# Patent

## Title
Compositions and methods for producing recombinant proteins

## Abstract

A class of integral membrane proteins, referred to as Mistic polypeptides, their variants, fusion proteins including a Mistic polypeptide domain, and nucleic acid molecules encoding Mistic polypeptides and Mistic fusion proteins are disclosed herein. Also described are methods of using Mistic polypeptides and Mistic fusion proteins to produce and/or isolate recombinant proteins (including without limitation classes of eukaryotic proteins that have previously been intractable to recombinant bacterial expression, such as, eukaryotic integral membrane proteins).

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 60/639,174, filed Dec. 22, 2004, which application is incorporated herein in its entirety.


STATEMENT OF GOVERNMENT SUPPORT
This invention was made with United States government support pursuant to grant GM 56653 from the National Institutes of Health. The United States government has certain rights in the invention.


REFERENCE TO SEQUENCE INFORMATION ON COMPACT DISC
This application contains sequence information submitted on duplicate compact discs (named SeqList1 and SeqList2, respectively), each of which compact discs contains one text document file, each such file written to disk on May 24, 2006 (last modified on Mar. 18, 2006), and identified as “Sequence Listing” (667 KB), the sequences in each such file are incorporated herein by reference in their entirety.
FIELD OF THE DISCLOSURE
This disclosure relates to membrane-associating proteins, including fusion proteins thereof, and to nucleic acid sequences encoding such proteins. Methods of using the disclosed proteins (including fusion proteins) and corresponding nucleic acid sequences are also disclosed.
BACKGROUND
The vast numbers of candidate proteins generated from genomics programs are creating enormous opportunities in the biotechnology sector. However, efficient and rapid expression of genes in homologous and heterologous expression systems, and high-level expression and efficient isolation of proteins encoded by such genes are often major bottlenecks. In many circumstances, the practical and/or cost-effective expression of recombinant proteins in amounts large enough to allow for subsequent characterization and evaluation is prohibitive. Expression and isolation of recombinant integral membrane (IM) proteins provide one of many examples.
IM proteins constitute nearly 30% of all open reading frames in fully sequenced eukaryotic genomes. They play central roles in living cells with respect to transport processes, intercellular signaling, and regulation of cell growth. Under native conditions, eukaryotic IM proteins are synthesized and integrated cotranslationally into the membrane of the endoplasmic reticulum (ER) through an aqueous hole known as the translocon (Schatz and Dobberstein, Science, 271(5255):1519-1526, 1996). Eukaryotic membrane proteins are usually expressed at low levels and they are often modified (N- and O-glycosylation, palmitoylation, prenylation, myristolation, GPI-modification, protease cleavage) inside the ER lumen. Within the ER lumen, disulfide bonds also can be formed as part of the folding process by a set of disulfide reductases and oxidases.
In principle, both prokaryotic and eukaryotic expression systems can be used for heterologous expression of eukaryotic IM proteins. A variety of expression hosts have been explored for this purpose, including Escherichia coli, Halobacterium, Lactococcus lactis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, baculovirus-infected Sf9 insect cells, mammalian cell lines transfected stably or transiently by expression vectors, and easily grown small animals. Traditionally, bacterial expression hosts, such as E. coli, have been the preferred platform for heterologous high-level expression of recombinant proteins for biochemical and structural research. The reasons for the popularity of these organisms include culture affordability, ease of genetic manipulation, and high yields of desired product. However, the application of this platform to IM proteins has met with limited success.
At least two complications arise with the production of IM proteins in bacteria. First, unlike soluble proteins, IM proteins must be trafficked to the membrane, involving targeting signals that may not be recognized by the machinery within the bacterial host. Second, even in cases where a signal sequence is discernable, high-level production of membrane proteins in E. coli can competitively exclude production of other vital membrane proteins in the E. coli, leading to toxicity. For this reason, traditional methods of IM protein expression in bacteria have utilized low copy-number plasmids with weak promoters that produce low levels of protein, compensated by extremely large culture volumes (Laage and Langosch, Traffic, 2:99-104, 2001). Alternatively, IM proteins can be purposefully targeted to bacterial inclusion bodies (Kiefer et al., Receptors Channels, 7:109-119, 2000). This process necessitates subsequent renaturation of the desired IM protein from these insoluble deposits, which is fraught with empirical difficulties and limited success rates. Both of these options fundamentally limit the application of traditional bacterial methods to production of high levels of recombinant IM proteins in their native conformations.
While fusion partner proteins have been used to assist in the successful production of soluble recombinant proteins, traditional fusion partners have not been useful in the production of IM proteins (Tucker and Grisshammer, Biochem J., 317:891-899, 1996). None of the currently available fusion proteins (e.g., glutathione-S-transferase, maltose binding protein, or thioredoxin) target the construct to the membrane and facilitate membrane insertion.
Compositions and methods that facilitate the production and isolation of recombinant proteins in heterologous expression systems are needed.
SUMMARY OF THE DISCLOSURE
This disclosure describes compositions and methods that further the goal of producing and/or isolating proteins (such as, eukaryotic IM proteins) in heterologous expression systems (such as, prokaryotic expression systems, like E. coli). For example, described is a newly discovered class of polypeptides that associate with a membrane (such as, a bacterial cell membrane) when expressed alone or as a chimera with a fusion partner (such as, an eukaryotic IM proteins). This class of polypeptides is referred to as Mistic polypeptides, where “Mistic” is an acronym for Membrane Integrating Sequence for Translation of IM protein Constructs. Amino acid sequences of, and nucleic acid sequences encoding, Mistic polypeptides and their variants (including, e.g., functional fragments), and methods of using the same (for example, to produce and/or isolate recombinant proteins) are disclosed herein.
The foregoing and other features and advantages will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 includes four panels (A-D) demonstrating various features of Mistic-L. FIG. 1A shows the primary sequence of Mistic-L (SEQ ID NO: 2). Wild-type Cys 3 and residues mutated to cysteine for probing Mistic-L's orientation in the membrane are shown in outline, and residues mutated in structural disruption mutants are boxed. The secondary structural boundaries of Mistic-L are illustrated beneath the primary sequence. FIG. 1B shows an SDS-PAGE gel of Ni-NTA-eluted proteins present in the indicated fractions of a culture recombinantly expressing octa-histidine tagged Mistic-L. FIG. 1C shows a graph of multi-angle static light scattering results for Mistic-L in 3 mM lauryl-dimethylamine oxide (LDAO). FIG. 1D shows a nitrocellulose blot of biotinylated proteins isolated by Ni-NTA chromatography from 3-(N-maleimido-propinyl) biocytin (MPB)-labeled right-side-out vesicles from cells expressing wild-type Mistic-L (Cys3(Wt)) or the indicated Mistic-L mutants (with Cys3 mutated to Val).
FIG. 2 includes three panels (A-C) describing the secondary structure and long-range interactions of Mistic-L (SEQ ID NO: 222). FIG. 2A, first line shows 1HN protection from solvent exchange indicative for hydrogen bond formation (stars). The solvent protection is determined by the absence of a cross-peak between the chemical shifts of 1HN and water in the 15N-resolved TROSY-[1H, 1H]-NOESY spectrum. FIG. 2A, second and third lines show NOEs observed in the 15N-resolved TROSY-[1H, 1H]-NOESY. Thin, medium and thick bars represent weak (4.5 to 5.5 Å), medium (3 to 4.5 Å) and strong (<3 Å) sequential NOEs (dNN(i,i+1)). The medium-range NOEs dNN(i,i+2) are shown by lines starting and ending at the positions of the residues related by the NOE. FIG. 2A, fourth and fifth lines show deviation of the 13Cα chemical shifts from corresponding ‘random coil’ chemical shifts in 0 mM K+ (solid) and 100 mM K+ (outline), as independently assigned. Values larger than 1.5 ppm are indicative of an α-helical secondary structure, values smaller than −1.5 ppm are indicative of β-sheet secondary structure. FIG. 2B shows the 2D [15N, 1H]-TROSY spectrum of Mistic-L along with parts of the 2D [15N, 1H]-TROSY spectra in presence of paramagnetic spin-labels at positions Cys3, Thr30Cys, Ser58Cys, Asn88Cys, and Glu110Cys. Comparison of peaks heights between perturbed spectra and multiple reference spectra was used to obtain long-range distance restraints. FIG. 2C shows the collected upper limit long-range restraints mapped to the structure of Mistic-L.
FIG. 3 shows several representations of Mistic-L tertiary structure. FIG. 3A illustrates the superposition of 10 conformers representing the final Mistic-L NMR structure. The bundle is obtained by superimposing the backbone Cα carbons of residues 13-62 and 67-102. FIG. 3B is a ribbon diagram of the lowest energy conformer highlighting the four α-helix bundle. FIG. 3C is a surface representation of Mistic-L, oriented as in FIG. 3B, mapping electrostatic potential. Positive charges are shown hatched, negative charges are shown in striped grey, and neutral surface is shown in white. FIG. 3D is a representation of the residues forming the core of Mistic-L. Residues mutated in structural disruption studies are designated by arrows. FIG. 3E is a schematic representation of a top view of Mistic-L from the intracellular side of the membrane. FIG. 3F is a surface representation of the electrostatic potential of Mistic-L, viewed from the opposite face from that shown in FIG. 3C.
FIG. 4 shows four panels describing Mistic-L-detergent interactions. FIG. 4A is a surface representation of Mistic-L indicating observed NOE interactions between detergent molecules and the protein. Interactions observed between the head methyl (CH3) groups of LDAO and backbone amides (1HN) of the protein are shown in light grey; interactions observed between the hydrophobic CH3 end of LDAO and 1HN of the protein are shown in striped grey, and interactions observed between the LDAO chain (CH2) and 1HN are shown hatched. FIG. 4B shows a selection of intermolecular NOEs between LDAO and residues 37-43 and 58-67 of Mistic-L. 15N, 1H strips from the 15N-resolved TROSY [1H, 1H]-NOESY are shown. For the differentiation between intramolecular and intermolecular NOEs, a second NOESY experiment was measured without decoupling on 13C during 1H evolution, that yielded doublets for protein-protein NOEs, but single peaks for detergent-protein NOEs. Arg43 for this measurement is shown in FIG. 4C in comparison with Arg43 in FIG. 4B showing the presence of a protein-protein NOE at 0.8 ppm and the presence of a detergent-protein NOE at 1.2 ppm. FIG. 4D is a surface representation of Mistic-L indicating TROSY perturbations by a hydrophobically partitioning probe (striped grey) and a hydrophilically partitioning probe (dark grey). Residues that exhibited at least a 40% drop in 1HN peak area are indicated.
FIG. 5 shows two panels relating to Mistic-L-assisted eukaryotic IM protein expression. FIG. 5A are topological depictions of three protein classes fused to Mistic-L as disclosed herein: G-protein coupled receptors (GPCR); TGF-β family receptors; and voltage-gated K+ channels (Kv). FIG. 5B shows a series of SDS-PAGE gels of proteins captured by Ni-NTA affinity chromatography from LDAO-solubilized membrane fractions of cells expressing a fusion protein comprising Mistic-L and the indicated eukaryotic IM protein (RAI3, CRFR2b, ActR IIb, BMPR II, or aKv1.1). The Mistic-L-fused protein (open arrow) is shown in the left lane of each set and the final product (solid arrow) after removal of Mistic-L by thrombin digestion is shown in the right lane of each set.
FIG. 6 is a gel filtration profile of thrombin digested aKv1.1 run in 3mM LDAO on a Superose-6 column. aKv1.1 elutes as a detergent solubilized tetramer subsequent to Mistic-L removal. The inset shows that baseline separation between aKv1.1 (peak 1; lane 1) and Mistic-L (peak 2; lane 2) allows two-step purification of aKv1.1 to near homogeneity.
FIG. 7 shows three SDS-PAGE gels of Ni-NTA-isolated proteins from the indicated fraction of cells expressing wild-type or mutant Mistic-L (“Mistic-L Only”) or fusion proteins comprising a potassium channel domain (aKv1.1) and wild-type or mutant Mistic-L domain (“Mistic-L-aKv1.1).
FIGS. 8A and 8B show schematic representations of Mistic-L-aKv1.1 fusion proteins incorporated into cell membranes. FIGS. 8A and B show Mistic-L fused to truncated (i.e., lacking intracellular T1 and C-terminal domains) and wild-type aKv1.1, respectively. FIG. 8C shows an SDS-gel of Ni-NTA purified Mistic-L-wt aKv1.1 fusion proteins having variable length linkers (0, 5, 8, and 32 amino acids, as indicated above the gel) between the Mistic-L and aKv1.1 domains.
FIG. 9 shows schematic representations of GatewayTM-adapted Mistic polypeptide fusion vectors. The schematic diagrams in panel A depict the cloning regions of pMis2.1 and pMisT2.1 after recombination with a pENTR vector. A thrombin cleavage site is abbreviated “Thr.” An exogenous transmembrane (TM) helix (spiral bar) of pMisT2.1 is located between the sequences encoding a Mistic polypeptide and the 6 His tag. FIG. 9B shows another GatewayTM-adapted Mistic polypeptide fusion vector, which includes additional features as compared to pMis2.1 and pMisT2.1, including a C-terminal affinity tag utilizing a TEV protease cleavage motif enabling flexibility in purification strategies and additional open reading frame(s) (e.g., “Protein 2”) to allow di-cistronic protein expression. Specific non-limiting examples of pMis2.1 and pMisT2.1 including a Mistic-L domain are described; however, it is understood that any Mistic polypeptide (e.g., Mistic-L, M1, M2, M3, or M4) can serve as a “Mistic” domain in the exemplary vectors.
FIG. 10 shows two non-limiting representative hydropathy profiles (panels A and B) for a prototypical Mistic-L polypeptide.
FIG. 11 is a graph of 125I-BMP7 ligand binding for the indicated receptors in the absence (black bars) or presence (grey bars) of unlabeled BMP7.
FIG. 12 is a gel filtration elution profile a Mistic-L-KvPae fusion protein and a KvPae fusion partner domain after thrombin removal of the Mistic-L domain by overnight digestion at room temperature.
FIG. 13 is a sequence alignment of Mistic-L (including M1; SEQ ID NO: 189), M2 (SEQ ID NO: 191), M3 (SEQ ID NO: 193) and M4 (SEQ ID NO: 195). M1-M4 are each 84 residues in length. Mistic-L includes 26 N-terminal amino acids not found in the other Mistic polypeptides. Residues identical among the aligned sequences are labeled with a star (*) and overall percent identity to Mistic-L over the common 84 residues is indicated. Non-conservative substitutions are numbered 1-9.
FIG. 14 is a composite of several SDS gels showing the successful expression of M1, M2, M3 or M4 fusions with Alk3, BMPR11, or CRFR2β (columns marked “−”) and the equally successful cleavage of the particular cargo protein from its respective Mistic domain in the presence of thrombin (columns marked “+”).
FIG. 15 shows two schematic representations of proposed, non-limiting models of Mistic polypeptide function.



