.431
−10.012
−3.840
1.00
0.00


ATOM
32
HG13
VAL
2
10.211
−10.304
−2.285
1.00
0.00


ATOM
33
CG2
VAL
2
7.638
−11.275
−2.277
1.00
0.00


ATOM
34
HG21
VAL
2
6.763
−11.417
−1.660
1.00
0.00


ATOM
35
HG22
VAL
2
8.418
−11.948
−1.954
1.00
0.00


ATOM
36
HG23
VAL
2
7.389
−11.481
−3.308
1.00
0.00


ATOM
37
C
VAL
2
8.542
−7.967
−0.542
1.00
0.00


ATOM
38
O
VAL
2
7.869
−7.135
−1.120
1.00
0.00


ATOM
39
N
ASN
3
9.562
−7.624
0.206
1.00
0.00


ATOM
40
HN
ASN
3
10.089
−8.316
0.658
1.00
0.00


ATOM
41
CA
ASN
3
9.926
−6.186
0.370
1.00
0.00


ATOM
42
HA
ASN
3
9.083
−5.644
0.771
1.00
0.00


ATOM
43
CB
ASN
3
11.112
−6.067
1.329
1.00
0.00


ATOM
44
HB1
ASN
3
11.492
−5.057
1.310
1.00
0.00


ATOM
45
HB2
ASN
3
11.892
−6.752
1.025
1.00
0.00


ATOM
46
CG
ASN
3
10.654
−6.411
2.748
1.00
0.00


ATOM
47
OD1
ASN
3
9.911
−7.350
2.949
1.00
0.00


ATOM
48
ND2
ASN
3
11.070
−5.683
3.748
1.00
0.00


ATOM
49
HD21
ASN
3
11.669
−4.925
3.586
1.00
0.00


ATOM
50
HD22
ASN
3
10.781
−5.894
4.660
1.00
0.00


ATOM
51
C
ASN
3
10.308
−5.602
−0.991
1.00
0.00


ATOM
52
O
ASN
3
11.414
−5.782
−1.466
1.00
0.00


ATOM
53
N
GLN
4
9.395
−4.909
−1.623
1.00
0.00


ATOM
54
HN
GLN
4
8.512
−4.784
−1.217
1.00
0.00


ATOM
55
CA
GLN
4
9.688
−4.313
−2.957
1.00
0.00


ATOM
56
HA
GLN
4
10.705
−3.950
−2.973
1.00
0.00


ATOM
57
CB
GLN
4
9.509
−5.381
−4.038
1.00
0.00


ATOM
58
HB1
GLN
4
8.468
−5.655
−4.103
1.00
0.00


ATOM
59
HB2
GLN
4
10.096
−6.249
−3.779
1.00
0.00


ATOM
60
CG
GLN
4
9.977
−4.834
−5.389
1.00
0.00


ATOM
61
HG1
GLN
4
10.940
−4.358
−5.272
1.00
0.00


ATOM
62
HG2
GLN
4
9.260
−4.114
−5.754
1.00
0.00


ATOM
63
CD
GLN
4
10.101
−5.986
−6.387
1.00
0.00


ATOM
64
OE1
GLN
4
9.108
−6.515
−6.849
1.00
0.00


ATOM
65
NE2
GLN
4
11.285
−6.401
−6.744
1.00
0.00


ATOM
66
HE21
GLN
4
12.086
−5.975
−6.373
1.00
0.00


ATOM
67
HE22
GLN
4
11.374
−7.140
−7.381
1.00
0.00


ATOM
68
C
GLN
4
8.728
−3.150
−3.217
1.00
0.00


ATOM
69
O
GLN
4
7.710
−3.016
−2.564
1.00
0.00


ATOM
70
N
HIS
5
9.053
−2.304
−4.160
1.00
0.00


ATOM
71
HN
HIS
5
9.883
−2.434
−4.665
1.00
0.00


ATOM
72
CA
HIS
5
8.175
−1.136
−4.467
1.00
0.00


ATOM
73
HA
HIS
5
7.882
−0.660
−3.543
1.00
0.00


ATOM
74
CB
HIS
5
8.952
−0.133
−5.327
1.00
0.00


ATOM
75
HB1
HIS
5
8.355
0.752
−5.481
1.00
0.00


ATOM
76
HB2
HIS
5
9.190
−0.582
−6.281
1.00
0.00


ATOM
77
CG
HIS
5
10.221
0.235
−4.607
1.00
0.00


ATOM
78
ND1
HIS
5
10.323
1.365
−3.815
1.00
0.00


ATOM
79
HD1
HIS
5
9.636
2.051
−3.698
1.00
0.00


ATOM
80
CD2
HIS
5
11.431
−0.401
−4.505
1.00
0.00


ATOM
81
HD2
HIS
5
11.691
−1.314
−5.005
1.00
0.00


ATOM
82
CE1
HIS
5
11.553
1.366
−3.267
1.00
0.00


ATOM
83
HE1
HIS
5
11.906
2.105
−2.570
1.00
0.00


ATOM
84
NE2
HIS
5
12.270
0.312
−3.659
1.00
0.00


ATOM
85
C
HIS
5
6.922
−1.614
−5.206
1.00
0.00


ATOM
86
O
HIS
5
6.951
−2.590
−5.931
1.00
0.00


ATOM
87
N
LEU
6
5.819
−0.941
−5.005
1.00
0.00


ATOM
88
HN
LEU
6
5.824
−0.170
−4.403
1.00
0.00


ATOM
89
CA
LEU
6
4.546
−1.353
−5.667
1.00
0.00


ATOM
90
HA
LEU
6
4.633
−2.370
−6.017
1.00
0.00


ATOM
91
CB
LEU
6
3.398
−1.259
−4.658
1.00
0.00


ATOM
92
HB1
LEU
6
2.510
−1.694
−5.086
1.00
0.00


ATOM
93
HB2
LEU
6
3.213
−0.219
−4.425
1.00
0.00


ATOM
94
CG
LEU
6
3.766
−2.012
−3.378
1.00
0.00


ATOM
95
HG
LEU
6
4.726
−1.669
−3.022
1.00
0.00


ATOM
96
CD1
LEU
6
2.702
−1.755
−2.306
1.00
0.00


ATOM
97
HD11
LEU
6
2.518
−2.664
−1.752
1.00
0.00


ATOM
98
HD12
LEU
6
1.786
−1.429
−2.775
1.00
0.00


ATOM
99
HD13
LEU
6
3.052
−0.989
−1.629
1.00
0.00


ATOM
100
CD2
LEU
6
3.837
−3.510
−3.681
1.00
0.00


ATOM
101
HD21
LEU
6
4.620
−3.694
−4.401
1.00
0.00


ATOM
102
HD22
LEU
6
2.891
−3.840
−4.083
1.00
0.00


ATOM
103
HD23
LEU
6
4.050
−4.052
−2.771
1.00
0.00


ATOM
104
C
LEU
6
4.239
−0.428
−6.848
1.00
0.00


ATOM
105
O
LEU
6
3.838
0.706
−6.667
1.00
0.00


ATOM
106
N
CYS
7
4.408
−0.908
−8.052
1.00
0.00


ATOM
107
HN
CYS
7
4.721
−1.830
−8.171
1.00
0.00


ATOM
108
CA
CYS
7
4.113
−0.063
−9.246
1.00
0.00


ATOM
109
HA
CYS
7
3.509
0.781
−8.947
1.00
0.00


ATOM
110
HB1
CYS
7
5.206
1.052
−10.719
1.00
0.00


ATOM
111
HB2
CYS
7
6.026
−0.406
−10.157
1.00
0.00


ATOM
112
C
CYS
7
3.348
−0.893
−10.279
1.00
0.00


ATOM
113
O
CYS
7
3.726
−2.004
−10.597
1.00
0.00


ATOM
114
CB
CYS
7
5.422
0.438
−9.857
1.00
0.00


ATOM
115
SG
CYS
7
6.321
1.417
−8.626
1.00
0.00


ATOM
116
N
GLY
8
2.272
−0.359
−10.798
1.00
0.00


ATOM
117
HN
GLY
8
1.989
0.538
−10.520
1.00
0.00


ATOM
118
CA
GLY
8
1.470
−1.108
−11.809
1.00
0.00


ATOM
119
HA1
GLY
8
2.057
−1.925
−12.201
1.00
0.00


ATOM
120
HA2
GLY
8
1.195
−0.442
−12.616
1.00
0.00


ATOM
121
C
GLY
8
0.206
−1.665
−11.152
1.00
0.00


ATOM
122
O
GLY
8
−0.493
−0.968
−10.441
1.00
0.00


ATOM
123
N
SER
9
−0.091
−2.917
−11.390
1.00
0.00


ATOM
124
HN
SER
9
0.492
−3.453
−11.968
1.00
0.00


ATOM
125
CA
SER
9
−1.309
−3.534
−10.788
1.00
0.00


ATOM
126
HA
SER
9
−2.073
−2.781
−10.672
1.00
0.00


ATOM
127
CB
SER
9
−1.822
−4.640
−11.711
1.00
0.00


ATOM
128
HB1
SER
9
−1.181
−5.508
−11.621
1.00
0.00


ATOM
129
HB2
SER
9
−1.810
−4.293
−12.731
1.00
0.00


ATOM
130
OG
SER
9
−3.154
−4.977
−11.346
1.00
0.00


ATOM
131
HG
SER
9
−3.683
−5.002
−12.147
1.00
0.00


ATOM
132
C
SER
9
−0.972
−4.134
−9.415
1.00
0.00


ATOM
133
O
SER
9
−1.845
−4.340
−8.594
1.00
0.00


ATOM
134
N
HIS
10
0.284
−4.419
−9.164
1.00
0.00


ATOM
135
HN
HIS
10
0.969
−4.248
−9.841
1.00
0.00


ATOM
136
CA
HIS
10
0.677
−5.009
−7.849
1.00
0.00


ATOM
137
HA
HIS
10
0.159
−5.946
−7.710
1.00
0.00


ATOM
138
CB
HIS
10
2.188
−5.262
−7.841
1.00
0.00


ATOM
139
HB1
HIS
10
2.488
−5.607
−6.863
1.00
0.00


ATOM
140
HB2
HIS
10
2.705
−4.342
−8.070
1.00
0.00


ATOM
141
CG
HIS
10
2.548
−6.305
−8.872
1.00
0.00


ATOM
142
ND1
HIS
10
1.608
−6.886
−9.715
1.00
0.00


ATOM
143
HD1
HIS
10
0.648
−6.690
−9.733
1.00
0.00


ATOM
144
CD2
HIS
10
3.750
−6.882
−9.204
1.00
0.00


ATOM
145
HD2
HIS
10
4.697
−6.655
−8.738
1.00
0.00


ATOM
146
CE1
HIS
10
2.255
−7.766
−10.500
1.00
0.00


ATOM
147
HE1
HIS
10
1.775
−8.370
−11.255
1.00
0.00


ATOM
148
NE2
HIS
10
3.562
−7.802
−10.231
1.00
0.00


ATOM
149
C
HIS
10
0.314
−4.052
−6.702
1.00
0.00


ATOM
150
O
HIS
10
0.243
−4.455
−5.557
1.00
0.00


ATOM
151
N
LEU
11
0.089
−2.792
−6.994
1.00
0.00


ATOM
152
HN
LEU
11
0.156
−2.485
−7.921
1.00
0.00


ATOM
153
CA
LEU
11
−0.264
−1.813
−5.918
1.00
0.00


ATOM
154
HA
LEU
11
0.542
−1.764
−5.202
1.00
0.00


ATOM
155
CB
LEU
11
−0.474
−0.420
−6.548
1.00
0.00


ATOM
156
HB1
LEU
11
−1.303
−0.471
−7.239
1.00
0.00


ATOM
157
HB2
LEU
11
0.417
−0.143
−7.090
1.00
0.00


ATOM
158
CG
LEU
11
−0.771
0.668
−5.484
1.00
0.00


ATOM
159
HG
LEU
11
−0.601
1.639
−5.929
1.00
0.00


ATOM
160
CD1
LEU
11
−2.235
0.588
−5.040
1.00
0.00


ATOM
161
HD11
LEU
11
−2.305
0.002
−4.134
1.00
0.00


ATOM
162
HD12
LEU
11
−2.823
0.121
−5.817
1.00
0.00


ATOM
163
HD13
LEU
11
−2.609
1.584
−4.853
1.00
0.00


ATOM
164
CD2
LEU
11
0.146
0.514
−4.260
1.00
0.00


ATOM
165
HD21
LEU
11
−0.214
−0.294
−3.640
1.00
0.00


ATOM
166
HD22
LEU
11
0.144
1.432
−3.691
1.00
0.00


ATOM
167
HD23
LEU
11
1.151
0.296
−4.589
1.00
0.00


ATOM
168
C
LEU
11
−1.543
−2.259
−5.207
1.00
0.00


ATOM
169
O
LEU
11
−1.542
−2.522
−4.020
1.00
0.00


ATOM
170
N
VAL
12
−2.636
−2.322
−5.922
1.00
0.00


ATOM
171
HN
VAL
12
−2.608
−2.089
−6.874
1.00
0.00


ATOM
172
CA
VAL
12
−3.931
−2.729
−5.298
1.00
0.00


ATOM
173
HA
VAL
12
−4.203
−1.999
−4.550
1.00
0.00


ATOM
174
CB
VAL
12
−5.020
−2.770
−6.379
1.00
0.00


ATOM
175
HB
VAL
12
−5.052
−1.816
−6.884
1.00
0.00


ATOM
176
CG1
VAL
12
−4.715
−3.872
−7.406
1.00
0.00


ATOM
177
HG11
VAL
12
−3.689
−4.192
−7.295
1.00
0.00


ATOM
178
HG12
VAL
12
−4.864
−3.485
−8.403
1.00
0.00


ATOM
179
HG13
VAL
12
−5.373
−4.713
−7.245
1.00
0.00


ATOM
180
CG2
VAL
12
−6.378
−3.035
−5.720
1.00
0.00


ATOM
181
HG21
VAL
12
−6.885
−2.097
−5.560
1.00
0.00


ATOM
182
HG22
VAL
12
−6.230
−3.531
−4.772
1.00
0.00


ATOM
183
HG23
VAL
12
−6.975
−3.662
−6.365
1.00
0.00


ATOM
184
C
VAL
12
−3.795
−4.105
−4.629
1.00
0.00


ATOM
185
O
VAL
12
−4.496
−4.415
−3.684
1.00
0.00


ATOM
186
N
GLU
13
−2.897
−4.928
−5.112
1.00
0.00


ATOM
187
HN
GLU
13
−2.343
−4.655
−5.873
1.00
0.00


ATOM
188
CA
GLU
13
−2.716
−6.281
−4.505
1.00
0.00


ATOM
189
HA
GLU
13
−3.650
−6.802
−4.509
1.00
0.00


ATOM
190
CB
GLU
13
−1.675
−7.072
−5.302
1.00
0.00


ATOM
191
HB1
GLU
13
−1.299
−7.882
−4.696
1.00
0.00


ATOM
192
HB2
GLU
13
−0.860
−6.418
−5.577
1.00
0.00


ATOM
193
CG
GLU
13
−2.319
−7.642
−6.567
1.00
0.00


ATOM
194
HG1
GLU
13
−2.926
−6.883
−7.038
1.00
0.00


ATOM
195
HG2
GLU
13
−2.939
−8.488
−6.306
1.00
0.00


ATOM
196
CD
GLU
13
−1.225
−8.091
−7.536
1.00
0.00


ATOM
197
OE1
GLU
13
−0.270
−8.698
−7.080
1.00
0.00


ATOM
198
OE2
GLU
13
−1.359
−7.821
−8.718
1.00
0.00


ATOM
199
C
GLU
13
−2.254
−6.129
−3.066
1.00
0.00


ATOM
200
O
GLU
13
−2.698
−6.838
−2.188
1.00
0.00


ATOM
201
N
ALA
14
−1.378
−5.204
−2.821
1.00
0.00


ATOM
202
HN
ALA
14
−1.044
−4.644
−3.552
1.00
0.00


ATOM
203
CA
ALA
14
−0.885
−4.986
−1.439
1.00
0.00


ATOM
204
HA
ALA
14
−0.531
−5.919
−1.027
1.00
0.00


ATOM
205
CB
ALA
14
0.254
−3.979
−1.475
1.00
0.00


ATOM
206
HB1
ALA
14
−0.159
−2.981
−1.506
1.00
0.00


ATOM
207
HB2
ALA
14
0.855
−4.148
−2.355
1.00
0.00


ATOM
208
HB3
ALA
14
0.862
−4.092
−0.591
1.00
0.00


ATOM
209
C
ALA
14
−2.010
−4.427
−0.563
1.00
0.00


ATOM
210
O
ALA
14
−1.948
−4.511
0.644
1.00
0.00


ATOM
211
N
LEU
15
−3.018
−3.829
−1.154
1.00
0.00


ATOM
212
HN
LEU
15
−3.038
−3.744
−2.129
1.00
0.00


ATOM
213
CA
LEU
15
−4.114
−3.237
−0.335
1.00
0.00


ATOM
214
HA
LEU
15
−3.678
−2.805
0.553
1.00
0.00


ATOM
215
CB
LEU
15
−4.815
−2.131
−1.125
1.00
0.00


ATOM
216
HB1
LEU
15
−5.783
−1.935
−0.691
1.00
0.00


ATOM
217
HB2
LEU
15
−4.936
−2.445
−2.153
1.00
0.00


ATOM
218
CG
LEU
15
−3.969
−0.854
−1.077
1.00
0.00


ATOM
219
HG
LEU
15
−2.977
−1.073
−1.450
1.00
0.00


ATOM
220
CD1
LEU
15
−4.617
0.221
−1.951
1.00
0.00


ATOM
221
HD11
LEU
15
−5.679
0.032
−2.029
1.00
0.00


ATOM
222
HD12
LEU
15
−4.176
0.199
−2.938
1.00
0.00


ATOM
223
HD13
LEU
15
−4.457
1.192
−1.507
1.00
0.00


ATOM
224
CD2
LEU
15
−3.873
−0.343
0.370
1.00
0.00


ATOM
225
HD21
LEU
15
−3.530
0.683
0.370
1.00
0.00


ATOM
226
HD22
LEU
15
−3.175
−0.955
0.927
1.00
0.00


ATOM
227
HD23
LEU
15
−4.847
−0.396
0.835
1.00
0.00


ATOM
228
C
LEU
15
−5.129
−4.291
0.108
1.00
0.00


ATOM
229
O
LEU
15
−5.380
−4.409
1.290
1.00
0.00


ATOM
230
N
TYR
16
−5.728
−5.057
−0.787
1.00
0.00


ATOM
231
HN
TYR
16
−5.524
−4.964
−1.743
1.00
0.00


ATOM
232
CA
TYR
16
−6.725
−6.069
−0.290
1.00
0.00


ATOM
233
HA
TYR
16
−7.334
−5.578
0.452
1.00
0.00


ATOM
234
CB
TYR
16
−7.655
−6.628
−1.371
1.00
0.00


ATOM
235
HB1
TYR
16
−8.424
−5.905
−1.580
1.00
0.00


ATOM
236
HB2
TYR
16
−8.115
−7.527
−1.002
1.00
0.00


ATOM
237
CG
TYR
16
−6.942
−6.954
−2.641
1.00
0.00


ATOM
238
CD1
TYR
16
−6.878
−5.998
−3.640
1.00
0.00


ATOM
239
HD1
TYR
16
−7.282
−5.020
−3.467
1.00
0.00


ATOM
240
CD2
TYR
16
−6.414
−8.230
−2.846
1.00
0.00


ATOM
241
HD2
TYR
16
−6.462
−8.969
−2.059
1.00
0.00


ATOM
242
CE1
TYR
16
−6.290
−6.298
−4.856
1.00
0.00


ATOM
243
HE1
TYR
16
−6.249
−5.541
−5.617
1.00
0.00


ATOM
244
CE2
TYR
16
−5.805
−8.541
−4.063
1.00
0.00


ATOM
245
HE2
TYR
16
−5.385
−9.525
−4.220
1.00
0.00


ATOM
246
CZ
TYR
16
−5.748
−7.574
−5.078
1.00
0.00


ATOM
247
OH
TYR
16
−5.159
−7.874
−6.289
1.00
0.00


ATOM
248
HH
TYR
16
−5.854
−7.948
−6.946
1.00
0.00


ATOM
249
C
TYR
16
−5.992
−7.206
0.394
1.00
0.00


ATOM
250
O
TYR
16
−6.522
−7.835
1.293
1.00
0.00


ATOM
251
N
LEU
17
−4.758
−7.455
0.025
1.00
0.00


ATOM
252
HN
LEU
17
−4.336
−6.915
−0.674
1.00
0.00


ATOM
253
CA
LEU
17
−3.980
−8.527
0.713
1.00
0.00


ATOM
254
HA
LEU
17
−4.465
−9.481
0.569
1.00
0.00


ATOM
255
CB
LEU
17
−2.558
−8.573
0.151
1.00
0.00


ATOM
256
HB1
LEU
17
−2.022
−7.685
0.448
1.00
0.00


ATOM
257
HB2
LEU
17
−2.602
−8.628
−0.925
1.00
0.00


ATOM
258
CG
LEU
17
−1.837
−9.805
0.688
1.00
0.00


ATOM
259
HG
LEU
17
−1.862
−9.793
1.769
1.00
0.00


ATOM
260
CD1
LEU
17
−2.536
−11.064