SEQUENCE LISTING
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. Amino acid mutations and the corresponding positions are shown in parentheses using standard one-letter amino acid designations (unless expressly stated otherwise). In the accompanying sequence listing:
SEQ ID NO: 1 shows a B. subtillus wild-type Mistic-L nucleic acid sequence.
SEQ ID NO: 2 shows a B. subtillus wild-type Mistic-L amino acid sequence.
SEQ ID NO: 3 shows a nucleic acid sequence of pMistic, which encodes an octa-histidine domain (residues 1-60) fused to a B. subtillus wild-type Mistic-L domain (residues 61-390).
SEQ ID NO: 4 shows an amino acid sequence of pMistic.
SEQ ID NO: 5 shows a nucleic acid sequence of pMistic (W13A).
SEQ ID NO: 6 shows an amino acid sequence of pMistic (W13A).
SEQ ID NO: 7 shows a nucleic acid sequence of pMistic (Q36E).
SEQ ID NO: 8 shows an amino acid sequence of pMistic (Q36E).
SEQ ID NO: 9 shows a nucleic acid sequence of pMistic (M75A).
SEQ ID NO: 10 shows an amino acid sequence of pMistic (M75A).
SEQ ID NO: 11 shows a nucleic acid sequence of pMistic (C3V).
SEQ ID NO: 12 shows an amino acid sequence of pMistic (C3V).
SEQ ID NO: 13 shows a nucleic acid sequence of pMistic (C3L).
SEQ ID NO: 14 shows an amino acid sequence of pMistic (C3L).
SEQ ID NO: 15 shows a nucleic acid sequence of pMistic (C3I).
SEQ ID NO: 16 shows an amino acid sequence of pMistic (C3I).
SEQ ID NO: 17 shows a nucleic acid sequence of pMistic (C3S).
SEQ ID NO: 18 shows an amino acid sequence of pMistic (C3S).
SEQ ID NO: 19 shows a nucleic acid sequence of pMistic (C3V, T30C).
SEQ ID NO: 20 shows an amino acid sequence of pMistic (C3V, T30C).
SEQ ID NO: 21 shows a nucleic acid sequence of pMistic (C3V, S58C).
SEQ ID NO: 22 shows an amino acid sequence of pMistic (C3V, S58C).
SEQ ID NO: 23 shows a nucleic acid sequence of pMistic (C3V, N88C).
SEQ ID NO: 24 shows an amino acid sequence of pMistic (C3V, N88C).
SEQ ID NO: 25 shows a nucleic acid sequence of pMistic (C3V, E110C).
SEQ ID NO: 26 shows an amino acid sequence of pMistic (C3V, E110C).
SEQ ID NO: 27 shows a nucleic acid sequence of pMistic (EK), wherein “EK” denotes mutations of nucleotide residues 373-384 to encode Asp-Asp-Asp-Asp (instead of Glu-Glu-Gly-Glu) at positions 105-108 of the wild-type Mistic-L amino acid sequence.
SEQ ID NO: 28 shows an amino acid sequence of pMistic (EK).
SEQ ID NO: 29 shows a nucleic acid sequence of pMistic (EK, C3S).
SEQ ID NO: 30 shows an amino acid sequence of pMistic (EK, C3S).
SEQ ID NO: 31 shows a nucleic acid sequence of pMistic (EK, W13A).
SEQ ID NO: 32 shows an amino acid sequence of pMistic (EK, W13A).
SEQ ID NO: 33 shows a nucleic acid sequence of pMistic (EK, Q36E).
SEQ ID NO: 34 shows an amino acid sequence of pMistic (EK, Q36E).
SEQ ID NO: 35 shows a nucleic acid sequence of pMistic (EK, M75A).
SEQ ID NO: 36 shows an amino acid sequence of pMistic (EK, M75A).
SEQ ID NO: 37 shows a nucleic acid sequence encoding a peptide (or fusion protein domain) that includes a thrombin-cleavage site (“Thr”).
SEQ ID NO: 38 shows an amino acid sequence of a peptide (or fusion protein domain) that includes a thrombin (“Thr”) cleavage site.
SEQ ID NO: 39 shows a nucleic acid sequence of a linker (“Link”).
SEQ ID NO: 40 shows an amino acid sequence of a linker (“Link”).
SEQ ID NO: 41 shows a nucleic acid sequence of a linker (“L”).
SEQ ID NO: 42 shows an amino acid sequence of a linker (“L”).
SEQ ID NO: 43 shows a nucleic acid sequence of a linker (“LI”).
SEQ ID NO: 44 shows an amino acid sequence of a linker (“LI”).
SEQ ID NO: 45 shows a nucleic acid sequence of a linker (“LINKER”).
SEQ ID NO: 46 shows an amino acid sequence of a linker (“LINKER”).
SEQ ID NO: 47 shows a nucleic acid sequence of a linker (“LINK”).
SEQ ID NO: 48 shows an amino acid sequence of a linker (“LINK”).
SEQ ID NO: 49 shows a nucleic acid sequence of a linker (“LINK2”).
SEQ ID NO: 50 shows an amino acid sequence of a linker (“LINK2”).
SEQ ID NO: 51 shows a nucleic acid sequence of pMistic-KchBsu265.
SEQ ID NO: 52 shows an amino acid sequence of pMistic-KchBsu265.
SEQ ID NO: 53 shows a nucleic acid sequence of pMistic(EK)-KchBsu265.
SEQ ID NO: 54 shows an amino acid sequence of pMistic(EK)-KchBsu265.
SEQ ID NO: 55 shows a nucleic acid sequence of pMistic(EK)-KchXfa297.
SEQ ID NO: 56 shows an amino acid sequence of pMistic(EK)-KchXfa297.
SEQ ID NO: 57 shows a nucleic acid sequence of pMistic(EK)-Link-KchMja209.
SEQ ID NO: 58 shows an amino acid sequence of pMistic(EK)-Link-KchMja209.
SEQ ID NO: 59 shows a nucleic acid sequence of pMistic(EK)-Link-KchPae283.
SEQ ID NO: 60 shows an amino acid sequence of pMistic(EK)-Link-KchPae283.
SEQ ID NO: 61 shows a nucleic acid sequence of pMistic-Link-KchPae283.
SEQ ID NO: 62 shows an amino acid sequence of pMistic-Link-KchPae283.
SEQ ID NO: 63 shows a nucleic acid sequence of pMistic-Thr-KchPae283.
SEQ ID NO: 64 shows an amino acid sequence of pMistic-Thr-KchPae283.
SEQ ID NO: 65 shows a nucleic acid sequence of pMistic(EK/C3S)-Link-KchPae283.
SEQ ID NO: 66 shows an amino acid sequence of pMistic(EK/C3S)-Link-KchPae283.
SEQ ID NO: 67 shows a nucleic acid sequence of pMistic(C3V)-Thr-KchPae283.
SEQ ID NO: 68 shows an amino acid sequence of pMistic(C3V)-Thr-KchPae283.
SEQ ID NO: 69 shows a nucleic acid sequence of pMistic(C3I)-Thr-KchPae283.
SEQ ID NO: 70 shows an amino acid sequence of pMistic(C3I)-Thr-KchPae283.
SEQ ID NO: 71 shows a nucleic acid sequence of pMistic(C3L)-Thr-KchPae283.
SEQ ID NO: 72 shows an amino acid sequence of pMistic(C3L)-Thr-KchPae283.
SEQ ID NO: 73 shows a nucleic acid sequence of pMistic(C3V/T30C)-Thr-KchPae283.
SEQ ID NO: 74 shows an amino acid sequence of pMistic(C3V/T30C)-Thr-KchPae283.
SEQ ID NO: 75 shows a nucleic acid sequence of pMistic(C3V/S58C)-Thr-KchPae283.
SEQ ID NO: 76 shows an amino acid sequence of pMistic(C3V/S58C)-Thr-KchPae283.
SEQ ID NO: 77 shows a nucleic acid sequence of pMistic(C3V/N88C)-Thr-KchPae283.
SEQ ID NO: 78 shows an amino acid sequence of pMistic(C3V/N88C)-Thr-KchPae283.
SEQ ID NO: 79 shows a nucleic acid sequence of pMistic(C3V/E110C)-Thr-KchPae283.
SEQ ID NO: 80 shows an amino acid sequence of pMistic(C3V/E110C)-Thr-KchPae283.
SEQ ID NO: 81 shows a nucleic acid sequence of pMistic(EK)-aKv1.1ΔT1.
SEQ ID NO: 82 shows an amino acid sequence of pMistic(EK)-aKv1.1ΔT1.
SEQ ID NO: 83 shows a nucleic acid sequence of pMistic(EK/W13A)-aKv1.1ΔT1.
SEQ ID NO: 84 shows an amino acid sequence of pMistic(EK/W13A)-aKv1.1ΔT1.
SEQ ID NO: 85 shows a nucleic acid sequence of pMistic(EK/Q36E)-aKv1.1ΔT1.
SEQ ID NO: 86 shows an amino acid sequence of pMistic(EK/Q36E)-aKv1.1ΔT1.
SEQ ID NO: 87 shows a nucleic acid sequence of pMistic(EK/M75A)-aKv1.1ΔT1.
SEQ ID NO: 88 shows an amino acid sequence of pMistic(EK/M75A)-aKv1.1ΔT1.
SEQ ID NO: 89 shows a nucleic acid sequence of pMistic(EK)-aKv1.1.
SEQ ID NO: 90 shows an amino acid sequence of pMistic(EK)-aKv1.1.
SEQ ID NO: 91 shows a nucleic acid sequence of pMistic(EK)-L-aKv1.1(Δ1-6).
SEQ ID NO: 92 shows an amino acid sequence of pMistic(EK)-L-aKv1.1(Δ1-6).
SEQ ID NO: 93 shows a nucleic acid sequence of pMistic(EK)-LI-aKv1.1(Δ1).
SEQ ID NO: 94 shows an amino acid sequence of pMistic(EK)-LI-aKv1.1(Δ1).
SEQ ID NO: 95 shows a nucleic acid sequence of pMistic(EK)-LINKER-aKv1.1.
SEQ ID NO: 96 shows an amino acid sequence of pMistic(EK)-LINKER-aKv1.1.
SEQ ID NO: 97 shows a nucleic acid sequence of pMistic(EK)-LINK-hKv1.5.
SEQ ID NO: 98 shows an amino acid sequence of pMistic(EK)-LINK-hKv1.5.
SEQ ID NO: 99 shows a nucleic acid sequence of pMistic(EK)-LINK-rKv2.1.
SEQ ID NO: 100 shows an amino acid sequence of pMistic(EK)-LINK-rKv2.1.
SEQ ID NO: 101 shows a nucleic acid sequence of pMistic(EK)-LINK-rKv3.1.
SEQ ID NO: 102 shows an amino acid sequence of pMistic(EK)-LINK-rKv3.1.
SEQ ID NO: 103 shows a nucleic acid sequence of pMistic(EK)-LINK-rKv1.2.
SEQ ID NO: 104 shows an amino acid sequence of pMistic(EK)-LINK-rKv1.2.
SEQ ID NO: 105 shows a nucleic acid sequence of pMistic(EK)-LINK2-rKv1.2.
SEQ ID NO: 106 shows an amino acid sequence of pMistic(EK)-LINK2-rKv1.2.
SEQ ID NO: 107 shows a nucleic acid sequence of pMistic(EK)-LINK-GABABR1.
SEQ ID NO: 108 shows an amino acid sequence of pMistic(EK)-LINK-GABABR1.
SEQ ID NO: 109 shows a nucleic acid sequence of pMistic(EK)-LINK-VIPR2.
SEQ ID NO: 110 shows an amino acid sequence of pMistic(EK)-LINK-VIPR2.
SEQ ID NO: 111 shows a nucleic acid sequence of pMis, which encodes an octa-histidine sequence (residues 1-60), a B. subtillus wild-type Mistic-L sequence (residues 61-390), and a linker sequence (residues 391-492).
SEQ ID NO: 112 shows an amino acid sequence of pMis.
SEQ ID NO: 113 shows a nucleic acid sequence of pMisT, which encodes an octa-histidine sequence (residues 1-60), a B. subtillus wild-type Mistic-L sequence (residues 61-390), an exogenous helix (residues 391-552), and a linker sequence (residues 553-654).
SEQ ID NO: 114 shows an amino acid sequence of pMisT.
SEQ ID NO: 115 shows a nucleic acid sequence of pMis-Alk2.
SEQ ID NO: 116 shows an amino acid sequence of pMis-Alk2.
SEQ ID NO: 117 shows a nucleic acid sequence of pMisT-Alk2.
SEQ ID NO: 118 shows an amino acid sequence of pMisT-Alk2.
SEQ ID NO: 119 shows a nucleic acid sequence of pMis-Alk3.
SEQ ID NO: 120 shows an amino acid sequence of pMis-Alk3.
SEQ ID NO: 121 shows a nucleic acid sequence of pMisT-Alk3.
SEQ ID NO: 122 shows an amino acid sequence of pMisT-Alk3.
SEQ ID NO: 123 shows a nucleic acid sequence of pMis-Alk5.
SEQ ID NO: 124 shows an amino acid sequence of pMis-Alk5.
SEQ ID NO: 125 shows a nucleic acid sequence of pMisT-Alk5.
SEQ ID NO: 126 shows an amino acid sequence of pMisT-Alk5.
SEQ ID NO: 127 shows a nucleic acid sequence of pMis-Alk6.
SEQ ID NO: 128 shows an amino acid sequence of pMis-Alk6.
SEQ ID NO: 129 shows a nucleic acid sequence of pMisT-Alk6.
SEQ ID NO: 130 shows an amino acid sequence of pMisT-Alk6.
SEQ ID NO: 131 shows a nucleic acid sequence of pMis-ActRII.
SEQ ID NO: 132 shows an amino acid sequence of pMis-ActRII.
SEQ ID NO: 133 shows a nucleic acid sequence of pMisT-ActRII.
SEQ ID NO: 134 shows an amino acid sequence of pMisT-ActRII.
SEQ ID NO: 135 shows a nucleic acid sequence of pMis-ActRIIb.
SEQ ID NO: 136 shows an amino acid sequence of pMis-ActRIIb.
SEQ ID NO: 137 shows a nucleic acid sequence of pMisT-ActRIIb.
SEQ ID NO: 138 shows an amino acid sequence of pMisT-ActRIIb.
SEQ ID NO: 139 shows a nucleic acid sequence of pMis-BMPRII.
SEQ ID NO: 140 shows an amino acid sequence of pMis-BMPRII.
SEQ ID NO: 141 shows a nucleic acid sequence of pMisT-BMPRII.
SEQ ID NO: 142 shows an amino acid sequence of pMisT-BMPRII.
SEQ ID NO: 143 shows a nucleic acid sequence of pMis-CRFR1.
SEQ ID NO: 144 shows an amino acid sequence of pMis-CRFR1.
SEQ ID NO: 145 shows a nucleic acid sequence of pMisT-CRFR1.
SEQ ID NO: 146 shows an amino acid sequence of pMisT-CRFR1.
SEQ ID NO: 147 shows a nucleic acid sequence of pMis-CRFR2β.
SEQ ID NO: 148 shows an amino acid sequence of pMis-CRFR2β.
SEQ ID NO: 149 shows a nucleic acid sequence of pMisT-CRFR2β.
SEQ ID NO: 150 shows an amino acid sequence of pMisT-CRFR2β.
SEQ ID NO: 151 shows a nucleic acid sequence of pMis-CD97.
SEQ ID NO: 152 shows an amino acid sequence of pMis-CD97.
SEQ ID NO: 153 shows a nucleic acid sequence of pMisT-CD97.
SEQ ID NO: 154 shows an amino acid sequence of pMisT-CD97.
SEQ ID NO: 155 shows a nucleic acid sequence of pMis-CCR5.
SEQ ID NO: 156 shows an amino acid sequence of pMis-CCR5.
SEQ ID NO: 157 shows a nucleic acid sequence of pMisT-CCR5.
SEQ ID NO: 158 shows an amino acid sequence of pMisT-CCR5.
SEQ ID NO: 159 shows a nucleic acid sequence of pMis-RAI3.
SEQ ID NO: 160 shows an amino acid sequence of pMis-RAI3.
SEQ ID NO: 161 shows a nucleic acid sequence of pMisT-RAI3.
SEQ ID NO: 162 shows an amino acid sequence of pMisT-RAI3.
SEQ ID NO: 163 shows a nucleic acid sequence of pMis-GPRC5B.
SEQ ID NO: 164 shows an amino acid sequence of pMis-GPRC5B.
SEQ ID NO: 165 shows a nucleic acid sequence of pMisT-GPRC5B.
SEQ ID NO: 166 shows an amino acid sequence of pMisT-GPRC5B.
SEQ ID NO: 167 shows a nucleic acid sequence of pMis-ETL.
SEQ ID NO: 168 shows an amino acid sequence of pMis-ETL.
SEQ ID NO: 169 shows a nucleic acid sequence of pMisT-ETL.
SEQ ID NO: 170 shows an amino acid sequence of pMisT-ETL.
SEQ ID NO: 171 shows a nucleic acid sequence of pMis-GABABR1.
SEQ ID NO: 172 shows an amino acid sequence of pMis-GABABR1.
SEQ ID NO: 173 shows a nucleic acid sequence of pMisT-GABABR1.
SEQ ID NO: 174 shows an amino acid sequence of pMisT-GABABR1.
SEQ ID NO: 175 shows a nucleic acid sequence of pMis-VIPR2.
SEQ ID NO: 176 shows an amino acid sequence of pMis-VIPR2.
SEQ ID NO: 177 shows a nucleic acid sequence of pMisT-VIPR2.
SEQ ID NO: 178 shows an amino acid sequence of pMisT-VIPR2.
SEQ ID NO: 179 shows a forward Mistic-L primer.
SEQ ID NO: 180 shows a reverse Mistic-L primer.
SEQ ID NOs: 181-183 show amino acid sequences of exemplar exogenous helices.
SEQ ID NOs: 184 and 185 show a nucleic acid and an amino acid sequence, respectively, of rKv4.2
SEQ ID NOs: 186 and 187 show the nucleic acid sequences of a representative pair of “MisticSeeker” primers for amplification from genomic DNA of nucleic acid sequences encoding Mistic polypeptides.
SEQ ID NOs: 188 and 189 show a nucleic acid and amino acid sequence, respectively, of M1.
SEQ ID NOs: 190 and 191 show a nucleic acid and amino acid sequence, respectively, of M2.
SEQ ID NOs: 192 and 193 show a nucleic acid and amino acid sequence, respectively, of M3.
SEQ ID NOs: 194 and 195 show a nucleic acid and amino acid sequence, respectively, of M4.
SEQ ID NOs: 196-201 show nucleic acid sequences encoding, and amino acid sequences of, fusion proteins comprising M1 and Alk3, BMPRII, or CRFR2β.
SEQ ID NOs: 202-207 show nucleic acid sequences encoding, and amino acid sequences of, fusion proteins comprising M2 and Alk3, BMPRII, or CRFR2β.
SEQ ID NOs: 208-213 show nucleic acid sequences encoding, and amino acid sequences of, fusion proteins comprising M3 and Alk3, BMPRII, or CPFR2β.
SEQ ID NOs: 218-219 show nucleic acid sequences encoding, and amino acid sequences of, fusion proteins comprising M4 and Alk3, BMPRII, or CRFR2β.
SEQ ID NOs: 220 and 221 show representative Mistic polypeptide consensus amino acid sequences.
DETAILED DESCRIPTION
I. Overview
Disclosed herein are isolated Mistic polypeptides, which include (or have) an amino acid sequence as set forth in SEQ ID NO: 2, 189, 191, 193, or 195, or which are capable of associating with a membrane and include (or have) at least 80% sequence identity to SEQ ID NO: 2, 189, 191, 193, or 195, or differ from SEQ ID NO: 2, 189, 191, 193, or 195 by one or more conservative amino acid substitutions (such as no more than about 50 highly conserved amino acid substitutions), or are functional fragments of SEQ ID NO: 2, 189, 191, 193, or 195. In specific embodiments, such polypeptide is no more than about 125 amino acids in length, and/or has no more than about 35% hydrophobic residues. In other embodiments, the isolated polypeptide has at least three (such as three or four) alpha helices, each from about 10 to about 25 amino acid residues in length and, in some embodiments, oriented anti-parallel to each other. In more particular embodiments, an alpha helix of the polypeptide is formed by about residue 8 to about residue 22, about residue 32 to about residue 55, about residue 67 to about residue 81, and about residue 89 to about residue 102 of the polypeptide. In still other embodiments, the isolated polypeptide has the tertiary structure characterized by the atomic structure coordinates set forth in PDB Accession No. 1YGM (release date Mar. 1, 2005) or in Table 4. Other isolated polypeptide embodiments, include an amino acid sequence as set forth in SEQ ID NO: 220 or 221, which, in some instances, are no more than about 125 amino acid residues in length and/or include at least three alpha helices (such as the alpha helices described above).
Also provided herein are recombinant fusion proteins including a cargo protein domain and a Mistic domain containing an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 2, 189, 191, 193, or 195 (and, in particular examples, contain the amino acid sequence of SEQ ID NO: 2, 189, 191, 193, or 195). In other fusion protein examples, a Mistic domain includes an amino acid sequence as set forth SEQ ID NO: 220 or 221 and, in some case, such Mistic domain is no more than about 125 amino acid residues in length and/or forms at least three (in some cases, anti-parallel) alpha helices. In some examples, the cargo protein domain contains an integral membrane protein or a portion thereof (such as, a potassium channel protein, a G-protein coupled receptor protein, or a TGF-β family receptor protein). Specific fusion protein embodiments include (or have) the amino acid sequence set forth in SEQ ID NO: 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, or 219. In some cases, the Mistic domain of the fusion protein is located N-terminal of the cargo protein domain, C-terminal of the cargo domain, or within the cargo protein domain. In other instances, the fusion protein includes a linker between the cargo protein domain and the Mistic domain (such as, a linker from 1 to 100 amino acids), which linker may include (or have) an amino acid sequence as set forth in SEQ ID NO: 40, 42, 44, 46, 48, or 50). Other exemplar fusion proteins include a protease-recognition site between the cargo protein domain and the Mistic domain. In specific examples, the protease-recognition site is capable of being cleaved by thrombin, chymotrypsin, trypsin, plasmin, papain, pepsin, subtilisin, enterokinase or TEV protease, and/or is located in the linker. Still other fusion protein embodiments include a peptide tag, which, in some instances, may be located at the N-terminus of the Mistic domain, the C-terminus of the Mistic domain, the N-terminus of the cargo protein domain, or the C-terminus of the cargo protein domain. A peptide tag may include a FLAG tag, a His tag, a HA tag, a streptactin tag, or a biotinylation peptide (BioTagTM). Some fusion proteins can include at least one exogenous helix domain (for example, located between the Mistic domain and the cargo protein domain). In particular examples, a disclosed fusion protein includes one or more of a peptide tag, a linker, and a protease-recognition site, each of which is located between the Mistic domain and the cargo protein domain.
This disclosure further contemplates isolated nucleic acid molecules encoding a disclosed Mistic polypeptide or fusion protein (such as any of those more particularly described in the immediately preceding paragraphs of this section). In some embodiments, an isolated nucleic acid molecule encodes a membrane-associated protein and includes (or has) a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 1, 188, 190, 192, or 194. In other embodiments, an isolated nucleic acid molecule includes (or has) the nucleic acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, 8, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 188, 190, 192, or 194. In still other embodiments, an isolated nucleic acid molecule encodes a membrane-associated protein and hybridizes under high-stringency conditions with a nucleic acid probe comprising at least 30 contiguous nucleotides of SEQ ID NO: 1, 188, 190, 192, or 194. Some nucleic acid molecule embodiments, which encode Mistic fusion proteins, include (or have) a nucleic acid sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 8, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 51, 52, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, or 218. The nucleic acid molecules contemplated by this disclosure also include a vector containing a promoter sequence operably linked to any of the disclosed nucleic acid molecules (such as those described with more particularity in the immediately preceding sentences of this paragraph). Cells transformed with such a vector are also contemplated. In some examples, the transformed cell is a prokaryotic cell (such as, a protease-deficient bacterial strain).
Methods of producing a recombinant fusion protein are also described herein. Some such methods involve expressing a recombinant fusion protein, which includes a cargo protein domain and a Mistic domain having at least 80% sequence identity to SEQ ID NO: 2, 189, 191, 193, or 195, in a cell (such as a prokaryotic cell), such that at least a portion of the fusion protein is incorporated into the cell membrane of the cell. In some instances, the cell is a prokaryotic cell (such as a bacterium). In more specific examples, the cell is a protease-deficient bacterium, such as any one of E. coli strains B1-21, B1-21 (DE3), B1-21 (DE3) pLysS, Origami B, OmpT-defective CD41, CD43 (DE3), and phosphatidylenthanolamine (PE)-deficient AD93. In some method embodiments, the cargo protein domain of the expressed fusion protein is an integral membrane protein comprising at least one transmembrane domain. In more specific embodiments, at least one of the transmembrane domains is incorporated into the cell membrane and the integral membrane protein substantially retains its native conformation.
Other methods disclosed herein include methods of producing an isolated recombinant protein. Such methods involve (i) expressing a recombinant fusion protein, which includes a cargo protein domain and a Mistic domain having at least 80% sequence identity to SEQ ID NO: 2, 189, 191, 193, or 195, in a cell (such as a prokaryotic cell), such that at least a portion of the fusion protein is incorporated into the cell membrane of the cell, and (ii) isolating from the cell a cell membrane fraction containing the fusion protein. More specific method embodiments, further involve isolating the fusion protein from the cell membrane fraction. In particular examples, the fusion protein includes a protease-recognition site between the Mistic domain and the cargo protein domain. In some of these examples, the cargo protein domain is not substantially incorporated into the cell membrane and is tethered to the cell membrane by portion of the fusion protein that is incorporated into the cell membrane and, in even more particular examples, the protease-recognition site is cleaved to release the cargo protein domain. In some examples, a release cargo protein domain is isolated. Some methods of producing an isolated recombinant protein involve a cargo protein domain that includes an integral membrane protein (such as, in particular examples, a potassium channel, a G-protein coupled receptor, or a TGF-β family receptor).
Also described herein are methods of isolating a recombinant fusion protein or domain thereof, involving (i) expressing a recombinant fusion protein, which includes a cargo protein domain and a Mistic domain having at least 80% sequence identity to SEQ ID NO: 2, 189, 191, 193, or 195, in a cell (such as a prokaryotic cell), such that at least a portion of the fusion protein is incorporated into the cell membrane of the cell; (ii) isolating a cell membrane fraction from the cell; and (iii) isolating the fusion protein or the cargo protein domain from the cell membrane fraction. In some examples, a yield of isolated fusion protein or isolated cargo protein domain from the cell is no less than 0.1 mg/L of cells, or no less than 1 mg/L of cells.
Disclosed methods of expressing a recombinant protein also involve (i) transfecting a cell with an expression vector encoding a recombinant fusion protein, which includes a cargo protein domain and a Mistic domain having at least 80% sequence identity to SEQ ID NO: 2, 189, 191, 193, or 195; and (ii) expressing the fusion protein in the cell, such that the amount of the cargo protein domain expressed in the cell is greater than the amount expressed in a control cell transfected with a control expression vector encoding the cargo protein domain alone. In particular examples of these methods, the amount of cargo protein domain expressed in the cell is at least 50-fold greater than the amount of cargo protein domain expressed in the control cell.
Methods of stabilizing the expression of a recombinant protein are also disclosed. Such methods involve co-expressing the recombinant protein with a disclosed Mistic polypeptide. In some embodiments, stabilizing the expression of the recombinant protein involves increasing the solubility of the recombinant protein or preventing the aggregation of the recombinant protein. In other embodiments, the recombinant protein and Mistic polypeptide are coexpressed as a fusion protein.
II. Abbreviation and Terms
