0.174
1.00
0.00


ATOM
261
HD11
LEU
17
−1.806
−11.843
0.011
1.00
0.00


ATOM
262
HD12
LEU
17
−3.044
−10.842
−0.759
1.00
0.00


ATOM
263
HD13
LEU
17
−3.259
−11.393
0.907
1.00
0.00


ATOM
264
CD2
LEU
17
−0.385
−9.787
0.207
1.00
0.00


ATOM
265
HD21
LEU
17
0.104
−8.898
0.578
1.00
0.00


ATOM
266
HD22
LEU
17
−0.363
−9.786
−0.873
1.00
0.00


ATOM
267
HD23
LEU
17
0.128
−10.661
0.578
1.00
0.00


ATOM
268
C
LEU
17
−3.939
−8.186
2.207
1.00
0.00


ATOM
269
O
LEU
17
−3.941
−9.052
3.060
1.00
0.00


ATOM
270
N
VAL
18
−3.940
−6.911
2.511
1.00
0.00


ATOM
271
HN
VAL
18
−3.959
−6.242
1.798
1.00
0.00


ATOM
272
CA
VAL
18
−3.943
−6.464
3.921
1.00
0.00


ATOM
273
HA
VAL
18
−3.358
−7.145
4.520
1.00
0.00


ATOM
274
CB
VAL
18
−3.370
−5.042
4.015
1.00
0.00


ATOM
275
HB
VAL
18
−4.051
−4.353
3.529
1.00
0.00


ATOM
276
CG1
VAL
18
−3.222
−4.647
5.484
1.00
0.00


ATOM
277
HG11
VAL
18
−4.150
−4.224
5.837
1.00
0.00


ATOM
278
HG12
VAL
18
−2.433
−3.917
5.584
1.00
0.00


ATOM
279
HG13
VAL
18
−2.980
−5.521
6.071
1.00
0.00


ATOM
280
CG2
VAL
18
−2.004
−4.964
3.327
1.00
0.00


ATOM
281
HG21
VAL
18
−1.904
−5.774
2.622
1.00
0.00


ATOM
282
HG22
VAL
18
−1.223
−5.030
4.066
1.00
0.00


ATOM
283
HG23
VAL
18
−1.924
−4.019
2.805
1.00
0.00


ATOM
284
C
VAL
18
−5.395
−6.419
4.421
1.00
0.00


ATOM
285
O
VAL
18
−5.660
−6.559
5.599
1.00
0.00


ATOM
286
N
CYS
19
−6.334
−6.183
3.528
1.00
0.00


ATOM
287
HN
CYS
19
−6.095
−6.041
2.586
1.00
0.00


ATOM
288
CA
CYS
19
−7.765
−6.079
3.939
1.00
0.00


ATOM
289
HA
CYS
19
−7.813
−5.715
4.955
1.00
0.00


ATOM
290
HB1
CYS
19
−9.470
−4.891
3.352
1.00
0.00


ATOM
291
HB2
CYS
19
−8.498
−5.465
2.012
1.00
0.00


ATOM
292
C
CYS
19
−8.452
−7.454
3.872
1.00
0.00


ATOM
293
O
CYS
19
−8.550
−8.144
4.870
1.00
0.00


ATOM
294
CB
CYS
19
−8.466
−5.071
3.012
1.00
0.00


ATOM
295
SG
CYS
19
−7.549
−3.504
2.992
1.00
0.00


ATOM
296
N
GLY
20
−8.933
−7.860
2.718
1.00
0.00


ATOM
297
HN
GLY
20
−8.849
−7.297
1.925
1.00
0.00


ATOM
298
CA
GLY
20
−9.613
−9.186
2.613
1.00
0.00


ATOM
299
HA1
GLY
20
−10.029
−9.451
3.574
1.00
0.00


ATOM
300
HA2
GLY
20
−8.895
−9.935
2.311
1.00
0.00


ATOM
301
C
GLY
20
−10.741
−9.110
1.580
1.00
0.00


ATOM
302
O
GLY
20
−10.548
−8.649
0.472
1.00
0.00


ATOM
303
N
GLU
21
−11.914
−9.569
1.937
1.00
0.00


ATOM
304
HN
GLU
21
−12.038
−9.939
2.837
1.00
0.00


ATOM
305
CA
GLU
21
−13.064
−9.539
0.983
1.00
0.00


ATOM
306
HA
GLU
21
−12.693
−9.624
−0.028
1.00
0.00


ATOM
307
CB
GLU
21
−14.004
−10.713
1.283
1.00
0.00


ATOM
308
HB1
GLU
21
−15.030
−10.392
1.186
1.00
0.00


ATOM
309
HB2
GLU
21
−13.831
−11.061
2.292
1.00
0.00


ATOM
310
CG
GLU
21
−13.736
−11.855
0.299
1.00
0.00


ATOM
311
HG1
GLU
21
−13.882
−12.801
0.797
1.00
0.00


ATOM
312
HG2
GLU
21
−12.719
−11.790
−0.060
1.00
0.00


ATOM
313
CD
GLU
21
−14.702
−11.748
−0.882
1.00
0.00


ATOM
314
OE1
GLU
21
−14.275
−11.299
−1.932
1.00
0.00


ATOM
315
OE2
GLU
21
−15.853
−12.118
−0.716
1.00
0.00


ATOM
316
C
GLU
21
−13.835
−8.223
1.132
1.00
0.00


ATOM
317
O
GLU
21
−14.459
−7.753
0.199
1.00
0.00


ATOM
318
N
ARG
22
−13.808
−7.631
2.302
1.00
0.00


ATOM
319
HN
ARG
22
−13.304
−8.035
3.040
1.00
0.00


ATOM
320
CA
ARG
22
−14.548
−6.350
2.524
1.00
0.00


ATOM
321
HA
ARG
22
−15.605
−6.522
2.398
1.00
0.00


ATOM
322
CB
ARG
22
−14.288
−5.853
3.944
1.00
0.00


ATOM
323
HB1
ARG
22
−14.444
−4.788
3.983
1.00
0.00


ATOM
324
HB2
ARG
22
−13.269
−6.080
4.225
1.00
0.00


ATOM
325
CG
ARG
22
−15.251
−6.543
4.912
1.00
0.00


ATOM
326
HG1
ARG
22
−14.802
−7.452
5.281
1.00
0.00


ATOM
327
HG2
ARG
22
−16.172
−6.778
4.395
1.00
0.00


ATOM
328
CD
ARG
22
−15.549
−5.610
6.085
1.00
0.00


ATOM
329
HD1
ARG
22
−16.250
−6.085
6.755
1.00
0.00


ATOM
330
HD2
ARG
22
−15.973
−4.689
5.714
1.00
0.00


ATOM
331
NE
ARG
22
−14.285
−5.315
6.818
1.00
0.00


ATOM
332
HE
ARG
22
−13.502
−4.985
6.331
1.00
0.00


ATOM
333
CZ
ARG
22
−14.224
−5.496
8.107
1.00
0.00


ATOM
334
NH1
ARG
22
−13.711
−6.595
8.589
1.00
0.00


ATOM
335
HH11
ARG
22
−13.363
−7.298
7.968
1.00
0.00


ATOM
336
HH12
ARG
22
−13.666
−6.735
9.578
1.00
0.00


ATOM
337
NH2
ARG
22
−14.679
−4.579
8.917
1.00
0.00


ATOM
338
HH21
ARG
22
−15.074
−3.737
8.547
1.00
0.00


ATOM
339
HH22
ARG
22
−14.634
−4.717
9.905
1.00
0.00


ATOM
340
C
ARG
22
−14.086
−5.290
1.520
1.00
0.00


ATOM
341
O
ARG
22
−14.890
−4.676
0.843
1.00
0.00


ATOM
342
N
GLY
23
−12.799
−5.074
1.417
1.00
0.00


ATOM
343
HN
GLY
23
−12.173
−5.585
1.972
1.00
0.00


ATOM
344
CA
GLY
23
−12.281
−4.057
0.456
1.00
0.00


ATOM
345
HA1
GLY
23
−13.077
−3.381
0.182
1.00
0.00


ATOM
346
HA2
GLY
23
−11.913
−4.556
−0.429
1.00
0.00


ATOM
347
C
GLY
23
−11.146
−3.266
1.102
1.00
0.00


ATOM
348
O
GLY
23
−10.816
−3.465
2.256
1.00
0.00


ATOM
349
N
PHE
24
−10.547
−2.370
0.361
1.00
0.00


ATOM
350
HN
PHE
24
−10.835
−2.236
−0.565
1.00
0.00


ATOM
351
CA
PHE
24
−9.425
−1.556
0.914
1.00
0.00


ATOM
352
HA
PHE
24
−9.307
−1.785
1.957
1.00
0.00


ATOM
353
CB
PHE
24
−8.124
−1.905
0.165
1.00
0.00


ATOM
354
HB1
PHE
24
−7.810
−2.901
0.433
1.00
0.00


ATOM
355
HB2
PHE
24
−7.355
−1.201
0.426
1.00
0.00


ATOM
356
CG
PHE
24
−8.358
−1.845
−1.319
1.00
0.00


ATOM
357
CD1
PHE
24
−8.560
−0.612
−1.925
1.00
0.00


ATOM
358
HD1
PHE
24
−8.529
0.279
−1.325
1.00
0.00


ATOM
359
CD2
PHE
24
−8.387
−3.019
−2.077
1.00
0.00


ATOM
360
HD2
PHE
24
−8.212
−3.981
−1.597
1.00
0.00


ATOM
361
CE1
PHE
24
−8.800
−0.532
−3.300
1.00
0.00


ATOM
362
HE1
PHE
24
−8.956
0.429
−3.768
1.00
0.00


ATOM
363
CE2
PHE
24
−8.625
−2.943
−3.457
1.00
0.00


ATOM
364
HE2
PHE
24
−8.657
−3.844
−4.051
1.00
0.00


ATOM
365
CZ
PHE
24
−8.833
−1.700
−4.068
1.00
0.00


ATOM
366
HZ
PHE
24
−9.018
−1.643
−5.130
1.00
0.00


ATOM
367
C
PHE
24
−9.755
−0.061
0.759
1.00
0.00


ATOM
368
O
PHE
24
−10.883
0.304
0.486
1.00
0.00


ATOM
369
N
PHE
25
−8.782
0.806
0.931
1.00
0.00


ATOM
370
HN
PHE
25
−7.883
0.493
1.152
1.00
0.00


ATOM
371
CA
PHE
25
−9.036
2.274
0.796
1.00
0.00


ATOM
372
HA
PHE
25
−10.093
2.450
0.662
1.00
0.00


ATOM
373
CB
PHE
25
−8.553
2.987
2.063
1.00
0.00


ATOM
374
HB1
PHE
25
−8.158
3.958
1.800
1.00
0.00


ATOM
375
HB2
PHE
25
−7.776
2.401
2.522
1.00
0.00


ATOM
376
CG
PHE
25
−9.693
3.162
3.040
1.00
0.00


ATOM
377
CD1
PHE
25
−9.908
4.410
3.637
1.00
0.00


ATOM
378
HD1
PHE
25
−9.267
5.244
3.392
1.00
0.00


ATOM
379
CD2
PHE
25
−10.524
2.082
3.360
1.00
0.00


ATOM
380
HD2
PHE
25
−10.359
1.119
2.900
1.00
0.00


ATOM
381
CE1
PHE
25
−10.955
4.579
4.550
1.00
0.00


ATOM
382
HE1
PHE
25
−11.118
5.542
5.010
1.00
0.00


ATOM
383
CE2
PHE
25
−11.570
2.252
4.271
1.00
0.00


ATOM
384
HE2
PHE
25
−12.210
1.421
4.514
1.00
0.00


ATOM
385
CZ
PHE
25
−11.787
3.500
4.867
1.00
0.00


ATOM
386
HZ
PHE
25
−12.594
3.629
5.572
1.00
0.00


ATOM
387
C
PHE
25
−8.263
2.828
−0.409
1.00
0.00


ATOM
388
O
PHE
25
−7.421
2.161
−0.979
1.00
0.00


ATOM
389
N
TYR
26
−8.540
4.051
−0.784
1.00
0.00


ATOM
390
HN
TYR
26
−9.216
4.567
−0.297
1.00
0.00


ATOM
391
CA
TYR
26
−7.825
4.671
−1.938
1.00
0.00


ATOM
392
HA
TYR
26
−6.785
4.381
−1.915
1.00
0.00


ATOM
393
CB
TYR
26
−8.463
4.196
−3.248
1.00
0.00


ATOM
394
HB1
TYR
26
−9.171
4.934
−3.593
1.00
0.00


ATOM
395
HB2
TYR
26
−8.975
3.259
−3.078
1.00
0.00


ATOM
396
CG
TYR
26
−7.391
4.000
−4.295
1.00
0.00


ATOM
397
CD1
TYR
26
−7.086
5.034
−5.188
1.00
0.00


ATOM
398
HD1
TYR
26
−7.617
5.973
−5.129
1.00
0.00


ATOM
399
CD2
TYR
26
−6.701
2.783
−4.373
1.00
0.00


ATOM
400
HD2
TYR
26
−6.936
1.985
−3.684
1.00
0.00


ATOM
401
CE1
TYR
26
−6.093
4.852
−6.159
1.00
0.00


ATOM
402
HE1
TYR
26
−5.858
5.650
−6.847
1.00
0.00


ATOM
403
CE2
TYR
26
−5.708
2.602
−5.344
1.00
0.00


ATOM
404
HE2
TYR
26
−5.177
1.663
−5.404
1.00
0.00


ATOM
405
CZ
TYR
26
−5.403
3.637
−6.236
1.00
0.00


ATOM
406
OH
TYR
26
−4.425
3.459
−7.192
1.00
0.00


ATOM
407
HH
TYR
26
−4.787
2.908
−7.890
1.00
0.00


ATOM
408
C
TYR
26
−7.934
6.195
−1.835
1.00
0.00


ATOM
409
O
TYR
26
−8.901
6.787
−2.277
1.00
0.00


ATOM
410
N
THR
27
−6.953
6.830
−1.241
1.00
0.00


ATOM
411
HN
THR
27
−6.191
6.325
−0.888
1.00
0.00


ATOM
412
CA
THR
27
−6.994
8.316
−1.090
1.00
0.00


ATOM
413
HA
THR
27
−7.965
8.612
−0.720
1.00
0.00


ATOM
414
CB
THR
27
−5.915
8.758
−0.091
1.00
0.00


ATOM
415
HB
THR
27
−6.127
8.330
0.876
1.00
0.00


ATOM
416
OG1
THR
27
−5.920
10.175
0.008
1.00
0.00


ATOM
417
HG1
THR
27
−6.818
10.458
0.194
1.00
0.00


ATOM
418
CG2
THR
27
−4.535
8.282
−0.562
1.00
0.00


ATOM
419
HG21
THR
27
−4.643
7.648
−1.431
1.00
0.00


ATOM
420
HG22
THR
27
−4.058
7.725
0.231
1.00
0.00


ATOM
421
HG23
THR
27
−3.926
9.137
−0.814
1.00
0.00


ATOM
422
C
THR
27
−6.743
8.988
−2.442
1.00
0.00


ATOM
423
O
THR
27
−6.319
8.357
−3.392
1.00
0.00


ATOM
424
N
ASP
28
−6.999
10.268
−2.527
1.00
0.00


ATOM
425
HN
ASP
28
−7.337
10.750
−1.743
1.00
0.00


ATOM
426
CA
ASP
28
−6.779
11.004
−3.806
1.00
0.00


ATOM
427
HA
ASP
28
−5.809
10.748
−4.207
1.00
0.00


ATOM
428
CB
ASP
28
−7.870
10.622
−4.812
1.00
0.00


ATOM
429
HB1
ASP
28
−8.163
11.495
−5.377
1.00
0.00


ATOM
430
HB2
ASP
28
−8.726
10.231
−4.281
1.00
0.00


ATOM
431
CG
ASP
28
−7.333
9.556
−5.771
1.00
0.00


ATOM
432
OD1
ASP
28
−6.648
9.924
−6.711
1.00
0.00


ATOM
433
OD2
ASP
28
−7.617
8.390
−5.549
1.00
0.00


ATOM
434
C
ASP
28
−6.837
12.509
−3.534
1.00
0.00


ATOM
435
O
ASP
28
−5.850
13.209
−3.651
1.00
0.00


ATOM
436
N
LYS
29
−7.992
13.007
−3.169
1.00
0.00


ATOM
437
HN
LYS
29
−8.770
12.418
−3.083
1.00
0.00


ATOM
438
CA
LYS
29
−8.126
14.466
−2.885
1.00
0.00


ATOM
439
HA
LYS
29
−7.666
15.029
−3.684
1.00
0.00


ATOM
440
CB
LYS
29
−9.614
14.835
−2.798
1.00
0.00


ATOM
441
HB1
LYS
29
−10.092
14.617
−3.741
1.00
0.00


ATOM
442
HB2
LYS
29
−9.708
15.890
−2.585
1.00
0.00


ATOM
443
CG
LYS
29
−10.293
14.027
−1.685
1.00
0.00


ATOM
444
HG1
LYS
29
−10.315
14.614
−0.779
1.00
0.00


ATOM
445
HG2
LYS
29
−9.741
13.116
−1.510
1.00
0.00


ATOM
446
CD
LYS
29
−11.725
13.685
−2.101
1.00
0.00


ATOM
447
HD1
LYS
29
−11.719
12.806
−2.728
1.00
0.00


ATOM
448
HD2
LYS
29
−12.149
14.515
−2.648
1.00
0.00


ATOM
449
CE
LYS
29
−12.565
13.413
−0.853
1.00
0.00


ATOM
450
HE1
LYS
29
−13.613
13.525
−1.093
1.00
0.00


ATOM
451
HE2
LYS
29
−12.297
14.115
−0.078
1.00
0.00


ATOM
452
NZ
LYS
29
−12.311
12.024
−0.375
1.00
0.00


ATOM
453
HZ1
LYS
29
−11.297
11.810
−0.453
1.00
0.00


ATOM
454
HZ2
LYS
29
−12.854
11.353
−0.957
1.00
0.00


ATOM
455
HZ3
LYS
29
−12.605
11.939
0.618
1.00
0.00


ATOM
456
C
LYS
29
−7.429
14.801
−1.562
1.00
0.00


ATOM
457
O
LYS
29
−6.940
15.899
−1.369
1.00
0.00


ATOM
458
N
ASP
30
−7.387
13.862
−0.651
1.00
0.00


ATOM
459
HN
ASP
30
−7.793
12.989
−0.833
1.00
0.00


ATOM
460
CA
ASP
30
−6.730
14.112
0.667
1.00
0.00


ATOM
461
HA
ASP
30
−7.187
14.969
1.138
1.00
0.00


ATOM
462
CB
ASP
30
−6.905
12.884
1.562
1.00
0.00


ATOM
463
HB1
ASP
30
−6.222
12.109
1.249
1.00
0.00


ATOM
464
HB2
ASP
30
−7.920
12.523
1.481
1.00
0.00


ATOM
465
CG
ASP
30
−6.612
13.262
3.016
1.00
0.00


ATOM
466
OD1
ASP
30
−6.960
14.367
3.402
1.00
0.00


ATOM
467
OD2
ASP
30
−6.045
12.442
3.719
1.00
0.00


ATOM
468
C
ASP
30
−5.238
14.384
0.456
1.00
0.00


ATOM
469
O
ASP
30
−4.735
15.434
0.811
1.00
0.00


ATOM
470
N
GLY
31
−4.526
13.444
−0.116
1.00
0.00


ATOM
471
HN
GLY
31
−4.956
12.607
−0.392
1.00
0.00


ATOM
472
CA
GLY
31
−3.066
13.643
−0.351
1.00
0.00


ATOM
473
HA1
GLY
31
−2.510
12.870
0.158
1.00
0.00


ATOM
474
HA2
GLY
31
−2.769
14.611
0.028
1.00
0.00


ATOM
475
C
GLY
31
−2.772
13.569
−1.851
1.00
0.00


ATOM
476
O
GLY
31
−3.342
14.300
−2.639
1.00
0.00


ATOM
477
N
LYS
32
−1.887
12.690
−2.246
1.00
0.00


ATOM
478
HN
LYS
32
−1.445
12.114
−1.589
1.00
0.00


ATOM
479
CA
LYS
32
−1.550
12.559
−3.694
1.00
0.00


ATOM
480
HA
LYS
32
−2.213
13.182
−4.274
1.00
0.00


ATOM
481
CB
LYS
32
−0.102
13.003
−3.922
1.00
0.00


ATOM
482
HB1
LYS
32
0.545
12.139
−3.924
1.00
0.00


ATOM
483
HB2
LYS
32
0.198
13.675
−3.131
1.00
0.00


ATOM
484
CG
LYS
32
0.004
13.719
−5.269
1.00
0.00


ATOM
485
HG1
LYS
32
−0.951
14.154
−5.522
1.00
0.00


ATOM
486
HG2
LYS
32
0.290