ER
endoplasmic reticulum



GPCR
G-protein coupled receptors



IM
integral membrane



IPTG
isopropyl-β-D-thiogalactopyranoside



Kν
voltage-gated K+ channel



LDAO
lauryl-dimethylamine oxide



LMPG
lyso-myristoyl-phosphotidyl-glycerol



Mistic
Membrane Integrating Sequence for Translation of




IM protein Constructs



MPB
3-(N-maleimido-propinyl) biocytin



MTSL
(1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)




methanethiosulfonate



NMR
nuclear magnetic resonance spectroscopy



NOE
nuclear Overhauser effect



ORF
open-reading-frame



PDC
protein-detergent complex



RMSD
squared root of mean square deviations



RSO
right-side-out [membrane]



TM
Transmembrane










Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs. Definitions of common terms relating to biochemistry and antibodies may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
As used herein, the singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. “Comprising” means “including.” Hence “comprising A or B” means “including A or B” or “including A and B.” All molecular weights, molecular mass values, or lengths given for nucleic acids or polypeptides are approximate, and are provided for description.
In order to facilitate review of the disclosed embodiments, the following explanations of specific terms are provided:
Alpha Helix: A particular helical folding of a polypeptide backbone in protein molecules, in which the carbonyl oxygens are hydrogen bonded to amide nitrogen atoms three residues along the chain. In a typical alpha helix, the translation of amino acid residues along the long axis of the helix is 0.15 nm and the rotation per residue is 100°; accordingly, there are 3.6 residues per turn. Side chains of helix-resident amino acids are arranged at the outside of the helix.
Associating [a Polypeptide] with a Membrane: The process of directing, targeting, or trafficking a polypeptide to a cell membrane or cell membrane-like structure so that the polypeptide is associated with the membrane or membrane-like structure. For example, a polypeptide is associated with a membrane or membrane-like structure if the polypeptide co-fractionates with the membrane or the membrane-like structure under conditions that maintain at least some of the structural integrity of the membrane or membrane-like structure (such as, in the absence of membrane-solubilizing agents). Methods of producing membrane fractions are commonly known in the art and exemplar methods are described herein. All or part of a polypeptide can be associated with a membrane or membrane-like structure by specific or non-specific interactions with the membrane or membrane-like structure, can be tethered to a membrane or membrane-like structure (either temporarily or permanently and with or without being incorporated to any degree into the membrane), and/or can be incorporated (in whole or in part) into the membrane or membrane-like structure. Exemplar membrane-like structures include, without limitation, micelles, liposomes, lipid rafts, or bicelles.
Cargo Protein: A polypeptide that is directed to a membrane as a result of an association with a disclosed Mistic polypeptide.
Encode: A polynucleotide is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those of ordinary skill in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof. The anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
Fusion protein: A polypeptide formed by the joining of two or more heterologous polypeptides through a peptide bond formed by the amino terminus of one polypeptide and the carboxyl terminus of the other polypeptide. Each polypeptide joined into a fusion protein may be referred to as a “domain” or “element” of the fusion protein. With respect to each other, heterologous polypeptides joined into a fusion protein are referred to as “fusion partners.”
G-protein Coupled Receptor Protein: A class of integral membrane proteins belonging to the “7TM” superfamily of transmembrane receptors, which is characterized by seven transmembrane helices. The extracellular portions of G-protein coupled receptors contain highly conserved cysteine residues which form disulfide bonds to stabilize the receptor structure. Unlike other types of IM protein receptors, whose ligand binding site is located in an extracellular domain, G-protein-coupled receptors ligands typically bind within the transmembrane domain. Upon ligand binding, G-protein coupled receptor proteins activate G proteins. Non-limiting examples of G-protein coupled receptors are listed in Table 3 and also include taste receptors, receptors of the olfactory epithelium, and receptors for acetylcholine, adenocorticotropin, rhodopsin, somatostatin, thyrotropin, vasopressin, VIP, GHRH, GABA and serotonin. Serotonin receptors are found in central and peripheral nervous systems, kidney, liver, pancreas, spleen, small intestine stomach, coronary and pulmonary arteries and aorta, heart and reproduction system.
Hybridization: Oligonucleotides and other nucleic acids hybridize by hydrogen bonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary bases. Generally, nucleic acid consists of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and the bonding of the pyrimidine to the purine is referred to as base pairing. More specifically, A will hydrogen bond to T or U, and G will bond to C. Complementary refers to the base pairing that occurs between two distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence.
“Specifically hybridizable” and “specifically complementary” are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between a first nucleic acid (such as, an oligonucleotide) and a DNA or RNA target. The first nucleic acid (such as, an oligonucleotide) need not be 100% complementary to its target sequence to be specifically hybridizable. A first nucleic acid (such as, an oligonucleotide) is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-specific binding of the first nucleic acid (such as, an oligonucleotide) to non-target sequences under conditions where specific binding is desired. Such binding is referred to as specific hybridization.
Integral Membrane Protein: A protein that has at least one domain that is incorporated into a membrane (such as, a cell membrane). Typically, a membrane-associated domain of an IM protein is hydrophobic and/or is sufficiently embedded in the membrane so that the IM protein remains associated with the membrane during biochemical purification schemes that substantially preserve membrane integrity (e.g., that do not involve detergent(s) or other membrane-disrupting agents). In some cases, an IM protein membrane-associated domain spans the membrane and may be referred to as a “transmembrane” domain. Often (although not always), IM proteins also have one or more extra-membrane domains, such as an extracellular and/or cytoplasmic domain, which extend beyond the surface of the membrane. Exemplar integral membrane proteins are described herein and include, without limitation, G-protein coupled receptor proteins, TGF-β family receptor proteins, and K+ channel proteins.
Isolated: An “isolated” biological component (such as a polynucleotide, polypeptide, or cell) has been purified away from other biological components in a mixed sample (such as a cell or nuclear extract). For example, an “isolated” polypeptide or polynucleotide is a polypeptide or polynucleotide that has been separated from the other components of a cell in which the polypeptide or polynucleotide was present (such as an expression host cell for a recombinant polypeptide or polynucleotide).
The term “purified” refers to the removal of one or more extraneous components from a sample. For example, where recombinant polypeptides are expressed in host cells, the polypeptides are purified by, for example, the removal of host cell proteins thereby increasing the percent of recombinant polypeptides in the sample. Similarly, where a recombinant polynucleotide is present in host cells, the polynucleotide is purified by, for example, the removal of host cell polynucleotides thereby increasing the percent of recombinant polynucleotide in the sample. Isolated polypeptides or nucleic acid molecules, typically, comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or even over 99% (w/w or w/v) of a sample.
Polypeptides and nucleic acid molecules are isolated by methods commonly known in the art and as described herein. Purity of polypeptides or nucleic acid molecules may be determined by a number of well-known methods, such as polyacrylamide gel electrophoresis for polypeptides, or agarose gel electrophoresis for nucleic acid molecules.
Linker: A relatively short series of amino acids that separates elements or domains of a fusion protein.
Membrane: A synthetic or naturally occurring, organized structure of lipids or other amphipathic molecules. Typically, naturally occurring membranes are relatively thin and structured bilayers of lipids (such as, phospholipids) that encapsulate cells and intracellular organelles. The term “cell membrane” specifically refers to a membrane encapsulating a cell. As used herein, the term “membrane” also contemplates membrane-like structures, such as micelles, bicelles, lipid rafts, or liposomes, which are comprised of amphipathic molecules and have a hydrophobic core and a hydrophilic surface. Unless the context requires otherwise, the term “membrane” encompasses membrane-like structures. Membrane-like structures are capable of incorporating an integral membrane protein in substantially the same conformation as such IM protein has in a lipid bilayer membrane, such as a cell membrane.
Ortholog: A gene from one species, for example Bacillus subtilis, that has a common origin and substantially similar function as a gene from another species, for example, E. coli, Drosophila, or yeast.
Peptide tag: A typically short amino acid sequence (for example, from 1 to 30 amino acids, such as from 4 to 20, or from 4 to 15 amino acid residues) that permits the tagged protein to be readily detected or purified, for example, by affinity purification.
Primer: An oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and of an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. In some examples, the primer is an oligodeoxyribonucleotide. A primer is of sufficient length to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method. A primer can be at least 15, at least 20, at least 23, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 150, at least 200, at least 250 or at least 300 consecutive nucleotides of a particular nucleotide sequence (such as a Mistic polypeptide-encoding nucleic acid sequence, including SEQ ID NO: 1, 188, 190, 192, or 194).
Methods for preparing and using primers are described, for example, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989), Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1998), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, Calif., 1990). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, © 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).
Probe: A detectable nucleic acid molecule that specifically hybridizes to another nucleic acid molecule. Probes are useful, for example, to detect, identify or isolated nucleic acid molecules to which the probe binds. A probe can be at least 15, at least 20, at least 23, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 150, at least 200, at least 250 or at least 300 consecutive nucleotides of a particular nucleotide sequence (such as a Mistic polypeptide-encoding nucleic acid sequence, including SEQ ID NO: 1, 188, 190, 192, or 194). Methods for preparing and using probes are described, for example, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989), Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1998), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, Calif., 1990).
A probe may be single stranded or double stranded. In some instances, a probe is directly attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g., in Sambrook et al. (In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989) and Ausubel et al. (In: Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992).
Protease-deficient [Bacterial Strain]: A bacterial strain (whether naturally occurring or genetically engineered) that fails to express one or more proteases expressed by a corresponding wild-type bacterium. Non-limiting exemplar protease-deficient bacterial strains are degP-deficient E. coli, ompT-deficient E. coli, and BL21 E. coli (and related strains). Representative protease-deficient bacterial strains are also described in U.S. Pat. Nos. 5,143,846; 5,508,192; 5,264,366; and 5,264,365; Intl. Pub. No. WO 88/05821; and Chaudhury and Smith, J. Bacteriol., 160:788-791, 1984; Elish, et al., J. Gen. Microbiol., 134:1355-1364, 1988; Baneyx and Georgiou, In: Stability of Protein Pharmaceuticals: Chemical and Physical Pathways of Protein Degradation, T. Akers and C. Manning (Eds), 1992; McIntosh, et al., J. Bacteriol., 137:653-657, 1979; Baneyx and Georgiou, Enzyme Microb. Technol., 11:559-567, 1989; Baneyx and Georgiou, J. Bacteriol., 172:491-494, 1990. Protease-deficient bacterial strains are commercially available from various suppliers, including without limitation American Type Culture Collection (e.g., ATCC Nos. 55039, 55040, 55099, and 55100), Novagen (e.g., Strain BL21(DE3), B21(DE3)-pLys, Origami (and related strains), Rosetta (and related strains)), Stratagene (e.g., BL21 CodonPlusTM), Invitrogen (e.g., BL21-SI, BL21-AI), or Amersham (e.g., BL21).
Potassium Channel: A common type of ion channel found in eukaryotes and prokaryotes, which forms a membrane-spanning, potassium-selective pore. Potassium channels are found in most cells and perform a variety of functions, including control of cell membrane electrical excitability and regulation of cellular processes (such as, the secretion of hormones). Potassium channels open or close in response to the transmembrane voltage, or the presence of calcium ions or other signaling molecules. Over 80 mammalian genes are known to encode potassium channel subunits. Potassium channels have a tetrameric arrangement with four subunits arranged around a central pore. Potassium channel subunits have a distinctive pore-loop structure that lines the top of the pore and is responsible for potassium selectivity. Exemplar potassium channel proteins are listed in Table 3.
Recombinant: The term “recombinant” refers to polypeptides or polynucleotides produced by molecular engineering. In most instances, a molecularly engineered polypeptide or polynucleotide has a sequence that is not naturally occurring. Molecular engineering can involve chemical synthesis of polypeptides or polynucleotides from corresponding peptides (or amino acids) or oligonucleotides (or nucleotides), respectively. Alternatively and more commonly, molecular engineering involves the manipulation of nucleic acid sequences using a myriad of now-common techniques, such as PCR, restriction digesting, ligation, DNA mutagenesis, and others. For example, a recombinant polynucleotide may be produced by combining (e.g., by ligation) two or more otherwise unrelated nucleic acid sequences to form a recombinant polynucleotide, for instance, which encodes a fusion protein. In another example, one or more nucleotides of a polynucleotide may be mutated (e.g., by site-directed mutagenesis) to form a recombinant polynucleotide, for instance, which encodes a recombinant mutant protein. In yet another example, a portion of a polynucleotide can be deleted (e.g., by PCR, or restriction enzyme digestion followed by re-ligation) to form a recombinant polynucleotide, for instance, which encodes another recombinant mutant protein. A recombinant nucleic acid sequence encodes a corresponding “recombinant” polypeptide. One of ordinary skill in the art will appreciate that many different recombinant polynucleotides and recombinant polypeptides may be created by molecular engineering.
Sequence identity: The similarity between two nucleic acid sequences or between two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences.
Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; Huang, et al., Computer Applications in the Biosciences 8:155-165, 1992; Pearson et al., Methods in Molecular Biology 24:307-331, 1994; Tatiana et al., (1999), FEMS Microbiol. Lett., 174:247-250, 1999. Altschul et al. present a detailed consideration of sequence-alignment methods and homology calculations (J. Mol. Biol. 215:403-410, 1990).
The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLASTTM, Altschul et al. J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence-analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the internet under the help section for BLASTTM.
For comparisons of amino acid sequences of greater than about 30 amino acids, the “Blast 2 sequences” function of the BLASTTM (Blastp) program is employed using the default BLOSUM62 matrix set to default parameters (cost to open a gap [default=5]; cost to extend a gap [default=2]; penalty for a mismatch [default=−3]; reward for a match [default=1]; expectation value (E) [default=10.0]; word size [default=3]; number of one-line descriptions (V) [default=100]; number of alignments to show (B) [default=100]). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity.
For comparisons of nucleic acid sequences, the “Blast 2 sequences” function of the BLASTTM (Blastn) program is employed using the default BLOSUM62 matrix set to default parameters (cost to open a gap [default=11]; cost to extend a gap [default=1]; expectation value (E) [default=10.0]; word size [default=11]; number of one-line descriptions (V) [default=100]; number of alignments to show (B) [default=100]). Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity.
TGF-β Family Receptor Protein: A receptor for the TGF-β superfamily of ligands. Members of the TGF-β family receptor proteins include, but are not limited to, Alk2 (Activin type Ia receptor); Alk3 (BMP type 1a receptor, also referred to as ALK3); Alk5 (TGB-beta type I receptor); Alk6 (BMP type Ib receptor); ActRII (Activin type II receptor); ActRIIb (Activin type IIb receptor); BMPRII (BMP type II receptor); TGFBR2 (TGF-beta receptor type II precursor); ALK1 (Serine/threonine-protein kinase receptor R3 precursor); ALK4 (Serine/threonine-protein kinase receptor R2); and ALK7 (Activin receptor-like kinase 7).
Vector: A nucleic acid molecule capable of transporting a non-vector nucleic acid sequence which has been introduced into the vector. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA into which non-plasmid DNA segments may be ligated. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments may be ligated into all or part of the viral genome. Viral vectors that infect bacterial cells are referred to as bacteriophages. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (for example, vectors having a bacterial origin of replication replicate in bacteria hosts). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell and are replicated along with the host genome. Some vectors contain expression control sequences (such as, promoters) and are capable of directing the transcription of an expressible nucleic acid sequence that has been introduced into the vector. Such vectors are referred to as “expression vectors.” A vector can also include one or more selectable marker genes and/or genetic elements known in the art.
Except as otherwise noted, methods and techniques for practice of the disclosed subject matter are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999. In addition, suitable methods and materials are described throughout the specification. The materials, methods, and examples are illustrative only and not intended to be limiting.
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety to the extent permitted by applicable law.
III. Mistic Nucleic Acid Molecules
This disclosure provides nucleic acid sequences encoding membrane-associating proteins, referred to as Mistic (for Membrane Integrating Sequence for Translation of IM protein Constructs) polypeptides and variants thereof. Representative Mistic polypeptide-encoding nucleic acid molecules (also referred to as Mistic nucleic acid molecules or Mistic nucleic acid sequences) and their corresponding amino acid sequence are shown in SEQ ID NOs: 1 and 2, SEQ ID NOs: 188 and 189, SEQ ID NOs: 190 and 191, SEQ ID NOs: 192 and 193, and SEQ ID NOs: 194 and 195, respectively.
With the provision herein of Mistic nucleic acid sequences, any method known to those of skill in the art may be used to isolate or produce such prototypic nucleic acid sequences and variants thereof. For example, in vitro nucleic acid amplification (such as polymerase chain reaction (PCR)) may be utilized as a simple method for producing Mistic nucleic acid sequences. PCR is a standard technique, which is described, for instance, in PCR Protocols: A Guide to Methods and Applications (Innis et al., San Diego, Calif.: Academic Press, 1990), or PCR Protocols, Second Edition (Methods in Molecular Biology, Vol. 22, ed. by Bartlett and Stirling, Humana Press, 2003).
A representative technique for producing a Mistic nucleic acid molecule by PCR involves preparing a sample containing a target nucleic acid molecule that encodes a Mistic polypeptide sequence (such as Mistic-L, M1, M2, M3, or M4). For example, DNA or RNA (such as mRNA or total RNA) may serve as a suitable target nucleic acid molecule for PCR reactions. Optionally, the target nucleic acid molecule is extracted from cells by any one of a variety of methods well known to those of ordinary skill in the art. Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992) provide descriptions of methods for DNA and RNA isolation. Target nucleic acid molecules encoding Mistic-L and Ml are found, at least, in Bacillus subtilis. Thus, in some examples, RNA or DNA may be extracted from B. subtilis cells. Target nucleic acid molecules encoding M2, M3, and M4 are found, at least, in B. licheniformis, B. mojavensis, and B. atrophaeus, respectively; thus, these bacteria may also serve as sources of RNA or DNA for preparing Mistic nucleic acid sequences. In examples where RNA is the initial target, the RNA is reverse transcribed (using one of a myriad of reverse transcriptases commonly known in the art) to produce a double-stranded template molecule for subsequent amplification. This particular method is known as reverse transcriptase (RT)-PCR. Representative methods and conditions for RT-PCR are described, for example, in Kawasaki et al. (In PCR Protocols, A Guide to Methods and Applications, Innis et al. (eds.), 21-27, Academic Press, Inc., San Diego, Calif., 1990).
The selection of amplification primers will be made according to the portion(s) of the target nucleic acid molecule that is to be amplified. In various embodiments, primers may be chosen to amplify all or part of a Mistic polypeptide-encoding sequence. Variations in amplification conditions may be required to accommodate primers and amplicons of differing lengths and composition; such considerations are well known in the art and are discussed for instance in Innis et al. (PCR Protocols, A Guide to Methods and Applications, San Diego, Calif.: Academic Press, 1990). By way of example, the portion of the B. subtilis genome encoding Mistic-L (approximately 333 base pairs) may be amplified using the following combination of primers:








(SEQ ID NO: 179)









5′-TCAGGGCCATGGCATGTTTTGTACATTTTTTG-3′ (forward)



 








(SEQ ID NO: 180)









5′-TCAGGAATTCAGCTTGATTCCGTT-3′ (reverse)







These primers are illustrative only; one skilled in the art will appreciate that many different primers may be derived from the provided Mistic nucleic sequence in order to amplify all or part of other Mistic polypeptide-encoding sequence (such as nucleic acid sequences encoding M1, M2, M3, or M4).

PCR primers will comprise at least 10 consecutive nucleotides of a Mistic nucleic acid sequence (e.g., a nucleic acid sequence encoding Mistic-L, M1, M2, M3 or M4). One of skill in the art will appreciate that sequence differences between a prototypical Mistic nucleic acid sequence and the target nucleic acid to be amplified may result in lower amplification efficiencies. To compensate for this, longer PCR primers or lower annealing temperatures may be used during the amplification cycle. Whenever lower annealing temperatures are used, sequential rounds of amplification using nested primer pairs may be used to enhance specificity.
Nucleotide variants of Mistic nucleic acid sequences are comprehended by this disclosure. Such nucleotide variants may be naturally occurring (such as orthologs from other organism) or produced using commonly known techniques, including without limitation site-directed mutagenesis or PCR. Standard techniques for DNA mutagenesis are provided, for instance, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, Ch. 15). In addition, numerous commercially available kits are available to perform DNA mutagenesis (see, for example, QuikchangeTM Site-Directed Mutagenesis Kit (Stratagene), GeneTailorTM Site-Directed Mutagenesis System (Invitrogen); GPSTM-Mutagenesis System (New England Biolabs, DiversifyTM PCR Random Mutagenesis Kit (BD Biosciences Clontech); Mutation Generation System (MJ Research); ExsiteTM PCR-Based Site-Directed Mutagenesis Kit (Stratagene); GeneMorphTM PCR Mutagenesis Kit (Stratagene); or LA PCR Mutagenesis Kit (Takara Mirus Bio)).
Variant Mistic nucleic acid sequences differ from a disclosed sequence by deletion, addition, or substitution of nucleotides, and encode a protein that retains at least one Mistic polypeptide function. Functions of a prototypic Mistic polypeptide include, without limitation, the ability to associate (for example, autonomously associate) with a membrane (such as, a bacterial cell membrane) or membrane-like structure (such as, a micelle or liposome), and/or to traffic a fusion partner (such as, an IM protein) to a cell membrane and/or to stabilize the structure of a fusion partner (for example, to prevent aggregation and/or facilitate solubilization of a detergent-solubilized IM fusion partner). In some embodiments, Mistic nucleic acid variants share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% nucleotide sequence identity with a disclosed Mistic nucleic acid sequence, such as SEQ ID NO: 1, 188, 190, 192 or 194. Alternatively, related nucleic acid molecules can have no more than 3, 5, 10, 20, 50, 75, or 100 nucleic acid changes compared to SEQ ID NO: 1, 188, 190, 192 or 194.
In one embodiment, Mistic nucleic acid sequence variants may differ from the disclosed sequences by alteration of the coding region to fit the codon usage bias of a particular organism, for example, an organism into which the nucleic acid molecule is to be introduced. In other embodiments, Mistic nucleic acid sequence variants are derived by taking advantage of the degeneracy of the genetic code to alter the Mistic coding sequence. In these embodiments, the variant nucleotide sequence may be substantially different from a prototypic Mistic nucleic acid sequence (e.g., SEQ ID NO: 1, 188, 190, 192 or 194) and, nevertheless, encode a protein having an amino acid sequence substantially similar to a disclosed Mistic polypeptide (e.g., Mistic-L, M1, M2, M3, or M4). For example, because of redundancy in the genetic code, any one of four nucleotide codons encode alanine (i.e., GCT, GCG, GCC or GCA); accordingly, the sequence encoding any alanine residue within a Mistic polypeptide could be changed to any of these alternative codons without affecting the amino acid composition or characteristics of the encoded protein. Analogous redundancies are well known for each amino acid. The genetic codes for a variety of organisms are publicly available on the National Center for Biotechnology Information (NCBI) Taxonomy website. In the vertebrate (“standard”) and bacterial genetic codes, methionine and tryptophan are the only two amino acids encoded by just one codon (ATG and TGG, respectively) (see also, Osawa et al., Microbiol. Rev., 56:229-264, 1992; Jukes and Osawa, Comp. Biochem. Physiol., 106B:489-494, 1993).
An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other. In certain embodiments, Mistic nucleic acid sequence variants hybridize to a disclosed Mistic nucleic acid sequences or fragments thereof (such as SEQ ID NO: 1, 188, 190, 192 or 194, or fragments thereof), for example, under low stringency, high stringency, or very high stringency conditions. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization, although wash times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (ed.), Molecular Cloning. A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11.
The following are representative hybridization conditions and are not meant to be limiting.












Very Hich Stringency (detects sequences that


share about 90% sequence identity)








Hybridization:
5x SSC at 65° C. for 16 hours


Wash twice:
2x SSC at room temperature (RT) for 15 minutes each


Wash twice:
0.5x SSC at 65° C. for 20 minutes each







High Stringency (detects sequences that


share about 80% sequence identity or greater)








Hybridization:
5x-6x SSC at 65° C.-70° C. for 16-20 hours


Wash twice:
2x SSC at RT for 5-20 minutes each


Wash twice:
1x SSC at 55° C.-70° C. for 30 minutes each







Low Stringency (detects sequences that share


greater than about 50% sequence identity)








Hybridization:
6x SSC at RT to 55° C. for 16-20 hours


Wash at least
2x-3x SSC at RT to 55° C. for 20-30 minutes each.


twice:









In some embodiments, a Mistic nucleic acid sequence variant is an ortholog of a disclosed Mistic polypeptide-encoding sequence. With the provision of the disclosed prototypic Mistic nucleic acid sequences, the cloning by standard methods of nucleic acid molecules (such as, genes, cDNAs, or other ORFs) that encode Mistic orthologs in other organisms (such as, other Bacillus species, B. subtilis subspecies or strains, B. mojavensis subspecies or strains, B. atrophaeus subspecies or strains, B. licheniformis subspecies or strains, or other genera of bacteria) is now enabled. As with other Mistic variants, Mistic orthologs of the disclosed Mistic nucleic acid molecules encode a polypeptide capable of associating with a membrane and/or trafficking a fusion partner to a membrane (and/or performing another Mistic polypeptide function), and will generally share at least 70% sequence identity with a disclosed Mistic nucleic acid sequence (for example, SEQ ID NO: 1, 188, 190, 192 or 194). Sequence identity will generally be greater in organisms more closely related to B. subtilis, B. mojavensis, B. atrophaeus, or B. licheniformis including, for example, other subspecies or strains of such Bacillus sp., other Bacillus species, and other genera of bacteria. In specific embodiments, orthologous Mistic molecules may share at least 60%, at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 93%, at least 95%, or at least 98% nucleic acid sequence identity with a disclosed Mistic nucleotide or amino acid sequences.
Any method known in the art may be used to isolate Mistic orthologs. For example, both conventional hybridization and PCR amplification procedures may be utilized to clone Mistic orthologs. Direct PCR amplification may be performed on cDNA or genomic libraries prepared from an organism having a Mistic ortholog (such as, other B. subtilis subspecies or strains), or RT-PCR may be performed using RNA extracted from such organism using standard methods. Exemplary methods for isolating Mistic orthologs are provided in Example 10.
For conventional hybridization techniques, a labeled probe derived from a Mistic nucleic acid sequence (such as SEQ ID NO: 1, 188, 190, 192 or 194) may be hybridized to a cDNA or genomic library prepared from an organism having a Mistic ortholog (such as, subspecies or strains of B. subtilis, B. mojavensis, B. atrophaeus, or B. licheniformis, other Bacillus sp. or other genera of bacteria). The hybridization probe is preferably conjugated with a detectable label such as a radioactive label. A hybridization signal may be detected using methods known in the art. The hybridizing colony or plaque (depending on the type of library used) is purified and the cloned sequence contained in that colony or plaque isolated and characterized.
Mistic orthologs may also be obtained by immunoscreening of an expression library. Antibodies (monoclonal or polyclonal) that are useful for performing such immunoscreening can be prepared using a Mistic polypeptide amino acid sequence provided herein. Methods for expressing and isolating such Mistic polypeptides are commonly known and specific exemplary methods are provided herein. Antibodies also may be raised against synthetic peptides derived from a Mistic amino acid sequence presented herein. Methods of raising antibodies are well known in the art and are described generally in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Springs Harbor, 1988.
Fragments of a disclosed Mistic nucleic acid sequence (e.g., SEQ ID NO: 1, 188, 190, 192 or 194) are encompassed by the present disclosure. Such nucleic acid fragments include, for instance, oligonucleotides (which are useful as, e.g., probes and/or primer) and nucleic acid sequences encoding functional fragments of a Mistic polypeptide. A functional Mistic polypeptide fragment is a portion of a disclosed Mistic polypeptide sequence that retains at least one functional activity of the full-length Mistic polypeptide from which the fragment is obtained (e.g., membrane-associating activity and/or fusion-partner-stabilizing activity). The functional properties of Mistic polypeptides are discussed in detail elsewhere in this disclosure.
In one embodiment, Mistic nucleic acid fragments (such as, oligonucleotides) may comprise a sequence of at least 10 consecutive nucleotides of a Mistic nucleic acid sequence. One of skill in the art will appreciate that Mistic-derived nucleic acid fragments (such as, oligonucleotides) of various lengths are useful for a variety purposes. For example, the specificity of an oligonucleotide probe or primer increases with its length. Thus, in some embodiments, an oligonucleotide (such as, a probe or primer) may comprise at least 15, at least 20, at least 23, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more consecutive nucleotides of Mistic nucleotide sequences. In other examples, Mistic nucleic acid fragments (such as, oligonucleotides, probes, or primers) can be at least 100, at least 150, at least 200, at least 250 or at least 300 consecutive nucleic acids of a disclosed Mistic sequence (e.g., SEQ ID NO: 1, 188, 190, 192 or 194).
Mistic nucleic acid fragments (such as an oligonucleotide or a nucleic acid sequence encoding a functional Mistic polypeptide fragment) may be obtained from any region of a disclosed Mistic nucleic acid sequence. By way of example, a Mistic nucleic acid sequence (such as, SEQ ID NO: 1, 188, 190, 192 or 194) may be apportioned into about halves, thirds or quarters based on sequence length, and the isolated nucleic acid molecules (e.g., oligonucleotides or a nucleic acid sequence encoding a functional Mistic polypeptide fragment) may be derived from the first or second halves of the molecules, from any of the three thirds, or from any of the four quarters. A Mistic nucleic acid sequence also could be divided into smaller regions, e.g. about eighths, sixteenths, twentieths, fiftieths and so forth, with similar effect. One of ordinary skill in the art can readily ascertain from the provided sequences which nucleotide numbers correspond to the described portions of a full-length Mistic sequence. For example, the first half of SEQ ID NO: 1 (not including any fractions of a nucleotide) corresponds to nucleotides 1-166 of SEQ ID NO: 1 and so forth. Some embodiments involve Mistic nucleic acid fragments encoding alpha helices 1, 2, 3 and 4, alpha helices 2, 3 and 4, alpha helices 2 and 3, or alpha helices 3 and 4 of a Mistic polypeptide (in each instance, including polypeptide loops between the respective helices so as to maintain the relative structural positions of the helices).
IV. Mistic Polypeptides
This disclosure further provides Mistic polypeptides (including Mistic polypeptide variants) and the nucleic acid sequence encoding such polypeptides. A panel of representative Mistic polypeptides is shown in SEQ ID NO: 2, 189, 191, 193 and 195. As described in detail in the Examples, Mistic-L (SEQ ID NO: 2) was produced and isolated by expressing the corresponding nucleic sequence, which was isolated from B. subtilis. M1 is a functional fragment (or an alternative-start-site variant) of Mistic-L, which lacks the 26 N-terminal amino acids of Mistic-L. Nucleic acid sequences encoding M2, M3 and M4 were isolated from the genomes of B. licheniformis, B. mojavensis, and B. atrophaeus, respectively, by amplifying the regions of those genomes that corresponds to the region in the B. subtilis genome from which Mistic-L was (or Ml can be) isolated. The sequences and relationship among these Mistic polypeptides is shown in the amino acid alignment of FIG. 13. From the large collective of Mistic polypeptides described herein, a wealth of structural and functional information concerning this class of polypeptides is provided.
As shown in FIG. 13, the functional M1 portion of Mistic-L, M2, M3, and M4 share considerable amino acid sequence identity (indicated by asterisks in FIG. 13). Where the sequences are not identical, there is generally conservative substitution of residues among these Mistic polypeptides. There are only nine non-conservative substitutions among the 89 residues of these polypeptides. For example, 30 of the N-terminal 39 amino acid residues of M1, M2, M3, and M4 are identical with only 3 non-conservative substitutions. In another example, 26 of the N-terminal 33 amino acid residues of M1, M2, M3, and M4 are identical with only 1 non-conservative substitution. Thus, in one embodiment, a class of Mistic polypeptides is identified as including the consensus sequence: M K V T X1 X2 E K E Q L S X3 A I D X4 M N E G L D X5 F I X6 X7 Y N E S E (SEQ ID NO: 220); wherein X1 is S or D; X2 is E, D or Q; X3 is T or A; X4 is R or K; X5 is A or V; X6 is any amino acid; and X7 is L or F. In other embodiments, a Mistic polypeptide comprises the consensus sequence set forth in SEQ ID NO: 220, except that the residues at X1-X7 correspond to the M1 Mistic polypeptide residues or a very highly conserved substitution, highly conserved substitution or conserved substitution (as set forth in Table 5) of such M1 residues. Nucleic acid sequences encoding such Mistic polypeptide consensus sequences are also contemplated by this disclosure.
In yet other embodiments, a Mistic polypeptide comprises the consensus sequence: M K V T (S/D) (E/D/Q) E K E Q L S (T/A) A I D (K/R) M N E G L D (A/V) F I (Xaa) (L/F) Y N E S E (Xaa) D E (Xaa) L I (Q/E) (L/F) (D/E) (D/E) (D/E) T A (E/D/K) (L/M) (M/I) (K/R) (Q/E) A (Xaa) (D/E) (Xaa) (Y/H) G (Q/K) (Xaa) (K/S) (Xaa) N (Q/E) (Xaa) L N (T/A) I I K (Q/E) I L S (I/F) S (V/L) (Xaa) (E/K) (E/D) G (E/K) (K/E) (Xaa) (SEQ ID NO: 221), wherein amino acid residues in parentheses indicate possible residues at that position and Xaa can be any amino acid residue. In other embodiments, each Xaa in SEQ ID NO: 221 is a very highly conserved substitution, highly conserved substitution, or conserved substitution (as set forth in Table 5) of the corresponding M1 residue.
In some embodiments, Mistic polypeptides are no more than 150, no more than 140, no more than 130, no more than 120, no more than 115, or no more than 110 amino acid residues. In a particular embodiment, a Mistic polypeptide contains about 110 amino acid residues. In other embodiments, a Mistic polypeptide has a molecular mass of no more than about 30 kDa, no more than about 25 kDa, no more than about 20 kDa, no more than about 15 kDa, or no more than about 13 kDa. In a specific embodiment, a Mistic polypeptide has a molecular mass of about 13 kDa.
In other embodiments, a Mistic polypeptide is hydrophilic with no more than about 25%, no more than about 30%, no more than about 35%, or no more than about 40% hydrophobic residues. Hydrophobic residues include, for example, Leu, Ile, Val, Met, Phe, or Trp. In one example, approximately 33% the residues of a Mistic polypeptide are hydrophobic. Some polypeptide embodiments have hydrophobic residues dispersed (such as, substantially evenly dispersed) throughout a Mistic polypeptide sequence. Non-limiting representative hydropathy profiles for a Mistic polypeptide are shown in FIGS. 10A and 10B. In some embodiments, a Mistic polypeptide lacks any substantial stretches of contiguous hydrophobic amino acids; thus, for example, a Mistic polypeptide will have no more than about 10, no more than about 8 or no more than about 6 contiguous hydrophobic residues. A hydropathy profile of some Mistic polypeptide embodiments indicates a complete absence of predictable transmembrane helices; however, such Mistic polypeptide will still be capable of associating with a membrane or membrane-like structure.
In some examples, a Mistic polypeptide (or a functional fragment thereof) is monomeric in solubilizing detergent solution and comprises four alpha helices with up-down-up-down topology (e.g, Mistic-L) or three alpha helices with down-up-down topology (e.g., M1, M2, M3, or M4). Exemplary alpha helices include from about 10 to about 40 residues; for example, from about 10 to about 36 residues, from about 10 to about 32 residues, from about 10 to about 29 residues or from about 10 to about 25 residues. In one example, each Mistic alphahelix includes at least 10 amino acid residues and no more than 25 amino acid residues. In other examples, an alpha helix is formed by about residue 8 to about residue 22, about residue 32 to about residue 55, about residue 67 to about residue 81, and/or about residue 89 to about residue 102 of a Mistic polypeptide (in each instance±up to about 5 residues, such as ±2 residues or ±3 residues). In more particular embodiments, an alpha helix of a Mistic polypeptide (e.g., M1, M2, M3, or M4) is formed by about residue 6 to about residue 29, about residue 41 to about residue 55, and/or about residue 63 to about residue 76 of the polypeptide (in each instance±up to about 5 residues, such as ±2 residues or ±3 residues).
The tertiary structure of a prototypical Mistic polypeptide is also disclosed herein. Atomic structural coordinates of the representative Mistic-L are listed in Table 4 (preceding the claims) and deposited as PDB Accession No. 1YGM (release date Mar. 1, 2005). The tertiary structures of M1, M2, M3, and M4 are expected to be closely related to that of Mistic-L.
Mistic polypeptides as disclosed herein have at least one function of the Mistic prototype protein (e.g., Mistic-L, M1, M2, M3, or M4). Such Mistic polypeptide functions include, without limitation, the ability to associate (for example, autonomously associate) with a membrane (such as, a bacterial cell membrane) or a membrane-like structure (such as, a micelle or liposome), and/or to traffic a fusion partner (such as, an IM protein) to a cell membrane, and/or to stabilize the structure of a fusion partner (for example, to prevent aggregation or facilitate solubilization of an IM fusion partner).
With the provision of Mistic amino acid sequences and the corresponding nucleic acid sequences herein, the creation of Mistic polypeptide variants is now enabled. Mistic variants include polypeptides that differ in amino acid sequence from a disclosed Mistic polypeptide sequence, but that substantially retain a wild-type function and/or three-dimensional structure (as disclosed elsewhere herein). In some embodiments, Mistic variants include polypeptides that share at least 60% amino acid sequence identity with a Mistic polypeptide sequence provided herein; for example, some Mistic variants will share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity with a disclosed Mistic sequence (for example, SEQ ID NO: 2, 189, 191, 193 or 195).
Mistic variants can be naturally occurring or produced by any method known in the art for making polypeptide variants. In some embodiments, a Mistic variant is produced by manipulation of a disclosed Mistic nucleotide sequence using standard procedures, including without limitation site-directed mutagenesis or PCR. Techniques for DNA mutagenesis have been described previously herein. Naturally occurring Mistic variants can be isolated using any of a myriad of protein purification techniques known in the art (for example, Scopes, Protein Purification: Principles and Practice, 3rd Edition, New York: Springer-Verlag, 1994; Protein Purification Techniques, 2nd Edition, ed. by Simon Roe, New York:Oxford University Press, 2001; Membrane Protein Purification and Crystallization, 2nd Edition, ed. by Hunte et al., San Diego:Academic Press, 2003). A nucleic acid sequence that encodes all or part of a Mistic variant can be readily determined simply by applying a genetic code to the respective portion of the variant's amino acid sequence. The nucleic acid sequence of a variant, then, can be isolated using methods described elsewhere in this specification.
Non-limiting examples of disclosed Mistic polypeptide variants include the following Mistic-L polypeptide variants: Trp13Ala (residues 21-130 of SEQ ID NO: 6); Gln36Glu (residues 21-130 SEQ ID NO: 8); Met75Ala (residues 21-130 of SEQ ID NO: 10); Cys3Val (residues 21-130 of SEQ ID NO: 12); Cys3Leu (residues 21-130 of SEQ ID NO: 14); Cys3Ile (residues 21-130 of SEQ ID NO: 16); Cys3Ser (residues 21-130 of SEQ ID NO: 18); Cys3Val/Thr30Cys (residues 21-130 of SEQ ID NO: 20); Cys3Val/Ser58Cys (residues 21-130 of SEQ ID NO: 22); Cys3Val/Asn88Cys (residues 21-130 of SEQ ID NO: 24); Cys3Val/Glu110Cys (residues 21-130 of SEQ ID NO: 26); EEGE105-108DDDD (where E=Glu, G=Gly, and D=Asp), which is collectively referred to as an “EK” variant (residues 21-130 of SEQ ID NO: 28); EK/Cys3Ser (residues 21-130 of SEQ ID NO: 30); EK/Trp13Ala residues 21-130 of SEQ ID NO: 32); EK/Gln36Glu (residues 21-130 of SEQ ID NO: 34); and EK/Met75Ala (residues 21-130 of SEQ ID NO: 36).
In some embodiments, Mistic polypeptide variants involve the substitution of one or several amino acids for amino acids having similar biochemical properties (so-called conservative substitutions). Conservative amino acid substitutions are likely to have minimal impact on the activity of the resultant protein. Further information about conservative substitutions can be found, for instance, in Ben Bassat et al. (J. Bacteriol., 169:751-757, 1987), O'Regan et al. (Gene, 77:237-251, 1989), Sahin-Toth et al. (Protein Sci., 3:240-247, 1994), Hochuli et al. (Bio/Technology, 6:1321-1325, 1988) and in widely used textbooks of genetics and molecular biology. The Blosum matrices are commonly used for determining the relatedness of polypeptide sequences. The Blosum matrices were created using a large database of trusted alignments (the BLOCKS database), in which pairwise sequence alignments related by less than some threshold percentage identity were counted (Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992). A threshold of 90% identity was used for the highly conserved target frequencies of the BLOSUM90 matrix. A threshold of 65% identity was used for the BLOSUM65 matrix. Scores of zero and above in the Blosum matrices are considered “conservative substitutions” at the percentage identity selected. The following table shows exemplary conservative amino acid substitutions:










TABLE 5







Highly Conserved




Very Highly -
Substitutions
Conserved Substitutions


Original
Conserved
(from the
(from


Residue
Substitutions
Blosum90 Matrix)
the Blosum65 Matrix)







Ala
Ser
Gly, Ser, Thr
Cys, Gly, Ser, Thr, Val


Arg
Lys
Gln, His, Lys
Asn, Gln, Glu, His, Lys


Asn
Gln; His
Asp, Gln, His, Lys,
Arg, Asp, Gln, Glu, His,




Ser, Thr
Lys, Ser, Thr


Asp
Glu
Asn, Glu
Asn, Gln, Glu, Ser


Cys
Ser
None
Ala


Gln
Asn
Arg, Asn, Glu, His,
Arg, Asn, Asp, Glu, His,




Lys, Met
Lys, Met, Ser


Glu
Asp
Asp, Gln, Lys
Arg, Asn, Asp, Gln, His,





Lys, Ser


Gly
Pro
Ala
Ala, Ser


His
Asn; Gln
Arg, Asn, Gln, Tyr
Arg, Asn, Gln, Glu, Tyr


Ile
Leu; Val
Leu, Met, Val
Leu, Met, Phe, Val


Leu
Ile; Val
Ile, Met, Phe, Val
Ile, Met, Phe, Val


Lys
Arg; Gln; Glu
Arg, Asn, Gln, Glu
Arg, Asn, Gln, Glu, Ser,


Met
Leu; Ile
Gln, Ile, Leu, Val
Gln, Ile, Leu, Phe, Val


Phe
Met; Leu; Tyr
Leu, Trp, Tyr
Ile, Leu, Met, Trp, Tyr


Ser
Thr
Ala, Asn, Thr
Ala, Asn, Asp, Gln, Glu,





Gly, Lys, Thr


Thr
Ser
Ala, Asn, Ser
Ala, Asn, Ser, Val


Trp
Tyr
Phe, Tyr
Phe, Tyr


Tyr
Trp; Phe
His, Phe, Trp
His, Phe, Trp


Val
Ile; Leu
Ile, Leu, Met
Ala, Ile, Leu, Met, Thr









In some examples, Mistic variants can have no more than about 3, 5, 10, 15, 20, 25, 30, 40, or 50 conservative amino acid changes (such as, very highly conserved or highly conserved amino acid substitutions) as compared to SEQ ID NO: 2, 189, 191, 193, or 195. In other examples, one or several hydrophobic residues (such as Leu, Ile, Val, Met, Phe, or Trp) in a Mistic sequence can be replaced with a different hydrophobic residue (such as Leu, Ile, Val, Met, Phe, or Trp) to create a variant functionally similar to a disclosed Mistic polypeptide. Substitution of hydrophobic residues in a Mistic sequence can be dispersed throughout all or most of the sequence. Alternative embodiments involve hydrophobic substitutions for hydrophobic residues in a particular region of a Mistic primary sequence (such as the N-terminus (e.g., first one-third), C-terminus (e.g., last one-third) or interior (e.g., middle one-third) portions of a Mistic sequence). Still other embodiments involve substitution of hydrophobic residues with other hydrophobic residues in particular domains of a Mistic tertiary structure (for example, in the core of the Mistic-L protein, including without limitation residues 17, 44, 75 and 98, as illustrated in FIG. 3D).
Mistic-L protein function (such as, autonomous association with a membrane, protein trafficking, and/or protein stabilizing functions) is substantially maintain in, at least, the following variants: Cys3Ser (residues 21-130 of SEQ ID NO: 18); Cys3Val (residues 21-130 of SEQ ID NO: 12); Cys3Ile (residues 21-130 of SEQ ID NO: 16; Cys3Leu (residues 21-130 of SEQ ID NO: 14); Trp13Ala (residues 21-130 of SEQ ID NO: 6); Gln36Glu (residues 21-130 SEQ ID NO: 8); Cys3Val/Ser58Cys (residues 21-130 of SEQ ID NO: 22); Cys3Val/Asn88Cys (residues 21-130 of SEQ ID NO: 24); Cys3Val/Glu110Cys (residues 21-130 of SEQ ID NO: 26); and EEGE105-108DDDD (where E=Glu, G=Gly, and D=Asp) (residues 21-130 of SEQ ID NO: 28). As is readily apparent from this panel of Mistic-L mutants, residues dispersed throughout the 110 amino acid prototypic Mistic-L polypeptide can be modified without substantial effect on protein function. Moreover, as demonstrated, even non-conservative mutations at various sites throughout the prototypic Mistic-L sequence are well tolerated. In comparison, a Met75Ala Mistic-L mutant loses, at least, its ability to traffic a fusion partner (also referred to as a “cargo protein”) to the membrane (see Example 5). Thus, Mistic-L variants preferably retain Met75.
From the amino acid sequence alignment of Mistic-L (including M1), M2, M3, and M4 (see FIG. 13), the residues in M1, M2, M3, and M4 corresponding to the Mistic-L point mutants readily can be determined. It is expected that analogous amino acid substitutions can be made in M1, M2, M3, and/or M4 with the corresponding effect (typically, no substantial effect) on a function of those Mistic polypeptides.
It is expected that mutations that substantially maintain the three-dimensional structure of a Mistic polypeptide will have little effect on its function. The provision of the Mistic-L three-dimensional structure herein enables the design of structurally equivalent, functional Mistic polypeptide variants. As shown, for instance, in Example 3, Mistic-L comprises four, anti-parallel alpha helices. M1, M2, M3 and M4 lack the N-terminus (including helix 1) of Mistic-L (see, e.g., FIG. 13) and are believed to comprise three, anti-parallel alpha helices. As known to those of skill in the art, alpha helices are destabilized by (i) the substitution of Pro for any helix-resident amino acid, (ii) Asp adjacent to Glu in a helix, or (iii) a cluster of Ile residues (such as, 3 or more contiguous Ile residues) in a helix. Accordingly, Mistic polypeptide variants preferably avoid helix destabilizing mutations.
As demonstrated herein, at least the N-terminal 26 amino acids of Mistic-L (or any smaller subset thereof) are not necessary for a function of the resultant, M1 Mistic polypeptide. Accordingly, Mistic variants that are functional fragments of a full-length Mistic polypeptide are also envisioned herein. In one embodiment, a functional fragment of a Mistic polypeptide comprises (or consists of) a Mistic-L sequence (e.g., SEQ ID NO: 2) that lacks up to 2, up to 5, up to 8, up to 10, up to 12, up to 15, up to 18, up to 20, up to 22, or up to all 26 of the N-terminal amino acids. Moreover, because it tolerates non-conservative substitutions, it is believed that the C-terminal non-helical “tail” of Mistic-L, M1, M2, M3, and M4 can be removed without substantial adverse effect on a function of the corresponding Mistic polypeptide. Thus, in another embodiment, a functional fragment of a Mistic polypeptide lacks from up to 2, up to 5, up to 6, up to 8, up to 10, up to 12, up to 15, up to 20, or up to 25 C-terminal amino acids. A functional fragment of a Mistic polypeptide retains at least one functional activity of the polypeptide from which the fragment is obtained, including, e.g., membrane-associating activity and/or the ability to stabilize a fusion partner.
Fusion proteins comprising Mistic polypeptides (also referred to as “Mistic fusion proteins”) are also contemplated by this disclosure. As demonstrated herein, such fusion proteins are useful, at least, for guiding a variety of Mistic fusion partners to a membrane (such as, a cell membrane) or a membrane-like structure (such as, a micelle or liposome). Among other things, this Mistic polypeptide trafficking function enables new methods of producing or isolating recombinant proteins (as discussed below).
Any protein can be fused to all or part of a disclosed Mistic polypeptide (referred to as a “Mistic domain”). In some embodiments, a soluble protein or a membrane protein (such as, an IM protein) is fused to a Mistic domain. One of ordinary skill in the art will recognize that a soluble protein is not substantially incorporated into a membrane or membrane-like structure even though under native conditions a soluble protein may interact, for instance, with elements of the cellular cytoskeleton and not be freely diffusible within a cell. Similarly, the ordinarily skilled artisan will understand that a membrane protein (such as, an IM protein) may have domains that are located outside the lipid bilayer, for example, in an intra- or extra-cellular space.
In some embodiments, a Mistic domain is fused (directly or indirectly) to all or part of an IM protein, including a K+ channel protein, a G-protein coupled receptor, or a TGF-β family receptor protein. In more specific embodiments, a K+ channel protein includes those listed in Table 3. In other specific embodiments, a G-protein coupled receptor includes those listed in Table 3. In still other specific embodiments, a TGF-β family receptor protein includes those listed in Table 3.
The various elements or domains of a Mistic fusion protein can be arranged in any order between the N-terminal and C-terminal ends of the fusion protein. An element or domain that is closer to the N-terminus of a Mistic fusion protein than another element or domain is said to be “N-terminal” of the other element or domain. Similarly, an element or domain that is closer to the C-terminus of a Mistic fusion protein than another element or domain is said to be “C-terminal” of the other element or domain. Unless expressly stated otherwise, different elements or domains of a Mistic fusion protein need not (but may) be adjacent (that is, without one or more intervening elements or domains).
In some Mistic fusion protein embodiments, a Mistic domain (e.g., Mistic-L, M1, M2, M3, or M4, or a functional fragment of any thereof) is N-terminal or C-terminal with respect to a fusion partner (e.g., cargo protein) domain. In other embodiments, a fusion partner protein may be interrupted or divided by a Mistic domain to form two separate domains of a Mistic fusion protein; for example, a Mistic domain may be located between particular functional regions of a particular fusion partner protein, or may divide a fusion partner protein into approximately equal halves, or may be inserted approximately one-fourth, one-third, two-thirds, or three-quarters into the fusion partner protein sequence.
A Mistic fusion protein can include one or more optional elements, such as one or more linker(s), peptide tags (such as, epitope tags), protease-recognition site(s), and/or exogenous helix(ces). A linker is a relatively short series of amino acids that separates other elements or domains of a Mistic fusion protein. For example, a linker can separate a Mistic domain (e.g., Mistic-L, M1, M2, M3, or M4, or a functional fragment of any thereof) and a fusion partner (e.g., cargo protein) domain. In some instances, a linker may be referred to as including another fusion protein element, such as a protease-recognition site or a peptide tag. In some embodiments, a linker is from 1 to 100 amino acids in length; for example, from 5 to 75, from 10 to 60, from 15 to 50, from 15 to 40, or from 1 to 50 amino acids in length. In specific embodiments, a linker has a sequence such as set forth in SEQ ID NO: 40, 42, 44, 46, 48, or 50.
A peptide tag is a, typically short, amino acid sequence (for example, from 1 to 30 amino acids, such as from 4 to 20, or from 4 to 15 amino acid residues) that permits the tagged protein to be readily detected or purified, for example, by affinity purification. Any peptide tag can be used as long as it is capable of being expressed as an element of a Mistic fusion protein and is capable of, for instance, facilitating detection or purification of the fusion protein. A peptide tag is generally (but need not be) placed at or near the N- or C-terminus of a Mistic fusion protein domain, such as a Mistic domain (e.g., Mistic-L, M1, M2, M3, or M4, or a functional fragment of any thereof) or a fusion partner domain. In particular embodiments, a tag is placed at or near the N-terminus of a Mistic domain and/or at or near the C-terminus of a fusion partner (e.g., cargo protein) domain. Various peptide tags are well known in the art. Non-limiting examples include poly-histidine tag (e.g., 4 to 15 consecutive His residues, such as 8 consecutive His residues), poly-histidine-glycine tag; HA tag (e.g., Field et al., Mol. Cell. Biol., 8:2159, 1988), c-myc tag (e.g., Evans et al., Mol. Cell. Biol., 5:3610, 1985), Herpes simplex virus glycoprotein D (gD) tag (e.g., Paborsky et al., Protein Engineering, 3:547, 1990), FLAG tag (e.g., Hopp et al., BioTechnology, 6:1204, 1988; U.S. Pat. Nos. 4,703,004 and 4,851,341), KT3 epitope tag (e.g., Martine et al., Science, 255:192, 1992), tubulin epitope tag (e.g., Skinner, Biol. Chem., 266:15173, 1991), T7 gene 10 protein peptide tag (e.g., Lutz-Freyemuth et al., Proc. Natl. Acad. Sci. USA, 87:6393, 1990), streptavidin tag (StrepTagTM or StrepTagIITM; see, e.g., Schmidt et al., J. Mol. Biol., 255(5):753-766, 1996 or U.S. Pat. No. 5,506,121; also commercially available from Sigma-Genosys), or a biotinylation peptide tag (BioTagTM, which can be specifically biotinylated in vivo or in vitro at a single lysine residue within the tag; e.g., U.S. Pat. Nos. 5,723,584; 5,874,239; and 5,932,433; and U.K Pat. No. GB2370039). Numerous other tag moieties are known to, and can be envisioned by, the ordinarily skilled artisan, and are contemplated herein. An “epitope tag” is a particular type of peptide tag that adds a recognizable epitope (antibody binding site) to the Mistic fusion protein to provide binding of a corresponding antibody; thereby allowing identification or affinity purification of the tagged protein.
A protease-recognition site is an amino acid sequence specifically recognized for cleavage by a particular protease. Protease recognition sites are useful, for example, to cleave one or more Mistic fusion protein domain(s) from each other and/or to isolate a particular Mistic fusion domain (such as, a fusion partner (e.g., cargo protein) domain). Protease-recognition sites are known in the art and proteases specific for such protease-recognition sites are commercially available. Exemplar proteases (and their corresponding recognition sites) include, for example, Tobacco Etch Virus (TEV) NIa protease (Glu-X-X-Tyr-X-Gln/Ser, wherein cleavage occurs between the conserved Gln and Ser residues; Dougherty et al., Virol., 171:356-364, 1989); factor Xa protease (Ile-Glu-Gly-Arg, wherein the C-terminal peptide bond is cleaved); PreScissionTM protease (Leu-Glu-Val-Leu-Phe-Gln/Gly-Pro) (available from Amersham Biosciences); serine protease enterokinase (EK) (Asp-Asp-Asp-Asp-Lys, wherein the C-terminal peptide bond is cleaved) (available from Stratagene), or thrombin (Arg-Gly). Other useful proteases, whose recognition sites are known in the art, include chymotyypsin, trypsin, plasmin, papain, pepsin, and subtilisin.
An exogenous helix is an optional alpha helical domain of a Mistic fusion protein that is distinct from either a Mistic domain or a fusion partner domain. An exogenous helix can facilitate an appropriate orientation of a Mistic domain and its fusion partner domain in a membrane. This is useful, for example, in a situation where the fused termini of Mistic and its fusion partner are naturally located on opposite sides of a membrane.
An exogenous helix includes any series of amino acids that fold into an alpha helix when expressed as a Mistic fusion protein domain. In some examples, an exogenous helix is of sufficient length to traverse a membrane (such as, a cell membrane); for example, an exogenous helix may be from about 5 to about 25 amino acids in length. An exogenous helix sequence can be synthetic or derived from a naturally occurring protein. In some examples, the amino acid sequence of an exogenous helix differs from the sequence of an alpha helix of a Mistic domain or a fusion partner (e.g., cargo protein) domain. In particular embodiments, an exogenous helix has the sequence of a Pseudomonas K+ channel S1 domain, or as set forth in SEQ ID NO: 181. In other embodiments, a synthetic exogenous helix has a sequence set forth in residues 131-184 of SEQ ID NO: 114 or in SEQ ID NOs: 182-183 (see also, Wimley and White, Biochem., 39:4432-4442, 2000).
Representative Mistic fusion proteins include, for instance, the polypeptides (and corresponding nucleic acid sequences) shown in the following Table 1.