13.010
−6.032
1.00
0.00


ATOM
487
CD
LYS
32
1.058
14.827
−5.180
1.00
0.00


ATOM
488
HD1
LYS
32
2.023
14.427
−5.452
1.00
0.00


ATOM
489
HD2
LYS
32
1.097
15.203
−4.168
1.00
0.00


ATOM
490
CE
LYS
32
0.695
15.967
−6.137
1.00
0.00


ATOM
491
HE1
LYS
32
−0.301
15.816
−6.527
1.00
0.00


ATOM
492
HE2
LYS
32
1.401
15.990
−6.953
1.00
0.00


ATOM
493
NZ
LYS
32
0.746
17.263
−5.402
1.00
0.00


ATOM
494
HZ1
LYS
32
1.615
17.304
−4.833
1.00
0.00


ATOM
495
HZ2
LYS
32
−0.083
17.339
−4.777
1.00
0.00


ATOM
496
HZ3
LYS
32
0.741
18.048
−6.082
1.00
0.00


ATOM
497
C
LYS
32
−1.712
11.100
−4.127
1.00
0.00


ATOM
498
O
LYS
32
−1.028
10.629
−5.015
1.00
0.00


ATOM
499
N
GLY
33
−2.614
10.385
−3.504
1.00
0.00


ATOM
500
HN
GLY
33
−3.152
10.792
−2.792
1.00
0.00


ATOM
501
CA
GLY
33
−2.831
8.956
−3.872
1.00
0.00


ATOM
502
HA1
GLY
33
−2.770
8.848
−4.945
1.00
0.00


ATOM
503
HA2
GLY
33
−3.808
8.643
−3.532
1.00
0.00


ATOM
504
C
GLY
33
−1.758
8.085
−3.216
1.00
0.00


ATOM
505
O
GLY
33
−0.590
8.419
−3.217
1.00
0.00


ATOM
506
N
ILE
34
−2.151
6.971
−2.656
1.00
0.00


ATOM
507
HN
ILE
34
−3.101
6.728
−2.667
1.00
0.00


ATOM
508
CA
ILE
34
−1.166
6.063
−1.993
1.00
0.00


ATOM
509
HA
ILE
34
−0.651
6.600
−1.211
1.00
0.00


ATOM
510
CB
ILE
34
−1.905
4.872
−1.389
1.00
0.00


ATOM
511
HB
ILE
34
−1.184
4.158
−1.016
1.00
0.00


ATOM
512
CG1
ILE
34
−2.780
4.208
−2.463
1.00
0.00


ATOM
513
HG11
ILE
34
−2.453
4.521
−3.443
1.00
0.00


ATOM
514
HG12
ILE
34
−3.812
4.497
−2.320
1.00
0.00


ATOM
515
CG2
ILE
34
−2.783
5.348
−0.237
1.00
0.00


ATOM
516
HG21
ILE
34
−3.214
4.491
0.255
1.00
0.00


ATOM
517
HG22
ILE
34
−3.572
5.977
−0.621
1.00
0.00


ATOM
518
HG23
ILE
34
−2.185
5.906
0.466
1.00
0.00


ATOM
519
CD1
ILE
34
−2.660
2.686
−2.356
1.00
0.00


ATOM
520
HD11
ILE
34
−3.421
2.221
−2.965
1.00
0.00


ATOM
521
HD12
ILE
34
−2.791
2.387
−1.326
1.00
0.00


ATOM
522
HD13
ILE
34
−1.685
2.376
−2.700
1.00
0.00


ATOM
523
C
ILE
34
−0.146
5.536
−3.011
1.00
0.00


ATOM
524
O
ILE
34
0.905
5.052
−2.641
1.00
0.00


ATOM
525
N
VAL
35
−0.460
5.593
−4.280
1.00
0.00


ATOM
526
HN
VAL
35
−1.324
5.962
−4.555
1.00
0.00


ATOM
527
CA
VAL
35
0.479
5.063
−5.316
1.00
0.00


ATOM
528
HA
VAL
35
0.679
4.022
−5.111
1.00
0.00


ATOM
529
CB
VAL
35
−0.172
5.185
−6.695
1.00
0.00


ATOM
530
HB
VAL
35
−0.280
6.229
−6.951
1.00
0.00


ATOM
531
CG1
VAL
35
0.706
4.491
−7.739
1.00
0.00


ATOM
532
HG11
VAL
35
0.080
4.041
−8.496
1.00
0.00


ATOM
533
HG12
VAL
35
1.299
3.725
−7.259
1.00
0.00


ATOM
534
HG13
VAL
35
1.360
5.218
−8.198
1.00
0.00


ATOM
535
CG2
VAL
35
−1.551
4.518
−6.665
1.00
0.00


ATOM
536
HG21
VAL
35
−1.505
3.626
−6.058
1.00
0.00


ATOM
537
HG22
VAL
35
−1.847
4.255
−7.669
1.00
0.00


ATOM
538
HG23
VAL
35
−2.273
5.204
−6.245
1.00
0.00


ATOM
539
C
VAL
35
1.802
5.840
−5.303
1.00
0.00


ATOM
540
O
VAL
35
2.820
5.331
−5.726
1.00
0.00


ATOM
541
N
GLU
36
1.799
7.063
−4.833
1.00
0.00


ATOM
542
HN
GLU
36
0.965
7.458
−4.504
1.00
0.00


ATOM
543
CA
GLU
36
3.066
7.861
−4.814
1.00
0.00


ATOM
544
HA
GLU
36
3.623
7.664
−5.718
1.00
0.00


ATOM
545
CB
GLU
36
2.737
9.359
−4.745
1.00
0.00


ATOM
546
HB1
GLU
36
2.149
9.635
−5.608
1.00
0.00


ATOM
547
HB2
GLU
36
3.657
9.926
−4.745
1.00
0.00


ATOM
548
CG
GLU
36
1.940
9.671
−3.469
1.00
0.00


ATOM
549
HG1
GLU
36
1.907
8.794
−2.840
1.00
0.00


ATOM
550
HG2
GLU
36
0.934
9.957
−3.739
1.00
0.00


ATOM
551
CD
GLU
36
2.607
10.817
−2.704
1.00
0.00


ATOM
552
OE1
GLU
36
2.693
11.902
−3.257
1.00
0.00


ATOM
553
OE2
GLU
36
3.017
10.591
−1.577
1.00
0.00


ATOM
554
C
GLU
36
3.931
7.469
−3.607
1.00
0.00


ATOM
555
O
GLU
36
5.117
7.727
−3.586
1.00
0.00


ATOM
556
N
GLN
37
3.350
6.870
−2.599
1.00
0.00


ATOM
557
HN
GLN
37
2.392
6.686
−2.626
1.00
0.00


ATOM
558
CA
GLN
37
4.148
6.481
−1.397
1.00
0.00


ATOM
559
HA
GLN
37
4.977
7.165
−1.285
1.00
0.00


ATOM
560
CB
GLN
37
3.261
6.559
−0.153
1.00
0.00


ATOM
561
HB1
GLN
37
2.696
5.644
−0.056
1.00
0.00


ATOM
562
HB2
GLN
37
2.583
7.396
−0.247
1.00
0.00


ATOM
563
CG
GLN
37
4.141
6.748
1.085
1.00
0.00


ATOM
564
HG1
GLN
37
4.996
7.357
0.829
1.00
0.00


ATOM
565
HG2
GLN
37
4.479
5.783
1.435
1.00
0.00


ATOM
566
CD
GLN
37
3.337
7.438
2.188
1.00
0.00


ATOM
567
OE1
GLN
37
3.428
8.637
2.363
1.00
0.00


ATOM
568
NE2
GLN
37
2.549
6.726
2.946
1.00
0.00


ATOM
569
HE21
GLN
37
2.476
5.760
2.806
1.00
0.00


ATOM
570
HE22
GLN
37
2.033
7.159
3.658
1.00
0.00


ATOM
571
C
GLN
37
4.689
5.053
−1.548
1.00
0.00


ATOM
572
O
GLN
37
5.778
4.749
−1.099
1.00
0.00


ATOM
573
N
CYS
38
3.930
4.169
−2.150
1.00
0.00


ATOM
574
HN
CYS
38
3.049
4.430
−2.488
1.00
0.00


ATOM
575
CA
CYS
38
4.396
2.753
−2.299
1.00
0.00


ATOM
576
HA
CYS
38
5.053
2.514
−1.480
1.00
0.00


ATOM
577
HB1
CYS
38
3.526
0.793
−2.375
1.00
0.00


ATOM
578
HB2
CYS
38
2.520
2.059
−3.077
1.00
0.00


ATOM
579
C
CYS
38
5.164
2.558
−3.610
1.00
0.00


ATOM
580
O
CYS
38
6.166
1.873
−3.639
1.00
0.00


ATOM
581
CB
CYS
38
3.188
1.813
−2.264
1.00
0.00


ATOM
582
SG
CYS
38
2.305
1.990
−0.686
1.00
0.00


ATOM
583
N
CYS
39
4.717
3.135
−4.695
1.00
0.00


ATOM
584
HN
CYS
39
3.906
3.683
−4.666
1.00
0.00


ATOM
585
CA
CYS
39
5.459
2.950
−5.982
1.00
0.00


ATOM
586
HA
CYS
39
5.633
1.897
−6.130
1.00
0.00


ATOM
587
HB1
CYS
39
4.453
4.551
−6.994
1.00
0.00


ATOM
588
HB2
CYS
39
3.678
2.985
−7.182
1.00
0.00


ATOM
589
C
CYS
39
6.813
3.671
−5.901
1.00
0.00


ATOM
590
O
CYS
39
7.723
3.370
−6.650
1.00
0.00


ATOM
591
CB
CYS
39
4.626
3.499
−7.149
1.00
0.00


ATOM
592
SG
CYS
39
5.492
3.260
−8.732
1.00
0.00


ATOM
593
N
THR
40
6.958
4.611
−4.994
1.00
0.00


ATOM
594
HN
THR
40
6.216
4.837
−4.397
1.00
0.00


ATOM
595
CA
THR
40
8.256
5.337
−4.866
1.00
0.00


ATOM
596
HA
THR
40
8.733
5.391
−5.833
1.00
0.00


ATOM
597
CB
THR
40
8.000
6.751
−4.343
1.00
0.00


ATOM
598
HB
THR
40
8.940
7.258
−4.199
1.00
0.00


ATOM
599
OG1
THR
40
7.305
6.680
−3.107
1.00
0.00


ATOM
600
HG1
THR
40
7.327
7.550
−2.702
1.00
0.00


ATOM
601
CG2
THR
40
7.164
7.527
−5.364
1.00
0.00


ATOM
602
HG21
THR
40
7.651
7.496
−6.326
1.00
0.00


ATOM
603
HG22
THR
40
7.066
8.554
−5.042
1.00
0.00


ATOM
604
HG23
THR
40
6.183
7.080
−5.442
1.00
0.00


ATOM
605
C
THR
40
9.166
4.582
−3.891
1.00
0.00


ATOM
606
O
THR
40
10.293
4.253
−4.215
1.00
0.00


ATOM
607
N
SER
41
8.686
4.300
−2.702
1.00
0.00


ATOM
608
HN
SER
41
7.775
4.572
−2.467
1.00
0.00


ATOM
609
CA
SER
41
9.524
3.562
−1.709
1.00
0.00


ATOM
610
HA
SER
41
10.499
3.375
−2.134
1.00
0.00


ATOM
611
CB
SER
41
9.670
4.394
−0.435
1.00
0.00


ATOM
612
HB1
SER
41
8.877
4.137
0.255
1.00
0.00


ATOM
613
HB2
SER
41
9.604
5.441
−0.679
1.00
0.00


ATOM
614
OG
SER
41
10.936
4.128
0.153
1.00
0.00


ATOM
615
HG
SER
41
10.794
3.925
1.080
1.00
0.00


ATOM
616
C
SER
41
8.860
2.225
−1.377
1.00
0.00


ATOM
617
O
SER
41
7.796
1.921
−1.867
1.00
0.00


ATOM
618
N
ILE
42
9.489
1.418
−0.567
1.00
0.00


ATOM
619
HN
ILE
42
10.357
1.676
−0.193
1.00
0.00


ATOM
620
CA
ILE
42
8.897
0.088
−0.235
1.00
0.00


ATOM
621
HA
ILE
42
8.391
−0.300
−1.106
1.00
0.00


ATOM
622
CB
ILE
42
10.011
−0.883
0.172
1.00
0.00


ATOM
623
HB
ILE
42
10.391
−0.610
1.147
1.00
0.00


ATOM
624
CG1
ILE
42
11.145
−0.836
−0.860
1.00
0.00


ATOM
625
HG11
ILE
42
11.496
0.180
−0.962
1.00
0.00


ATOM
626
HG12
ILE
42
10.777
−1.186
−1.813
1.00
0.00


ATOM
627
CG2
ILE
42
9.443
−2.298
0.224
1.00
0.00


ATOM
628
HG21
ILE
42
9.306
−2.663
−0.783
1.00
0.00


ATOM
629
HG22
ILE
42
8.494
−2.287
0.737
1.00
0.00


ATOM
630
HG23
ILE
42
10.131
−2.941
0.750
1.00
0.00


ATOM
631
CD1
ILE
42
12.301
−1.729
−0.400
1.00
0.00


ATOM
632
HD11
ILE
42
12.203
−2.705
−0.854
1.00
0.00


ATOM
633
HD12
ILE
42
12.275
−1.825
0.675
1.00
0.00


ATOM
634
HD13
ILE
42
13.239
−1.285
−0.700
1.00
0.00


ATOM
635
C
ILE
42
7.888
0.233
0.907
1.00
0.00


ATOM
636
O
ILE
42
8.249
0.470
2.044
1.00
0.00


ATOM
637
N
CYS
43
6.621
0.082
0.604
1.00
0.00


ATOM
638
HN
CYS
43
6.363
−0.113
−0.322
1.00
0.00


ATOM
639
CA
CYS
43
5.569
0.198
1.656
1.00
0.00


ATOM
640
HA
CYS
43
5.746
1.082
2.251
1.00
0.00


ATOM
641
HB1
CYS
43
3.453
−0.182
1.629
1.00
0.00


ATOM
642
HB2
CYS
43
4.214
−0.215
0.039
1.00
0.00


ATOM
643
C
CYS
43
5.621
−1.042
2.550
1.00
0.00


ATOM
644
O
CYS
43
5.914
−2.131
2.093
1.00
0.00


ATOM
645
CB
CYS
43
4.189
0.289
0.993
1.00
0.00


ATOM
646
SG
CYS
43
3.742
2.028
0.741
1.00
0.00


ATOM
647
N
SER
44
5.342
−0.887
3.820
1.00
0.00


ATOM
648
HN
SER
44
5.111
0.002
4.163
1.00
0.00


ATOM
649
CA
SER
44
5.377
−2.057
4.746
1.00
0.00


ATOM
650
HA
SER
44
5.988
−2.837
4.317
1.00
0.00


ATOM
651
CB
SER
44
5.970
−1.626
6.089
1.00
0.00


ATOM
652
HB1
SER
44
7.044
−1.758
6.065
1.00
0.00


ATOM
653
HB2
SER
44
5.555
−2.229
6.880
1.00
0.00


ATOM
654
OG
SER
44
5.652
−0.261
6.327
1.00
0.00


ATOM
655
HG
SER
44
6.453
0.254
6.213
1.00
0.00


ATOM
656
C
SER
44
3.958
−2.582
4.966
1.00
0.00


ATOM
657
O
SER
44
2.988
−1.872
4.776
1.00
0.00


ATOM
658
N
LEU
45
3.831
−3.821
5.374
1.00
0.00


ATOM
659
HN
LEU
45
4.633
−4.366
5.523
1.00
0.00


ATOM
660
CA
LEU
45
2.479
−4.408
5.619
1.00
0.00


ATOM
661
HA
LEU
45
1.902
−4.381
4.707
1.00
0.00


ATOM
662
CB
LEU
45
2.642
−5.866
6.086
1.00
0.00


ATOM
663
HB1
LEU
45
2.210
−5.981
7.070
1.00
0.00


ATOM
664
HB2
LEU
45
3.693
−6.109
6.130
1.00
0.00


ATOM
665
CG
LEU
45
1.940
−6.828
5.113
1.00
0.00


ATOM
666
HG
LEU
45
2.092
−7.842
5.453
1.00
0.00


ATOM
667
CD1
LEU
45
0.437
−6.533
5.085
1.00
0.00


ATOM
668
HD11
LEU
45
0.254
−5.543
5.475
1.00
0.00


ATOM
669
HD12
LEU
45
−0.083
−7.259
5.692
1.00
0.00


ATOM
670
HD13
LEU
45
0.074
−6.591
4.068
1.00
0.00


ATOM
671
CD2
LEU
45
2.530
−6.674
3.700
1.00
0.00


ATOM
672
HD21
LEU
45
1.731
−6.619
2.977
1.00
0.00


ATOM
673
HD22
LEU
45
3.154
−7.528
3.478
1.00
0.00


ATOM
674
HD23
LEU
45
3.126
−5.774
3.650
1.00
0.00


ATOM
675
C
LEU
45
1.763
−3.590
6.700
1.00
0.00


ATOM
676
O
LEU
45
0.555
−3.464
6.698
1.00
0.00


ATOM
677
N
TYR
46
2.512
−3.032
7.616
1.00
0.00


ATOM
678
HN
TYR
46
3.486
−3.149
7.586
1.00
0.00


ATOM
679
CA
TYR
46
1.903
−2.213
8.702
1.00
0.00


ATOM
680
HA
TYR
46
1.032
−2.718
9.092
1.00
0.00


ATOM
681
CB
TYR
46
2.945
−2.040
9.826
1.00
0.00


ATOM
682
HB1
TYR
46
3.877
−1.704
9.396
1.00
0.00


ATOM
683
HB2
TYR
46
3.100
−2.989
10.316
1.00
0.00


ATOM
684
CG
TYR
46
2.474
−1.024
10.846
1.00
0.00


ATOM
685
CD1
TYR
46
1.239
−1.187
11.485
1.00
0.00


ATOM
686
HD1
TYR
46
0.620
−2.044
11.256
1.00
0.00


ATOM
687
CD2
TYR
46
3.274
0.084
11.139
1.00
0.00


ATOM
688
HD2
TYR
46
4.228
0.205
10.644
1.00
0.00


ATOM
689
CE1
TYR
46
0.806
−0.239
12.419
1.00
0.00


ATOM
690
HE1
TYR
46
−0.147
−0.362
12.913
1.00
0.00


ATOM
691
CE2
TYR
46
2.843
1.031
12.074
1.00
0.00


ATOM
692
HE2
TYR
46
3.462
1.886
12.298
1.00
0.00


ATOM
693
CZ
TYR
46
1.608
0.871
12.714
1.00
0.00


ATOM
694
OH
TYR
46
1.182
1.807
13.635
1.00
0.00


ATOM
695
HH
TYR
46
1.440
2.676
13.317
1.00
0.00


ATOM
696
C
TYR
46
1.489
−0.848
8.130
1.00
0.00


ATOM
697
O
TYR
46
0.498
−0.273
8.537
1.00
0.00


ATOM
698
N
GLN
47
2.250
−0.329
7.202
1.00
0.00


ATOM
699
HN
GLN
47
3.049
−0.812
6.900
1.00
0.00


ATOM
700
CA
GLN
47
1.917
0.999
6.609
1.00
0.00


ATOM
701
HA
GLN
47
1.816
1.727
7.398
1.00
0.00


ATOM
702
CB
GLN
47
3.037
1.432
5.663
1.00
0.00


ATOM
703
HB1
GLN
47
2.649
2.158
4.965
1.00
0.00


ATOM
704
HB2
GLN
47
3.403
0.573
5.123
1.00
0.00


ATOM
705
CG
GLN
47
4.180
2.060
6.463
1.00
0.00


ATOM
706
HG1
GLN
47
5.107
1.930
5.927
1.00
0.00


ATOM
707
HG2
GLN
47
4.251
1.582
7.429
1.00
0.00


ATOM
708
CD
GLN
47
3.908
3.555
6.649
1.00
0.00


ATOM
709
OE1
GLN
47
2.774
3.961
6.811
1.00
0.00


ATOM
710
NE2
GLN
47
4.907
4.396
6.632
1.00
0.00


ATOM
711
HE21
GLN
47
5.821
4.070
6.501
1.00
0.00


ATOM
712
HE22
GLN
47
4.740
5.355
6.751
1.00
0.00


ATOM
713
C
GLN
47
0.606
0.914
5.822
1.00
0.00


ATOM
714
O
GLN
47
−0.167
1.855
5.797
1.00
0.00


ATOM
715
N
LEU
48
0.356
−0.193
5.164
1.00
0.00


ATOM
716
HN
LEU
48
0.999
−0.932
5.186
1.00
0.00


ATOM
717
CA
LEU
48
−0.898
−0.317
4.361
1.00
0.00


ATOM
718