TABLE 1







Representative Mistic Fusion Constructs










SEQ ID NO.












Fusion Protein
NT
AA















pMistic-KchBsu265
51
52



pMistic(EK)-KchBsu265
53
54



pMistic(EK)-KchXfa297
55
56



pMistic(EK)-Link-KchMja209
57
58



pMistic(EK)-Link-KchPae283
59
60



pMistic-Link-KchPae283
61
62



pMistic-Thr-KchPae283
63
64



pMistic(EK/C3S)-Link-KchPae283
65
66



pMistic(C3V)-Thr-KchPae283
67
68



pMistic(C3I)-Thr-KchPae283
69
70



pMistic(C3L)-Thr-KchPae283
71
72



pMistic(C3V/T30C)-Thr-KchPae283
73
74



pMistic(C3V/S58C)-Thr-KchPae283
75
76



pMistic(C3V/N88C)-Thr-KchPae283
77
78



pMistic(C3V/E110C)-Thr-KchPae283
79
80



pMistic(EK)-aKv1.1 ΔT1
81
82



pMistic(EK/W13A)-aKv1.1ΔT1
83
84



pMistic(EK/Q36E)-aKv1.1ΔT1
85
86



pMistic(EK/M75A)-aKv1.1ΔT1
87
88



pMistic(EK)-aKv1.1
89
90



pMistic(EK)-L-aKv1.1(Δ1-6)
91
92



pMistic(EK)-LI-aKv1.1(Δ1)
93
94



pMistic(EK)-LINKER-aKv1.1
95
96



pMistic(EK)-LINK-hKv1.5
97
98



pMistic(EK)-LINK-rKv2.1
99
100



pMistic(EK)-LINK-rKv3.1
101
102



pMistic(EK)-LINK-rKv1.2
103
104



pMistic(EK)-LINK2-rKv1.2
105
106



pMistic(EK)-LINK-GABABR1
107
108



pMistic(EK)-LINK-VIPR2
109
110



pMis-Alk2
115
116



pMisT-Alk2
117
118



pMis-Alk3
119
120



pMisT-Alk3
121
122



pMis-Alk5
123
124



pMisT-Alk5
125
126



pMis-Alk6
127
128



pMisT-Alk6
129
130



pMis-ActRII
131
132



pMisT-ActRII
133
134



pMis-ActRIIb
135
136



pMisT-ActRIIb
137
138



pMis-BMPRII
139
140



pMisT-BMPRII
141
142



pMis-CRFR1
143
144



pMisT-CRFR1
145
146



pMis-CRFR2β
147
148



pMisT-CRFR2β
149
150



pMis-CD97
151
152



pMisT-CD97
153
154



pMis-CCR5
155
156



pMisT-CCR5
157
158



pMis-RAI3
159
160



pMisT-RAI3
161
162



pMis-GPRC5B
163
164



pMisT-GPRC5B
165
166



pMis-ETL
167
168



pMisT-ETL
169
170



pMis-GABABR1
171
172



pMisT-GABABR1
173
174



pMis-VIPR2
175
176



pMisT-VIPR2
177
178



His/Linker-M1-Link-Alk3
196
197



His/Linker-M1-Link-BMPRII
198
199



His/Linker-M1-Link-CRFR2β
200
201



His/Linker-M2-Link-Alk3
202
203



His/Linker-M2-Link-BMPRII
204
205



His/Linker-M2-Link-CRFR2β
206
207



His/Linker-M3-Link-Alk3
208
209



His/Linker-M3-Link-BMPRII
210
211



His/Linker-M3-Link-CRFR2β
212
213



His/Linker-M4-Link-Alk3
214
215



His/Linker-M4-Link-BMPRII
216
217



His/Linker-M4-Link-CRFR2β
218
219










The fusion proteins in Table 1 are shown as domains (or modules) fused together with a hyphen (“−”) representing a linkage point between contiguous domains. A pMis, pMisT, pMistic, or pMistic( . . . ) module includes a Mistic-L amino acid sequence with or without additional functional elements, such as an octa-his tag (e.g., pMis, pMisT, pMistic, or pMistic(. . . )), a linker (e.g., pMis, or pMisT), an exogenous helix (e.g., pMisT), and/or an indicated mutation (e.g., pMistic(. . . )). A linker is represented in some of exemplary Mistic fusion proteins by “Link”, “L”, “LI”, “LINKER”, “LINK”, or “LINK2”. Some such linkers (e.g., Link, LINKER, LINK and LINK2) also include a thrombin cleavage site. A thrombin protease cleavage site module is denoted in some constructs as “Thr”. Numerous representative Mistic fusion partner domains (also referred to as cargo protein domains), including a variety of potassium channel proteins, G-protein linked receptor proteins, and TGF-β family receptor proteins, are shown above with designations well known in the art (see also, Table 3 herein). Such designations include KchBsu265, KchXfa297, KchMja209, KchPae283, aKv1.1ΔT1, aKv1.1DT1, aKv1.1, aKv1.1(Δ1-6), aKv1.1(Δ1), hKv1.5, rKv2.1, rKv3.1, rKv1.2, GABABR1, VIPR2, Alk2, Alk3, Alk5, Alk6, ActRII, ActRIIb, BMPRII, CRFR1, CRFR2β, CD97, CCR5, RAI3, GPRC5B, and ETL. The particular arrangement (and corresponding amino acid residues) of functional elements in the foregoing Mistic fusion proteins are shown with particularity in the Sequence Listing.
Methods of making fusion proteins are well known in the art. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the N-terminal fusion protein domain in frame with a nucleic acid sequence encoding the next fusion protein domain (or element) and so forth. Appropriate molecular biological techniques may be found in Sambrook et al. (Molecular Cloning; A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989). Specific examples of genetically engineered multi-domain fusion proteins can be found in U.S. Pat. Nos. 5,834,209; 5,821,082; 5,696,237; 5,668,255; and 5,587,455; and in International Pub. Nos. WO 98/17682 and WO 98/12328. Alternatively, a fusion protein can be produced chemically by crosslinking the fusion protein domains (and/or elements) one to the other in the desired sequence.
V. Heterologous Expression of Recombinant Mistic and Mistic Fusion Protein
Various commonly known systems are available for heterologous expression of the disclosed Mistic polypeptides (e.g., Mistic-L, M1, M2, M3, or M4, or variants or functional fragments of any thereof) and Mistic fusion proteins, including eukaryotic and prokaryotic expression systems, and cell-free translation systems.
Methods of expressing proteins in heterologous expression systems are well known in the art. Typically, a nucleic acid molecule encoding all or part of a protein of interest (such as a Mistic polypeptide or a Mistic fusion protein) is obtained using methods such as those described herein. The protein-encoding nucleic acid sequence is cloned into an expression vector that is suitable for the particular host cell of interest using standard recombinant DNA procedures. Expression vectors include (among other elements) regulatory sequences (e.g., promoters) that can be operably linked to the desired protein-encoding nucleic acid molecule to cause the expression of such nucleic acid molecule in the host cell. Together, the regulatory sequences and the protein-encoding nucleic acid sequence are an “expression cassette.” Expression vectors may also include an origin of replication, marker genes that provide phenotypic selection in transformed cells, one or more other promoters, and a polylinker region containing several restriction sites for insertion of heterologous nucleic acid sequences.
Expression vectors useful for expression of heterologous protein(s) in a multitude of host cells are well known in the art, and some specific examples are provided herein. The host cell is transfected with (or infected with a virus containing) the expression vector using any method suitable for the particular host cell. Such transfection methods are also well known in the art and non-limiting exemplar methods are described herein. The transfected (also called, transformed) host cell is capable of expressing the protein encoded by the corresponding nucleic acid sequence in the expression cassette. Transient or stable transfection of the host cell with one or more expression vectors is contemplated by the present disclosure.
Many different types of cells may be used to express heterologous proteins, such as bacteria, yeasts, fungi, insects, vertebrate cells (such as mammalian cells), and plant cells, including (as appropriate) primary cells and immortal cell lines. Numerous representatives of each cell type are commonly used and are available from a wide variety of commercial sources, including, for example, ATCC, Pharmacia, and Invitrogen.
Further details of some specific embodiments are discussed below.
A. Prokaryotes
Prokaryotes, such as bacteria, may be used as host cells. Prokaryotic expression systems are advantageous, at least, because of culture affordability, ease of genetic manipulation, and high yields of desired product(s). Suitable prokaryotic host cells include, without limitation, E. coli K12 strain 94 (ATCC No. 31,446), E. coli strain W3 110 (ATCC No. 27,325), E. coli X1776 (ATCC No. 31,537), E. coli B, and many other strains of E. coli, such as HB101, JM101, NM522, NM538, NM539, B1-21, B1-21 (DE3), B1-21 (DE3) pLysS, Origami B, OmpT-defective CD41, CD43 (DE3), and phosphatidylenthanolamine (PE)-deficient AD93. Similarly, other species and genera of prokaryotes including bacilli such as Bacillus subtilis, B. mojavensis, B. atrophaeus, or B. licheniformis, or other enterobacteriaceae, such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may all be used as prokaryotic expression hosts. Particular examples contemplate the use of protease-attenuated bacterial host strains such as membrane protease OmpT-defective E. coli (Quick and Wright, Proc. Natl. Acad. Sci. USA, 99:8597-8601, 2002).
Prokaryotic host cells or other host cells with rigid cell walls may be transformed using any method known in the art, including, for example, calcium phosphate precipitation, or electroporation. Representative prokaryote transformation techniques are described in Dower (Genetic Engineering, Principles and Methods, 12:275-296, Plenum Publishing Corp., 1990) and Hanahan et al. (Meth. Enzymol., 204:63, 1991).
Vectors typically used for transformation of E. coli include, without limitation, pBR322, pUC18, pUC19, pUC118, pUC119, Bluescript M13 and derivatives thereof. Numerous such plasmids are commercially available and are well known in the art. Representative promoters used in prokaryotic vectors include the β-lactamase (penicillinase) and lactose promoter systems (Chang et al., Nature, 375:615, 1978; Itakura et al., Science, 198:1056, 1977; Goeddel et al., Nature, 281:544, 1979), a tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res., 8:4057, 1980), and the alkaline phosphatase system.
Example 7 describes representative vectors useful for expression of disclosed Mistic polypeptides (including Mistic fusion proteins), for instance, in bacteria (such as E. coli).
B. Yeast
Various yeast strains and yeast-derived vectors are used commonly for the expression of heterologous proteins. For instance, Pichia pastoris expression systems, obtained from Invitrogen (Carlsbad, Calif.), may be used to express the disclosed Mistic polypeptides. Such systems include suitable Pichia pastoris strains, vectors, reagents, transformants, sequencing primers, and media. Available strains include KM71H (a prototrophic strain), SMD1168H (a prototrophic strain), and SMD1168 (a pep4 mutant strain) (Invitrogen).
Saccharomyces cerevisiae is another yeast that is commonly used in heterologous expression systems. The plasmid YRp7 (Stinchcomb et al., Nature, 282:39,1979; Kingsman et al., Gene, 7:141, 1979; Tschemper et al., Gene, 10:157, 1980) is commonly used as an expression vector in Saccharomyces. This plasmid contains the trp1 gene that provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, such as strains ATCC No. 44,076 and PEP4-1 (Jones, Genetics, 85:12, 1977). The presence of the trp1 lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
Yeast host cells can be transformed using the polyethylene glycol method, as described by Hinnen (Proc. Natl. Acad. Sci. USA, 75:1929, 1978). Additional yeast transformation protocols are set forth in Gietz et al. (Nucl. Acids Res., 20(17):1425, 1992) and Reeves et al. (FEMS, 99(2-3):193-197, 1992).
Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg., 7:149, 1968; Holland et al., Biochemistry, 17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In the construction of suitable expression vectors, the termination sequences associated with these genes are also ligated into the expression vector 3′) of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination. Other promoters that have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing yeast-compatible promoter, origin of replication and termination sequences is suitable.
C. Baculovirus-infected Insect Cells
Another representative eukaryotic expression system involves the recombinant baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV; Summers and Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, 1986; Luckow et al., Biotechnol., 6:47-55, 1987). Infection of insect cells (such as cells of the species Spodoptera frugiperda) with recombinant baculoviruses results in the expression Mistic polypeptides (e.g., Mistic-L, M1, M2, M3, or M4, or variants or functional fragments of any thereof) and Mistic fusion proteins in the insect cells. Baculoviruses do not infect humans and can therefore be safely handled in large quantities.
A baculovirus expression vector is prepared as previously described using standard molecular biology techniques. The vector may comprise the polyhedron gene promoter region of a baculovirus, the baculovirus flanking sequences necessary for proper crossover during recombination (the flanking sequences comprise about 200-300 base pairs adjacent to the promoter sequence) and a bacterial origin of replication which permits the construct to replicate in bacteria. In particular examples, the vector is constructed so that (i) a Mistic polypeptide-encoding nucleic acid sequence is operably linked to the polyhedron gene promoter (collectively, the “expression cassette”) and (ii) the expression cassette is flanked by the above-described baculovirus flanking sequences.
Insect host cells (such as, Spodoptera frugiperda cells) are infected with a recombinant baculovirus and cultured under conditions allowing expression of the baculovirus-encoded Mistic polypeptide (including Mistic fusion proteins). The expressed protein may, if desired, be extracted from the insect cells using methods known in the art or as described herein.
D. Mammalian Cells
Mammalian host cells may also be used for heterologous expression of a disclosed Mistic polypeptide (e.g., Mistic-L, M1, M2, M3, or M4, or variants or functional fragments of any thereof) and/or a Mistic fusion protein. Examples of suitable mammalian cell lines include, without limitation, monkey kidney CV1line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line 293S (Graham et al., J. Gen. Virol., 36:59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (Urlab and Chasin, Proc. Natl. Acad. Sci USA, 77:4216, 1980); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243, 1980); monkey kidney cells (CVI-76, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL 5 1); rat hepatoma cells (HTC, MI.54, Baumann et al., J. Cell Biol., 85:1, 1980); and TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44, 1982). Expression vectors for these cells ordinarily include (if necessary) DNA sequences for an origin of replication, a promoter located 5′ of the nucleic acid sequence to be expressed, a ribosome binding site, an RNA splice site, a polyadenylation site, and/or a transcription terminator site.
Promoters used in mammalian expression vectors can be of viral origin. Such viral promoters may be derived from polyoma virus, adenovirus 2, and simian virus 40 (SV40). The SV40 virus contains two promoters that are termed the early and late promoters. These promoters are useful because they are both easily obtained from the virus as one nucleic acid fragment that also contains the viral origin of replication (Fiers et al., Nature, 273:113, 1978). Smaller or larger SV40 DNA fragments may also be used, provided they contain the approximately 250-bp sequence extending from the HindIII site toward the BglI site located in the viral origin of replication. Alternatively, promoters that are naturally associated with the foreign gene (homologous promoters) may be used provided that they are compatible with the host cell line selected for transformation.
An origin of replication may be obtained from an exogenous source, such as SV40 or other virus (e.g., polyoma virus, adenovirus, VSV, BPV) and inserted into the expression vector. Alternatively, the origin of replication may be provided by the host cell chromosomal replication mechanism.
E. Cell-Free Translation
Cell-free translation systems are known in the art, and can be used to synthesize the disclosed Mistic polypeptides (including Mistic fusion proteins) (see, e.g., Kurland, Cell, 28:201-202, 1982; Pavlov and Ehrenberg, Arch. Biochem. Biophys., 328:9-16, 1996). The most frequently used cell-free translation systems consist of extracts from rabbit reticulocytes, wheat germ and E. coli. All are prepared as crude extracts containing all the macromolecular components (70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation and termination factors, etc.) required for translation of exogenous RNA. Each extract is supplemented with amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase for eukaryotic systems, and phosphoenol pyruvate and pyruvate kinase for the E. coli lysate), and other co-factors (Mg2+, K+, etc.) that facilitate the function of the particular translation machinery.
Either DNA or RNA can be used as the starting material for cell-free protein synthesis. However, DNA starting material is necessarily transcribed to RNA using a “coupled” or “linked” system. A “linked” system generally involves DNA transcription with a bacteriophage polymerase followed by translation in the rabbit reticulocyte lysate or wheat germ lysate. Unlike eukaryotic systems (such as, rabbit reticulocyte or wheat germ) where transcription and translation occur sequentially, transcription and translation occur simultaneously in E. coli. Thus, E. coli translation systems are “coupled” and can be performed in the same tube using either a DNA or RNA template. Methods of using E. coli cell-free systems have been described in detail (e.g., Kigawa et al., FEBS Lett., 442:15-19, 1999; Noren et al., Science, 244:182-188, 1989), Hanes and Plukthun, Proc. Natl. Acad. Sci. USA, 94:4937-4942, 1997, Wilson et al., Proc. Natl. Acad. Sci. USA 98, 3750-3755, 2001; Sawasaki, Proc. Natl. Acad. Sci. USA, 99(23): 14652-14657, 2002). In the E. coli system, it may be advantageous to place a Shine-Dalgamo ribosome binding site upstream of the initiator codon in a DNA template. In particular examples, an E. coli S30 extract system allows expression from DNA vectors containing natural E. coli promoter sequences (such as lac or tac).
Commercially available cell-free translation products (also referred to as in vitro translation products) and instructions for use may be purchased from Ambion (e.g., PROTEINscript-PROTM Kit, Retic Lysate IVTTM Kit), Roche Diagnostics (e.g., RTS 500 ProteoMaster E. coli HY Kit, RTS 9000 E. coli HY Kit), Qiagen (e.g., EasyXpressTM Protein Synthesis Kit), Promega (e.g., TNT® T7 Quick Coupled Transcription/Translation System), and numerous other suppliers.
In some embodiments, a membrane, membrane fragments, or membrane-like structures are added to, or are present in, the cell-free translation system to provide a hydrophobic structure with which a Mistic polypeptide (e.g., Mistic-L, M1, M2, M3, or M4, or variants or functional fragments of any thereof) or, as applicable, a Mistic fusion partner may associate.
VI. Methods of Using Mistic and Mistic Fusion Proteins
Disclosed herein are methods of using Mistic polypeptides (e.g., Mistic-L, M1, M2, M3, or M4, or variants or functional fragments of any thereof), including Mistic fusion proteins, and the corresponding nucleic acid sequences. Such methods contemplate the use of any of the disclosed Mistic polypeptides and/or corresponding nucleic acid sequences, such as those any of those described in the preceding sections or the Examples (below).
Methods of producing a recombinant protein are envisioned. Such methods involve expressing a recombinant fusion protein, which includes, at least, a Mistic domain and a cargo protein domain, in an expression system having a membrane or membrane-like structure, such that at least a portion of the fusion protein is associated with the membrane or membrane-like structure (for example, all or part of the fusion protein may be incorporated into the membrane or membrane-like structure). Representative expression systems, including, for example, bacteria (such as, E. coli) are described elsewhere in this specification. In some examples, a Mistic polypeptide or a Mistic fusion protein co-fractionates with its associated membrane under conditions that do not otherwise solubilize or destabilize the membrane or substantially disrupt the structural integrity of the membrane. Certain examples, where all or part of a polypeptide is incorporated in a membrane (or membrane-like structure), envision insertion of all or part of the polypeptide into or among the molecules (such as, lipids (e.g., phospholipids), or amphipathic molecules (e.g., detergents) that make up the membrane (or membrane-like structure).
In specific embodiments, substantially all or a portion of the Mistic domain is incorporated into the cell membrane (or membrane-like structure) (see, for example, FIG. 4A or 15); for example, at least one Mistic alpha helix is incorporated into the cell membrane (or, in more specific examples, at least four or at least three Mistic alpha helices (such as helices 1-4 or helices 2-4) are incorporated into the cell membrane). In some cases, less than about 50, less than about 35, less than about 25 of amino acids of the Mistic domain protrude from the membrane; for example, C- and/or N-terminal amino acids of the Mistic domain may protrude from the membrane. In other examples, all or part of a cargo protein domain will be incorporated into the membrane. The extent to which a particular cargo protein domain incorporates into a membrane likely will depend on the nature of the particular cargo protein domain. For example, a substantial portion of a cargo protein domain that includes integral membrane protein sequences may be incorporated into a membrane (such as, about 50%, about 60%, about 75%, about 90% or more of the cargo protein domain). A cargo protein domain that is an integral membrane protein typically will include at least one (often, two, three, four, six or more) transmembrane domains. Generally, at least one (or all) of such transmembrane domains will be incorporated within a membrane. In still other examples, all or part of the Mistic domain is incorporated into a membrane and its associated cargo protein domain is not substantially incorporated into the membrane (as may be expected, for example, for a soluble cargo protein domain). In these examples, the cargo protein domain is tethered to the membrane by its association with the membrane-bound Mistic domain and, unless the Mistic and cargo protein domains are cleaved from one another, both domains will fractionate with the membrane. In some embodiments, a cargo protein domain substantially maintains its native configuration either in solution or within a membrane, as applicable.
Membrane incorporation of all or part of a recombinant Mistic fusion protein can be determined, for example, by isolating a membrane fraction from the expression system using commonly known methods (including those described in the Examples herein), and identifying the proteins contained in the membrane fraction (for example, by gel electrophoresis or other known methods). A membrane-associated fusion protein will migrate with the membrane fraction with which it associates.
A Mistic domain includes, for example, an amino acid sequence of any Mistic polypeptide (or functional fragment or variant) described herein. In some embodiments, a Mistic domain autonomously associates with a membrane and, thereby, results in a cargo protein domain fused to the Mistic domain also becoming associated with the membrane. A cargo protein domain can be any polypeptide that can be included in a recombinant fusion protein that also contains a Mistic domain. Some method embodiments envision cargo protein domains that are all or part of an integral membrane or a soluble protein. Particular embodiments conceive a cargo protein domain as all or part of an integral membrane protein, such as a potassium channel, a G-protein linked receptor protein, a TGF-β family receptor protein. Specific examples of such integral membrane proteins are provide throughout this specification.
Integral membrane proteins had been thought to be difficult (often impossible) to successfully express in heterologous expression systems (for review, Tate, FEBS Lett., 504:94-98, 2001). As disclosed herein, a recombinant fusion protein including a Mistic domain and an integral membrane protein domain is reproducibly found within the membrane of a heterologous expression system (such as, E. coli). In particular examples, the disclosed methods may yield no less than about 1 mg/L cells (such as no less than about 0.5, about 0.25, or about 0.1 mg/L cells) of isolated cargo protein domain or isolated recombinant fusion protein. In some cases, an amount of a cargo protein domain (which is functional and/or expressed in a substantially native configuration) that is expressed as a domain of a recombinant Mistic fusion protein is at least 10×, 25×, 50×, 100×, 250×, 500× greater than the amount of a cargo protein domain expressed alone (e.g., without a Mistic fusion partner) in a comparable (e.g., control) expression system.
Certain disclosed methods are useful for isolating a recombinant fusion protein containing a Mistic domain and a cargo protein domain, or for isolating either a Mistic domain or a cargo protein domain of such recombinant fusion protein. In these methods, a recombinant Mistic fusion protein is expressed in an expression system as described above, a membrane fraction from the expression system is isolated, and a recombinant Mistic fusion protein or its Mistic domain or cargo protein domain(s) is isolated from the isolated membrane fraction. In particular examples, a recombinant Mistic fusion protein is expressed in a cell (such as, a bacterium, like E. coli) and the membrane is a cell membrane. In other examples, a recombinant Mistic fusion protein is expressed in a cell-free system and the membrane or membrane-like structure are, for example, micelles, liposomes, or lipid rafts, which are included in the cell-free expression system. A cell membrane fraction can include a plasma membrane, or any intracellular membrane into which a recombinant Mistic fusion protein can be incorporated.
Any technique for isolating membrane fractions may be used in the disclosed methods. Such techniques are well known in the art (e.g., Current Protocols in Cell Biology, New York: John Wiley and Sons, 2001, Chapter 3), and representative techniques are provided in the Examples herein. Typically a membrane fraction can be separated from soluble materials by centrifugation because the membranes are generally heavier than the soluble materials. For example, crude plasma membranes can be prepared by suspending cells in a saline buffer (such as, 10 mM HEPES, 10 mM NaCl, 1 mM KCl, 5 mM NaHCO3, 1 mM CaCl2, 0.5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride (PMSF), 100 U/mL aprotinin, and 5 mM EDTA) and disrupting the cells in a cell homogenizer (such as, a Dounce homogenizer). Gentle centrifugation of the homogenate (for example, at 1000×g) will pellet nuclei and intact cells, and crude plasma membranes will remain in a first supernatant. Subsequent centrifugation of the first supernatant at higher g-force (for example, at 15,000×g) will purify the plasma membranes (in the pellet) from soluble materials.
By virtue of its association with a membrane, a recombinant Mistic fusion protein will migrate with a membrane fraction. Consequently, all or part of a recombinant Mistic fusion protein (such as, a cargo protein domain or a Mistic domain) can be isolated from a membrane fraction. Isolation of all or part of a recombinant Mistic fusion protein from a membrane fraction can be accomplished using any method known to those of skill in the art. For example, immunopurification can be used. In particular examples, a recombinant Mistic fusion protein includes a tag (such as an epitope tag or a multimer-His tag) that can be recognized by a binding agent specific for the tag (such as an antibody or a nickel-containing column). By immobilizing the specific binding agent (for example, on beads or in a column), the tagged recombinant Mistic fusion protein (or a tagged portion of the fusion protein) can be captured and removed from non-bound materials. In some examples, a cleavable site (such as, a protease-sensitive site) is engineered into the recombinant Mistic fusion protein in a manner that permits a cargo protein domain to be cleaved from an isolated recombinant Mistic fusion protein. In particular examples, a tagged portion of a recombinant Mistic fusion protein (such as, a cargo protein domain) is cleaved from the fusion protein and isolated, for example, by immunopurification. In another particular example, a tethered cargo protein domain is fractionated with a membrane by virtue of its association with a membrane-bound Mistic domain, and is thereby separated from other expression system components (such as, soluble cellular components); thereafter, a tethered cargo protein domain can be cleaved from its Mistic domain fusion partner, for example, to isolate the cargo protein domain.
The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described.
EXAMPLES
Example 1
Isolation of Mistic-L, a Self-Integrating Integral Membrane Protein
This example describes the isolation of a Mistic-L-encoding nucleic acid sequence and the expression of the corresponding protein. Characterization of the Mistic-L primary sequence and its oligomerization state in the presence of detergent are also described. The provision of this and other Mistic polypeptide-encoding nucleic acids, at least, enables the construction of Mistic polypeptide-encoding nucleic acid and Mistic polypeptide variants using standard molecular techniques and the expression of Mistic polypeptides and their variants in a variety of host cells, as is now common practice. The example further emphasizes the unique properties of a representative Mistic polypeptide and the clear association of members of this class of polypeptides with membranes and membrane-like structures (such as micelles or liposomes).
A. Isolation of Mistic-L-Encoding Nucleic Acid Sequence
A nucleic acid sequence encoding a 110 amino acid (13 kDa) protein (FIG. 1A) was cloned by PCR from Bacillus subtilis (strain 168) genomic DNA. The primers used for PCR were:








(SEQ ID NO: 179)









5′-TCAGGGCCATGGCATGTTTTGTACATTTTTTG-3′ (forward)



 








(SEQ ID NO: 180)









5′-TCAGGAATTCAGCTTGATTCCGTT-3′ (reverse)







PCR was conducted with VENT thermostable polymerase for 30 cycles between 94° C. (1 minute), 50° C. (1 minute), and 72° C. (2 minutes). This amplification produced a single,  ̃1200 bp band, containing Mistic-L and a portion of a downstream K+ channel gene. This product was purified and digested with the restriction enzymes NcoI and EcoRI, then ligated using T4 ligase into an octa-histidine modified pET-28a plasmid (Novagen) that had been similarly digested. The ligation mixture was used to transform competent Nova Blue cells in accordance with the provided instructions (Stratagene). Cells were plated on kanamycin-laced agar and incubated overnight at 37° C. Individual colonies were cultured in 5 ml volumes of Terrific Broth and plasmid DNA was prepared using a Qiagen miniprep kit and provided instructions. Successful construction of the vector was verified by in-house DNA sequencing. Exemplary Mistic-L nucleic acid and amino acid sequences are provided in SEQ ID NOs: 1 and 2, respectively.