HA
LEU
48
−1.178
0.661
4.011
1.00
0.00


ATOM
719
CB
LEU
48
−0.653
−1.227
3.151
1.00
0.00


ATOM
720
HB1
LEU
48
−1.599
−1.466
2.688
1.00
0.00


ATOM
721
HB2
LEU
48
−0.178
−2.139
3.482
1.00
0.00


ATOM
722
CG
LEU
48
0.250
−0.524
2.127
1.00
0.00


ATOM
723
HG
LEU
48
1.213
−0.323
2.575
1.00
0.00


ATOM
724
CD1
LEU
48
0.434
−1.434
0.914
1.00
0.00


ATOM
725
HD11
LEU
48
−0.381
−1.278
0.222
1.00
0.00


ATOM
726
HD12
LEU
48
0.441
−2.465
1.236
1.00
0.00


ATOM
727
HD13
LEU
48
1.369
−1.202
0.427
1.00
0.00


ATOM
728
CD2
LEU
48
−0.391
0.793
1.667
1.00
0.00


ATOM
729
HD21
LEU
48
−0.293
1.532
2.447
1.00
0.00


ATOM
730
HD22
LEU
48
−1.437
0.629
1.455
1.00
0.00


ATOM
731
HD23
LEU
48
0.108
1.143
0.775
1.00
0.00


ATOM
732
C
LEU
48
−2.047
−0.892
5.201
1.00
0.00


ATOM
733
O
LEU
48
−3.135
−1.097
4.695
1.00
0.00


ATOM
734
N
GLU
49
−1.832
−1.150
6.468
1.00
0.00


ATOM
735
HN
GLU
49
−0.954
−0.979
6.864
1.00
0.00


ATOM
736
CA
GLU
49
−2.930
−1.700
7.315
1.00
0.00


ATOM
737
HA
GLU
49
−3.371
−2.553
6.822
1.00
0.00


ATOM
738
CB
GLU
49
−2.361
−2.134
8.669
1.00
0.00


ATOM
739
HB1
GLU
49
−3.148
−2.130
9.407
1.00
0.00


ATOM
740
HB2
GLU
49
−1.583
−1.445
8.968
1.00
0.00


ATOM
741
CG
GLU
49
−1.773
−3.547
8.552
1.00
0.00


ATOM
742
HG1
GLU
49
−0.698
−3.485
8.536
1.00
0.00


ATOM
743
HG2
GLU
49
−2.119
−4.009
7.640
1.00
0.00


ATOM
744
CD
GLU
49
−2.211
−4.392
9.751
1.00
0.00


ATOM
745
OE1
GLU
49
−2.509
−5.558
9.550
1.00
0.00


ATOM
746
OE2
GLU
49
−2.237
−3.860
10.848
1.00
0.00


ATOM
747
C
GLU
49
−4.006
−0.631
7.530
1.00
0.00


ATOM
748
O
GLU
49
−5.146
−0.942
7.823
1.00
0.00


ATOM
749
N
ASN
50
−3.655
0.624
7.393
1.00
0.00


ATOM
750
HN
ASN
50
−2.730
0.851
7.163
1.00
0.00


ATOM
751
CA
ASN
50
−4.655
1.714
7.596
1.00
0.00


ATOM
752
HA
ASN
50
−5.281
1.470
8.441
1.00
0.00


ATOM
753
CB
ASN
50
−3.919
3.024
7.881
1.00
0.00


ATOM
754
HB1
ASN
50
−4.525
3.856
7.552
1.00
0.00


ATOM
755
HB2
ASN
50
−2.979
3.032
7.348
1.00
0.00


ATOM
756
CG
ASN
50
−3.657
3.151
9.383
1.00
0.00


ATOM
757
OD1
ASN
50
−4.068
4.112
10.004
1.00
0.00


ATOM
758
ND2
ASN
50
−2.983
2.218
9.997
1.00
0.00


ATOM
759
HD21
ASN
50
−2.650
1.444
9.497
1.00
0.00


ATOM
760
HD22
ASN
50
−2.810
2.291
10.959
1.00
0.00


ATOM
761
C
ASN
50
−5.538
1.894
6.347
1.00
0.00


ATOM
762
O
ASN
50
−6.396
2.757
6.321
1.00
0.00


ATOM
763
N
TYR
51
−5.342
1.102
5.313
1.00
0.00


ATOM
764
HN
TYR
51
−4.649
0.413
5.342
1.00
0.00


ATOM
765
CA
TYR
51
−6.184
1.260
4.086
1.00
0.00


ATOM
766
HA
TYR
51
−6.744
2.180
4.159
1.00
0.00


ATOM
767
CB
TYR
51
−5.293
1.305
2.838
1.00
0.00


ATOM
768
HB1
TYR
51
−5.911
1.234
1.956
1.00
0.00


ATOM
769
HB2
TYR
51
−4.601
0.476
2.860
1.00
0.00


ATOM
770
CG
TYR
51
−4.525
2.597
2.799
1.00
0.00


ATOM
771
CD1
TYR
51
−5.154
3.766
2.364
1.00
0.00


ATOM
772
HD1
TYR
51
−6.189
3.738
2.061
1.00
0.00


ATOM
773
CD2
TYR
51
−3.187
2.625
3.189
1.00
0.00


ATOM
774
HD2
TYR
51
−2.706
1.719
3.518
1.00
0.00


ATOM
775
CE1
TYR
51
−4.442
4.970
2.323
1.00
0.00


ATOM
776
HE1
TYR
51
−4.924
5.873
1.980
1.00
0.00


ATOM
777
CE2
TYR
51
−2.470
3.824
3.147
1.00
0.00


ATOM
778
HE2
TYR
51
−1.435
3.843
3.448
1.00
0.00


ATOM
779
CZ
TYR
51
−3.098
4.998
2.716
1.00
0.00


ATOM
780
OH
TYR
51
−2.392
6.183
2.678
1.00
0.00


ATOM
781
HH
TYR
51
−2.342
6.527
3.574
1.00
0.00


ATOM
782
C
TYR
51
−7.163
0.085
3.961
1.00
0.00


ATOM
783
O
TYR
51
−7.191
−0.606
2.958
1.00
0.00


ATOM
784
N
CYS
52
−7.969
−0.146
4.964
1.00
0.00


ATOM
785
HN
CYS
52
−7.933
0.419
5.764
1.00
0.00


ATOM
786
CA
CYS
52
−8.945
−1.273
4.885
1.00
0.00


ATOM
787
HA
CYS
52
−9.067
−1.561
3.852
1.00
0.00


ATOM
788
HB1
CYS
52
−9.205
−3.211
5.770
1.00
0.00


ATOM
789
HB2
CYS
52
−8.165
−2.127
6.692
1.00
0.00


ATOM
790
C
CYS
52
−10.299
−0.835
5.445
1.00
0.00


ATOM
791
O
CYS
52
−10.402
0.139
6.168
1.00
0.00


ATOM
792
CB
CYS
52
−8.430
−2.462
5.700
1.00
0.00


ATOM
793
SG
CYS
52
−6.973
−3.180
4.902
1.00
0.00


ATOM
794
N
ASN
53
−11.337
−1.559
5.114
1.00
0.00


ATOM
795
HN
ASN
53
−11.218
−2.339
4.531
1.00
0.00


ATOM
796
CA
ASN
53
−12.699
−1.212
5.617
1.00
0.00


ATOM
797
HA
ASN
53
−12.919
−0.183
5.382
1.00
0.00


ATOM
798
CB
ASN
53
−13.733
−2.121
4.950
1.00
0.00


ATOM
799
HB1
ASN
53
−14.565
−2.272
5.621
1.00
0.00


ATOM
800
HB2
ASN
53
−13.279
−3.074
4.721
1.00
0.00


ATOM
801
CG
ASN
53
−14.234
−1.471
3.658
1.00
0.00


ATOM
802
OD1
ASN
53
−13.551
−0.657
3.069
1.00
0.00


ATOM
803
ND2
ASN
53
−15.407
−1.800
3.192
1.00
0.00


ATOM
804
HD21
ASN
53
−15.958
−2.456
3.669
1.00
0.00


ATOM
805
HD22
ASN
53
−15.737
−1.391
2.364
1.00
0.00


ATOM
806
C
ASN
53
−12.750
−1.410
7.133
1.00
0.00


ATOM
807
OT1
ASN
53
−12.763
−2.553
7.560
1.00
0.00


ATOM
808
OT2
ASN
53
−12.775
−0.416
7.840
1.00
0.00


END









Example 3
The insulin analogue precursor AspB28IP(AspGlyLys) was produced culturing yeast strain MT663 transformed with an expression plasmid expressing either a YAP3-TA39-EEGEPK(SEQ ID NO:8)-AspB28IP(DGK) fusion protein or a YAP3-TA57-EEGEPK(SEQ ID NO:8)-AspB28IP (DGK) fusion protein. TA39 is a pro-sequence QPIDDTESNTTSVNLMADDTESRFATNTTLAGGLDWNLISMAKR(SEQ ID NO:15). The sequence EEGEPK(SEQ ID NO:8) is an N-terminal extension to the B-chain of the insulin analogue. TA57 is a pro-sequence QPIDDTESQTTSVNLMADDTESAFATQTNSGGLDWGLISMAKR (SEQ ID NO:16). cDNA encoding the leader sequences YAP3-TA39 and YAP3-TA57 and cDNA encoding the AspB28IP(DGK) and the N-terminal extension were cloned into an expression vector of the C-POT type using standard techniques (Sambrook J, Fritsch E F and Maniatis T, Molecular cloning, Cold spring Harbour laboratory press, 1989). The DNA and inferred amino acids sequences are shown in FIG. 7 (SEQ ID NO:9 and 10) and FIG. 8 (SEQ ID NO:11 and 12).
Table 5 shows the yields. Fermentation was conducted at 30° C. for 72 h in 5 ml YPD. IP yield was determined by RP-HPLC of the culture supernatant and is expressed relative to the IP yield of the strain yJB155.












TABLE 5





Leader
Precursor
N-terminal extension
C-peptide
Yield*
SEQ ID





















α*-ex4
AspB28IP
GluGluAlaGluAlaGlu-
None
100 
SEQ ID NO:3





AlaProLys


 


YAP3-TA39
AspB28IP
GluGluGlyGluProLys
AspGlyLys
477%
SEQ ID NO:8


 


YAP3-TA57
AspB28IP
GluGluGlyGluProLys
AspGlyLys
306%
SEQ ID NO:8









Example 4
Construction of Human Insulin Precursors with Synthetic C-eptides with a Glycine Residue
Synthetic genes encoding fusion proteins consisting of the insulin precursor associated with a leader sequence consisting of a pre-peptide (signal peptide) and a propeptide, were constructed using PCR under standard conditions (Sambrook et al. (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press) and E.H.F. polymerase (Boehringer Mannheim GmbH, Sandhoefer Strasse 116, Mannheim, Germany). The resulting DNA fragments were isolated and digested with endonucleases and purified using the Gene Clean kit (Bio101 Inc., La Jolla, Calif., USA). Standard methods were used for DNA ligation and transformation of E. coli cells were performed by the CaCl2 method (Sambrook et al. (1989) supra). Plasmids were purified from transformed E. coli cells using QIAGEN columns (QIAGEN, Hilden, Germany). Nucleotide sequences were determined using the ALF Pharmacia Biotech DNA sequencing system with purified double-stranded plasmid DNA as template. Oligonucleotide primers for PCR were obtained from DNA technology (Århus, Denmark).
Secretion of the insulin precursor was facilitated by the TA57 leader (Kjeldsen et al., 1998. Protein Expression Purif. 14, 309-316), although a variety of known yeast leader sequences may be used.
As shown in FIG. 9 and 10, the pAK855 S. cerevisiae expression plasmid expressing the TA57 leader-GluGluGlyGluProLys(SEQ ID NO:8)-insulin precursor fusion protein was constructed based on the S. cerevisiae-E. coli shuttle POT plasmid (U.S. Pat. No. 5,871,957). L-IP indicates the fusion protein expression cassette encoding the leader-insulin precursor fusion protein, TPI-PROMOTER is the S. cerevisiae TPI1 promoter and TPI-TERMINATOR is the S. cerevisiae TPI1 terminator; TPI-POMBE indicates the S. pombe POT gene used for selection in S. cerevisiae; ORIGIN indicates a S. cerevisiae origin of replication derived from the 2 μm plasmid; AMP-R indicates the β-lactamase gene conferring resistance toward ampicillin, facilitating selection in E. coli and ORIGIN-PBR322 indicates an E. coli origin of replication.
DNA encoding a number of fusions proteins of leader sequences and insulin precursors with different mini C-peptides was generated by PCR using appropriate oligonucleotides as primers, as described below. Standard methods were used to subclone DNA fragments encoding the leader-insulin precursor fusion proteins into the CPOT expression vector in the following configuration: leader-Lys-Arg-spacer-insulin precursor, where Lys-Arg is a potential dibasic endoprotease processing site and spacer is an N-terminal extension. To optimize processing of the fusion protein by the S. cerevisiae Kex2 endoprotease, DNA encoding a spacer peptide (N-terminal extension), e.g. GluGluGlyGluProLys(SEQ ID NO:8) was inserted between the DNA encoding the leader and the insulin precursor (Kjeldsen et al. (1999b.) J. Biotechnology, 75 195-208). However, the present of the spacer peptide is not mandatory. The insulin precursor was secreted as a single-chain N-terminally extended insulin precursor with a mini C-peptide, connecting LysB29 and GlyA1. After purification of the insulin precursor and proteolytic removal of the N-terminal extension and the mini C-peptide, the amino acid ThrB30 can be added to LysB29 by enzyme-mediated transpeptidation, to generate human insulin (Markussen, et al. (1987) in “Peptides 1986” (Theodoropoulos, D., Ed.), pp. 189-194, Walter de Gruyter & Co., Berlin.).
Development of synthetic mini C-peptides was performed by randomization of one or more codon(s) encoding the amino acids in the mini C-peptide. The synthetic mini C-peptides feature typically an enzymatic processing site (Lys) at the C-terminus which allows enzymatic removal of the synthetic mini C-peptide. Randomization was performed using doped oligonucleotides which introduced codon(s) variations at one or more positions of the synthetic mini C-peptides. Typically one of the two primers (oligonucleotides) used for PCR was doped. Examples of primers are:








Primer A;









(SEQ ID NO:19)









5′-TTGCTTAAATCTATAACTAC-3′



 


Primer B:








(SEQ ID NO:20)









5′-TTAGTTTCTAGACTAGTTGCAGTAGTTTTCCAATTGGTACAAGGAG-



 


CAGATGGAGGTACAGCATTGTTCGACAATACCCTTACCMNNCTTAGGAG-


 


TGTAGAAGAAACC-3′


 


N = ACTG, M = GT






PCR was typically performed as indicated below: 5 μl Primer A (20 pmol), 5 μl Primer B (20 pmol), 10 μl 10× PCR buffer, 8 μl dNTP mix, 0.75 μl E.H.F. enzyme, 1 μl pAK885 plasmid as template (approximately 0.2 μg DNA), and 70.25 μl distilled water.
Typically between 10 and 15 cycles were performed, one cycle typically was 95° C. for 45 sec.; 55° C. for 1 min; 72° C. for 1.5 min. The PCR mixture was subsequently loaded onto an 2% agarose gel and electrophoresis was performed using standard techniques. The resulting DNA fragment was cut out of the agarose gel and isolated by the Gene Clean kit.
FIG. 9 shows the nucleotide sequence of the pAK855 DNA expression cassette used as template for PCR and inferred amino acids of the encoded fusion protein (TA57-leader-EEGEPK(SEQ ID NO:8)-insulin precursor of pAK855 (SEQ ID NO:17 and 18).
The purified PCR DNA fragment was dissolved in water and restriction endonucleases buffer and digested with suitable restriction endonucleases (e.g. Bgl II and Xba I) according to standard techniques. The BgIII-XbaI DNA fragments were subjected to agarose electrophoresis and purified using The Gene Clean Kit. The digested and isolated DNA fragments were ligated together with a suitable vector (e.g. of the CPOT type) using T4 DNA ligase and standard conditions. The ligation mix was subsequently transformed into a competent E. coli strain (R−, M+) followed by selection with ampicillin resistance. Plasmids from the resulting E. coli's were isolated using QIAGEN columns.
The plasmids were subsequently used for transformation of a suitable S. cerevisiae strainMT663 (MATa/MATα pep4-31pep4-3 HIS41his4 tpi::LEU21tpi::LEU2 Cir+). Individual transformed S. cerevisiae clones were grown in liquid culture, and the quantity of secreted insulin precursor the culture supernatants was determined by RP-HPLC. The DNA sequence encoding the synthetic mini C-peptide of the expression plasmids from S. cerevisiae clones secreting increased quantity of the insulin precursor were then determined.
Table 6 shows the insulin precursors generated by the above method and production yield expressed as a percent of control. Fermentation was conducted at 30° C. for 72 h in 5 ml YPD. Yield of the insulin precursor was determined by RP-HPLC of the culture supernatant, and is expressed relative to the yield of a control strain expressing a leader-insulin precursor fusion protein in which the B29 residue is linked to the A1 residue by a mini C-peptide Ala-Ala-Lys. YAP3 is the YAP3 signal sequence. The sequence EEGEPK (SEQ ID NO:8) is an N-terminal extension to the B-chain and TA57 is a synthetic prosequence QPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAKR (SEQ ID NO:16).











TABLE 6






N-terminal





Leader
extension
C-peptide
Yield*
SEQ ID







YAP3-TA57
GluGluGlyGluProLys
AlaAlaLys
100
SEQ ID




Control

NO:2


YAP3-TA57
GluGluGlyGluProLys
AspGlyLys
185
SEQ ID






NO:2


YAP3-TA57
GluGluGlyGluProLys
GluGlyLys
153
SEQ ID






NO:2

## Claims

1. A human insulin precursor or human insulin analog precursor comprising a sequence of formula:


B(1-27)-X3 -X2-X1-Y-A(1-21),


wherein X1 is 1-5 amino acid residues in length and comprises at least one Gly, X2 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain, X3 is one of Pro, Asp, Lys, or lie at position 28 of the B chain, and Y is Lys or Arg, and wherein X1-Y does not contain two adjacent basic amino acids.

2. A human insulin precursor comprising a sequence of formula:


B(1-29)-X1Y-A(1-21),


wherein X1 is of up to 5 amino acid residues in length of which at least one is Gly, Y is a cleavage site, and X1-Y does not contain two adjacent basic amino acids.

3. The human insulin precursor according to claim 2, wherein X1 is of 1-4 amino acid residues in length.

4. The human insulin precursor or human insulin precursor analog according to claim 1, wherein X1 is 1-4 amino acid residues in length.

5. The human insulin precursor or human insulin precursor analog according to claim 1, wherein X1 is 1-3 amino acid residues in length.

6. The human insulin precursor or human insulin precursor analog according to claim 1, wherein X1 contains up to three, preferably one Gly.

7. The human insulin precursor or human insulin precursor analog according to claim 1, wherein X3 is Asp and X2 is Lys.

8. The human insulin precursor or human insulin precursor analog according to claim 1, wherein X1-Y is selected from the group of: (a) Glu-Glu-Gly-Lys (SEQ ID NO:1), (b) Glu-Gly-Lys, (c) Ser-Gly-Lys, (d) Asn-Gly-Lys, (e) Thr-Gly-Lys, (f) Asp-Gly-Lys, (g) Me-Gly-Lys, (h) Ala-Gly-Lys, (i) His-Gly-Lys and (I) Gly-Lys.

9. The human insulin precursor or human insulin precursor analog according to claim 1, wherein one Gly is immediately N-terminal to Y.

10. The human insulin precursor or human insulin precursor analog according to claim 1, wherein the B27 (atom CG2) has a proximity to the A1 (atom CA) of less than 5 Å.

11. The human insulin precursor according to claim 2, wherein X1 is of 1-3 amino acid residues in length.

12. The human insulin precursor according to claim 2, wherein X1 is of 1-2 amino acid residues in length.