The fully sequenced B. subtilis (subspecies subtilis, strain 168) genome is known (see, for example, GenBank Accession No. NC 000964.2 (GI:50812173). The Mistic-L nucleotide sequence provide herein is not recognized in the B. subtilis genome as a protein coding gene. Instead, a Mistic-L-like nucleotide sequence is shown to a portion of the larger YugO gene open reading frame (see, for example, GenBank Accession Nos. NP 391010.2 (GI:50812283) and Z93936.1 (GI:1934801)). Derst and Karschin (J. Exp. Biol., 201:2791-2799, 1998) report that the YugO nucleotide sequence (GenBank Accession No. Z93936.1 (GI:1934801)) contained a sequence encoding a K+ channel (referred to as YugO-b) and a “putative N-terminal domain” (referred to as YugO-a). However, neither the nucleotide nor amino acid sequences of the putative N-terminal domain were specifically identified. Thus, until now, a Mistic-L-encoding nucleic acid sequence and its corresponding protein have not been known as separate and independent biological compounds.
As shown in FIG. 1A, Mistic-L nucleic acid sequence encodes a highly hydrophilic protein. Only 33% of the Mistic-L amino acid residues are hydrophobic and all such residues are spatially dispersed throughout the sequence. Mistic-L protein lacks any known signal sequences, such as a membrane-targeting sequence. The provision herein of the Mistic-L-encoding nucleic acid sequence enables, for example, the isolation of homologs from other species (see, e.g., Example 10).
B. Expression and Isolation of Recombinant His-tagged Mistic-L
Mistic-L and certain of its variants were expressed in bacteria using methods common in the art. Briefly, Mistic-L-encoding nucleic acids (such as, sequences encoding Mistic-L, its variants, and Mistic-L fusion proteins) were introduced into octa-histidine-tag modified pET-28a (Novagen) for expression of His-tagged Mistic-L proteins in bacterial host cells. Freshly transformed colonies were cultured in TB and induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at an O.D. of 1.0. Growth was continued overnight at 10-18 ° C. Cells were harvested and resuspended in 50 mM Tris pH 8.0, 300 mM KCl, 10% glycerol, 10 mM imidazole with 1 mg/ml lysozyme. Cells were disrupted by sonication on ice and membranes were pelleted by high speed centrifuigation. Membranes were solubilized by sonication in the above buffer with the addition of 20 mM LDAO. Insoluble material was removed by high speed centrifugation and the desired protein was purified from the resulting supernatant using Ni-NTA affinity chromatography (Qiagen). Further purification, when necessary, was conducted by FPLC gel filtration (Pharmacia) in 50 mM Tris pH 8.0, 300 mM KCl with 3 mM LDAO using either S200 SuperdexTM (for Mistic-L) or Superose-6TM (for eukaryotic IM proteins and Mistic-L fusions thereof) (Pharmacia).
As shown in FIG. 1B, Mistic-L associated tightly with the bacterial membrane when expressed recombinantly in E. coli. There was no appreciable accumulation of Mistic-L protein in any other bacterial compartment, including cytoplasm or inclusion bodies, and Mistic-L was not substantially secreted by the bacteria (see FIG. 1B). The association of Mistic-L with the bacterial membrane was (and is) a surprisingly unexpected result, at least, because Mistic-L is highly hydrophilic (see FIG. 1A) and lacks a recognizable membrane-targeting sequence. These particular properties support the non-binding theory that Mistic-L may insert and fold into the membrane in a unique fashion.
C. Mistic-L Oligomerization in Detergent
To further demonstrate the unexpected interaction of Mistic-L with hydrophobic structures (such as, cell membranes), the oligomerization state of Mistic-L solubilized in lauryl-dimethylamine oxide (LDAO) was determined utilizing static light scattering in combination with detection of UV absorption.
Static light scattering analysis was conducted on the eluent from a GFC-1300 analytical HPLC column (Supelco), monitored with a three-angle scattering detector (Wyatt Minidawn) and a photodiode array UV absorbance detector at 280 mn (Waters) according to the manufacturers' instructions. Detergent-solubilized Mistic-L bound tightly to micelles and aggregated rapidly when forcefully stripped of surfactant. The data was fitted with integral multimers of Mistic-L with the protein-detergent complex (PDC) extinction coefficient corrected for the relative percentage of non-absorbing LDAO, as provided by the manufacturers.
Only a monomeric model yielded a relatively equal mass ratio of protein to detergent, as observed for other typical PDCs, with the number of detergent molecules in the PDC (49) similar to the aggregation number for LDAO micelles (76). As shown in FIG. 1C, Mistic-L forms a PDC of approximately 25 kDa containing approximately 50 molecules of LDAO (MW=229.4) per Mistic-L (that is, a 13.4 kDa Mistic-L protein complexed with an 11.4 kDa micelle).
Gel filtration was performed using a SuperDexTM column (Amersham Biosciences) equilibrated with 300 mM KCl, 50 mM Tris (pH 8.0), and 3 mM LDAO and run at a rate of 1 ml per minute. Gel filtration sizing supported either a monomeric or dimeric complex. However, only a monomeric model was consistent with the observation that under oxidizing conditions, Mistic-L underwent a single, discreet shift in size as measured by size exclusion chromatography, presumably through intermolecular cross-linking via its only cysteine. A dimeric assembly would theoretically either not alter in apparent size (cis-oxidation) or polymerize (trans-oxidation).
Example 2
Mistic-L Mutants Reveal the Orientation of Mistic-L in the Membrane
This example demonstrates the orientation (also referred to as topology) of a representative Mistic polypeptide in a cell membrane model using a panel of mono-cysteine Mistic-L mutants. Among other things, these mutants define Mistic-L residues that can be modified without appreciable effect on Mistic-L membrane-associating function. From information provided herein, the corresponding residues of other Mistic polypeptides (e.g., M1, M2, M3 or M4) can be readily determined.
Mistic-L topology studies were conducted with a Mistic-L fusion to a bacterial 6TM potassium channel from Pseudomonas aeruginosa (KvPae) (GenBank Accession No. NP—250187). The KvPae channel, which was substantially identical in all constructs, served as an internal control for calibrating expression, extraction, biotinylation and detection efficiency between the samples. Mistic-L-KvPae fusion constructs were created by subcloning KvPae from a sequencing cosmid containing the gene into a pET-15b vector (Novagen) containing an N-terminal octa-His tag and Mistic-L sequence. Each Mistic-L-KvPae fusion protein contained a thrombin site between the two protein domains to facilitate cleavage of Mistic-L from KvPae as appropriate.
Four mono-cysteine Mistic-L mutants were engineered. In addition to the single natural occurring (wild-type) cysteine (residue 3), cysteine mutations were introduced individually at the C-terminus (residue 110) and in predicted loop regions at positions 30, 58, and 88 (FIG. 1A). In these cysteine variants, the naturally occurring cysteine was mutated to valine. These positions were chosen using NMR-derived knowledge of the secondary structural element boundaries of Mistic-L (see Example 3). All mutagenesis was conducted using QuickchangeTM (Stratagene) in conformance with manufacturer's instructions.
Mistic-L-KvPae fusion proteins were expressed in E. coli as described in Example 1. Approximately 10 hours after induction of fusion protein expression, right-side-out (RSO) vesicles were prepared as described by Kaback (Methods Enzymol., 22:99-120, 1971). Mistic-L (and KvPae) biotinylation was achieved by exposing the RSO vesicles to the membrane impermeable thiol biotinylating reagent, 3-(N-maleimido-propinyl) biocytin (MPB; Molecular Probes), as described by Ramamurthy and Oliver (J. Biol. Chem., 272:23239-23246, 1997). Subsequent to MPB labeling, vesicles were solubilized by sonication in 20 mM LDAO and His-tagged Mistic-L-KvPae fusion proteins were purified using Ni-NTA resin. Affinity purified proteins were digested with thrombin to cleave the Mistic-L and KvPae domains and were separated on SDS-PAGE gels. Streptavidin conjugated horseradish peroxidase was used to illuminate biotinylated products using standard ECLTM protocols (Amersham).
As shown in FIG. 1D, only the cysteine at position 110 of Mistic-L (in the Glu110Cys mutant) had sufficient periplasmic exposure in E. coli RSO vesicles to be reactive with MPB. Cysteines at positions 30, 58, and 88 (in mutants Thr30Cys, Ser58Cys, and Asn88Cys, respectively) were nonreactive with MPB, which demonstrates that these Mistic-L residues are chemically inaccessible from the exterior of RSO vesicles. This result is consistent with the residues at positions 30, 58, and 88 of Mistic-L being membrane embedded.
Regardless of which Mistic-L mutant was fused to KvPae, KvPae was consistently biotinylated in RSO vesicles (see FIG. 1D). KvPae is a transmembrane protein with Cys residues exposed in the E. coli periplasmic space. KvPae biotinylation in each sample indicates that no substantial differences between preparation or reaction conditions for the various samples. Moreover, because the Mistic-L and KvPae domains were fused at the time of membrane insertion, consistent KvPae biotinylation demonstrates that each of the corresponding Mistic-L fusion proteins retained membrane-associating function. Accordingly, none of the mono-cysteine Mistic-L mutations affected the membrane-associating capacity of the corresponding Mistic-L fusion protein.
Example 3
NMR Structure of Mistic-L
The ability to stably extract high yields of Mistic-L from E. coli membranes utilizing the detergent LDAO permitted the use of NMR for the de novo determination of Mistic-L structure. This example describes the secondary and tertiary (aka, three-dimensional or space-filling) structure of Mistic-L, as determined by NMR. The provision of the tertiary structure of Mistic-L, at least, enables rapid and relatively effortless determination of structural mutants that will or will not affect the membrane-associating function of Mistic-L or other Mistic polypeptides (such as, M1, M2, M3 and/or M4).
A. NMR Materials and Methods
Stable-isotope labeled protein was expressed utilizing established protocols (Marley et al., J. Biomol. NMR, 20:71-75, 2001). This procedure allowed straightforward adaptation of protein-specific expression protocols that use rich media and provided a several-fold reduction in isotope costs. Using this method, the incorporation of 15N and 13C was about 85% and deuteration level was about 70%. A series of [15N, 1H]-TROSY experiments of 15N, 2H-labeled Mistic-L in presence of the detergents lyso-myristoyl-phosphotidyl-glycerol (LMPG) and LDAO were measured allowing a qualitative comparison of the extent of peak overlap and the 15N/1H linewidths of cross peaks. The results suggested that structure determination could be best facilitated in a solution of 10 mM BisTris(HCl) pH 5.4, 95% H2O/5% D2O, in presence of approximately 50 mM LDAO with a protein concentration of 2 mM. All NMR spectra were recorded at 37° C. on Bruker 700 MHz spectrometer equipped with four radio-frequency channels and a triple resonance cryo-probe with a shielded z-gradient coil. 1H, 13C and 15N backbone resonances were assigned using the TROSY-based (Pervushin et al., Proc. Natl. Acad. Sci. USA., 94:12366-12371, 1997) triple resonance experiments HNCA (Grzesiek and Bax, J. Biomol. NMR, 9:207-211, 1997) and HNCAcodedCO (Ritter et al., J. Biomol. NMR, 28:289-294, 2004), and 3D 15N-resolved TROSY-[1H, 1H]-NOESY with amixing time of 200 ms. Partial side chain assignment was achieved with 3D H(CC-TOCSY-CO)-NH and 3D 15N-resolved TROSY-[1H, 1H]-NOESY experiments. Aromatic side chain assignments were obtained from 3D 13Caromatic resolved [1H, 1H]-NOESY and a high-resolution [13C, 1H]-HMQC using the C-C splitting for spin system identification. Distance constraints for the calculation of the 3D structure were derived from 3D 13C- or 15N-resolved [1H, 1H]-NOESY spectra recorded with a mixing time of 200 ms. Angle restraints were derived from the deviation of the 13Cα chemical shifts from ‘random coil’ chemical shifts (Luginbuhl et al., J. Biomol. NMR., 8:136-146, 1996; Erratum in: J. Biomol. NMR., 9:212, 1997). All experiments were optimized for sensitivity and set up in a water flip-back manner to enhance the longitudinal relaxation (Riek et al., J. Am. Chem. Soc., 124:12144-12153, 2002). The TROSY-HNCA formed the base for the sequential backbone assignment as the most sensitive triple resonance experiment that connects sequential residues through the 13Cα chemical shifts. Ambiguities were resolved with the TROSY-HNCAcodedCO, which has a two-fold increased resolution along the 13C frequency and contains correlations via the chemical shifts of both 13Cα, and 13Cγ advantages that compensate for a lower overall sensitivity as compared with the TROSY-HNCA. The TROSY-based 15N-resolved [1H, 1H]-NOESY was also used to resolve ambiguities in the assignment process by the collection of sequential amide-amide NOEs. Hence, the backbone assignment was established through three independent correlations, the 13Cα chemical shifts, the 13Cγ chemical shifts and the 1HN-1HN NOEs. Hydrophobicity-selective paramagnetic perturbation was conducted utilizing Gd3+DOTA-Amp and 16-doxyl-stearic acid (Hilty et al., Chembiochem., 5:467-473, 2004). Spectra were analyzed utilizing CARA with XEASY, 13Cα chemical shift deviations were measured with MAPPER (Guntert et al., J. Biomol. NMR, 18:129-137, 2000), and restraint models were built and assessed with CYANA (Guntert, Methods Mol. Biol., 278:353-378, 2004). Structural figures were made using either MOLSCRIPT (Esnouf, Acta Crystallogr. D. Biol. Crystallogr., 55:938-940, 1999) or MOLMOL (Koradi et al., J. Mol. Graph., 14:29-32, 1996).
B. NMR Determination of Mistic-L Secondary and Tertiary Structure
For the sequential backbone assignment, the TROSY-HNCA (Pervushin et al., Proc. Natl. Acad. Sci. USA., 94:12366-12371, 1997; Salzmann et al., J. Biomol. NMR, 15:181-184, 1999); TROSY-HNCAcodedCO (Ritter et al., J. Biomol. NMR, 28:289-294, 2004) and the TROSY-based 15N-resolved [1H, 1H]-NOESY (mixing time 200 ms) of a 2H, 15N, 13C-labeled sample was measured. The 13Cα chemical shift deviation from ‘random coil’ values, the observed NOE pattern, and slow 1HN exchange with solvent strongly indicates the presence of four helices comprising residues 8-22, 32-55, 67-81, and 89-102 (see FIG. 2A).
While intra-residue, sequential and medium range NOEs and angle restraints enabled the assignment of secondary structure, long range restraints are needed to determine the fold of the protein. Since a lack of unambiguous long range NOEs is common in a-helical, 2H, 15N, 13C-labeled membrane proteins, alternative tactics for collecting long range restraints were employed. The mono-cysteine mutant library described in Example 2 was used to incorporate site-directed spin-labels that perturb the NMR spectra in order to derive long-range distance restraints. It has long been recognized that distance-dependent line broadening of nuclear magnetic resonances can be observed in protein samples containing paramagnetic electrons (Kosen, Methods Enzymol., 177:86-121, 1989). However, only recently has the observed line broadening effect been translated into distances for structure determination (Battiste and Wagner, Biochemistry, 39:5355-5365, 2000).
[15N, 1H]-TROSY experiments were measured on Mistic-L samples modified with the thiol-reactive nitroxide label, (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl) methanethiosulfonate (MTSL) (see FIG. 2B). Paramagnetic labeling with MTSL was done in accordance with established protocols (Battiste and Wagner, Biochemistry, 39:5355-5365, 2000). Corresponding reference experiments were measured by quenching the nitroxide label with ascorbic acid, followed by additional reference experiments after removal of the quenched nitroxide label with reducing agents. The signal changes observed for the five spin-labeled samples were transformed into 197 long range upper distance and 290 lower distance restraints (see FIG. 2C).
Initial structure calculation was performed with CYANA (Guntert, Methods Mol. Biol., 278:353-378, 2004) using the collected NOE data, chemical shift-derived angle restraints, and restraints derived from spin-labeling. In addition, α-helical hydrogen bond restraints were implemented for residues that show all of the three following properties: slow HN exchange, a helical 13C chemical shift, and helical backbone NOEs (FIG. 2A). In an iterative process, the derived scaffold was used to collect long-range and medium range NOEs and to refine calibration of the spin-label restraints. Several rounds of structure calculation and collection of restraints were performed. The 29 collected long-range NOEs are of the type methyl/aromatic protons to amide protons. Since these distances are intrinsically large in a helical bundle and concomitantly result in weak NOEs, the use of a cryoprobe and long mixing times of 200 ms were used.
The final structure calculation was performed with 573 NOE distance restraints, 346 angle restraints from chemical shifts and NOE's, and 478 distance restraints from the spin-label experiments, as shown more particularly in the following Table 2.







TABLE 2





NMR Structure Statistics







Restraints











Hydrogen bonds

43



NOE distances
intraresidue
127




short range
248




medium range
169




long range
29



Dihedral angles

346



Spin label restraints
long range, upper distance
197




long range, lower distance
290







Residual upper limit constraint violations


(including spin label restraints)










Number > 0.5
3



Maximum
0.7 +/− 0.1







Residual dihedral angle constraint violations










Number > 5.0°
2



Maximum
6.7° +/− 4   







Backbone RMSD.










Residues 11-105
1.4



Residues 13-52, 67-102
1.0







Heavy Atom RMSD










Residues 11-105
2.0



Residues 13-52, 67-102
1.6







Ramachandran Plot










Residues in most favored regions
64.4%



Residues in allowed regions
31.8%










A total of 100 conformers were initially generated by CYANA and the bundle of 10 conformers with the lowest target function was used to represent the three-dimensional NMR structure (see FIG. 3A). The small residual constraint violations in the 10 refined conformers and the good coincidence of experimental NOEs show that the input data represent a self-consistent set and that the restraints are well satisfied in the calculated conformers. The deviations from ideal geometry were minimal, and similar energy values were obtained for all 10 conformers (Table 2). The high quality of the structure was also reflected by the small (approximately 1.0 Å) backbone RMSD values relative to the mean coordinates for residues 13-52, 67-102.
The resulting structure is a four-helical bundle with many highly unusual features for an IM protein (FIG. 3B, E). For example, all but the second helix (α2) are shorter (approximately 14 amino acids) than expected for a typical bilayer-traversing helix. This may be due to partial unraveling of the ends of the helices in the detergent micelle environment. Additionally, α2 possesses a significant kink, centrally positioned and putatively within the membrane. Also surprisingly, while assembled internally with a typical hydrophobic core and compensating polar interactions towards its surface (FIG. 3D), Mistic-L retains an unexpectedly hydrophilic surface for an IM protein (FIG. 3C, F) (Berman et al., Nucleic Acids Research, 28:235-242, 2000). The atomic coordinates for Mistic-L structure are listed in Table 4 (preceding the claims), and have been deposited as PDB Identification No. 1YGM (release date Mar. 1, 2005).
C. Interaction between Mistic-L and Hydrophobic Lipid Bilayer
Given the membrane-transversing topology demonstrated by MPB labeling (see Example 1 and FIG. 1D), the hydrophilic surface of Mistic-L was surprisingly unexpected. To confirm the orientation of Mistic-L with respect to the membrane and to understand the chemical nature of Mistic-L's interaction with a hydrophobic lipid bilayer, NOEs between Mistic-L and its solubilizing LDAO detergent micelle were measured and assigned.
When mapped to the surface of the Mistic-L structure, as expected for a membrane-integrated protein, a concentric ring of detergent interactions around the helical bundle was observed (FIG. 4A-C). Additionally, Mistic-L spectra was perturbed with paramagnetic probes that selectively partition to hydrophilic or hydrophobic environments as described by Hilty et al. (Chembiochem., 5:467-473, 2004). Results from this study (shown in FIG. 4D) also correlate well with the aforementioned detergent belt around Mistic-L and confirm that Mistic-L was embedded within the LDAO micelle.
This example demonstrates that Mistic-L is a hydrophilic protein that nevertheless traverses the hydrophobic environments of micelles cores and lipid bilayers.
Example 4
Proteins Fused to Mistic-L are Targeted to the Membrane
This example demonstrates that Mistic-L targets (i.e., cargoes or traffics) a variety of other proteins to which it is fused to a cell membrane. When fused to Mistic-L's C-terminus, a fusion partner protein (also referred to as a “cargo protein”) readily folds into its native, lipid bilayer-inserted conformation, apparently (though not necessarily) bypassing E. coli translocon and chaperone apparatus. Accordingly, Mistic-L fusion proteins provide, among other things, useful methods of producing and isolating Mistic-L fusion partners.
Mistic-L was subcloned along with the promoter and affinity tag into pET-15b (Novagen) (see, e.g., Examples 1 and 2) or GatewayTM destination (Invitrogen) vectors (in accordance with manufacturer's directions) for expression studies of Mistic-L-eukaryotic IM protein fusions. Mistic-L-assisted expression was determined for three topologically and structurally distinct classes of eukaryotic IM proteins: voltage-gated K+ channels, receptor serine kinases of the TGF-β superfamily, and G-protein coupled receptors (each shown schematically in FIG. 5A). Eukaryotic target genes were cloned by PCR and fused downstream of Mistic-L with a separation linker of 5-35 amino acids. The full length genes of cargo proteins (6 Kv channels, 9 GPCRs, 7 TGF-β receptors; see Table 3) were used, except that signal sequences were omitted and for Kv channels, flexible N-terminal ‘ball & chain’ motifs were also omitted. Representative Mistic-L fusion protein constructs are shown in Table 1 (together with sequence identifiers for the respective amino acid and nucleic acid sequences).







TABLE 3







Fusion Partner Proteins















Protein






Gene
Gene
MW
Membrane

Binding


Name
Length
(kDa)
Expression
Ligand
Assay
Full Namea










Potassium Channels













aKv1.1
1380
52
+


Aplysia Kv1.1 channel








(NT 391-1743 of SEQ ID NO: 89)








(AA 131-581 of SEQ ID NO: 90)


rKv1.2
1404
53
+
dendrotoxin

Rat Kv1.2 channel








(NT 436-1836 of SEQ ID NO: 103)








(AA 146-612 of SEQ ID NO: 104)


hKv1.5
1485
56
+


human Kv1.5 channel








(NT 436-1917 of SEQ ID NO: 97)








(AA 146-639 of SEQ ID NO: 98)


rKv2.1
2484
92
+


Rat Kv2.1 channel








(NT 436-2916 of SEQ ID NO: 99)








(AA 146-972 of SEQ ID NO: 100)


rKv3.1
1758
66
+


Rat Kv3.1 channel








(NT 436-2190 of SEQ ID NO: 101)








(AA 146-730 of SEQ ID NO: 102)


rKv4.2
1773
51
−


Rat Kv4.2 channel








(NT SEQ ID NO: 184)








(AA SEQ ID NO: 185)







Receptor Serine Kinases of the TGF-β Superfamily













Alk2
1461
55
+
Activin

Activin type Ia receptor








(NT 493-2260 of SEQ ID NO: 115)








(AA 165-655 of SEQ ID NO: 116)


Alk3
1527
58
+
BMP2/7
+
BMP type 1a receptor








(NT 493-2326 of SEQ ID NO: 119)








(AA 165-677 of SEQ ID NO: 120)


Alk5
1407
53
+
TGF-beta

TGB-beta type I receptor








(NT 493-2206 of SEQ ID NO: 123)








(AA 165-637 of SEQ ID NO: 124)


Alk6
1467
56
+
BMP2/7

BMP type Ib receptor








(NT 493-2266 of SEQ ID NO: 127)








(AA 165-657 of SEQ ID NO: 128)


ActRII
1482
56
+
Activin

Activin type II receptor








(NT 493-2281 of SEQ ID NO: 131)








(AA 165-662 of SEQ ID NO: 132)


ActRIIb
1545
59
+
Activin
−
Activin type IIb receptor








(NT 493-2344 of SEQ ID NO: 135)








(AA 165-683 of SEQ ID NO: 136)


BMPRII
1512
57
+
BMP2/7
+
BMP type II receptor








(NT 493-2311 of SEQ ID NO: 139)








(AA 165-672 of SEQ ID NO: 140)







G-protein Coupled Receptors













CRFR1
1176
46
+
Astressin
+
CRF receptor 1








(NT 493-1975 of SEQ ID NO: 143)








(AA 165-560 of SEQ ID NO: 144)


CRFR2β
1215
48
+
Astressin
+
CRF receptor 2 beta








(NT 493-2014 of SEQ ID NO: 147)








(AA 165-573 of SEQ ID NO: 148)


CD97
2166
80
+
CD55

CD97 antigen








(NT 493-2661 of SEQ ID NO: 151)








(AA 165-886 of SEQ ID NO: 152)


CCR5
1056
41
+
CC3

Chemokine (C-C motif) receptor 5








(NT 493-1855 of SEQ ID NO: 155)








(AA 165-520 of SEQ ID NO: 156)


RAI3
1071
40
+


Retinoic acid induced 3








(NT 493-1566 of SEQ ID NO: 159)








(AA 165-521 of SEQ ID NO: 160)


GPRC5B
1125
42
+


G protein-coupled receptor, family C,








grp 5, mbr B








(NT 493-1620 of SEQ ID NO: 163)








(AA 165-539 of SEQ ID NO: 164)


ETL
2013
76
−


EGF-TM7-latrophilin-related protein








(NT 493-2508 of SEQ ID NO: 167)








(AA 165-835 of SEQ ID NO: 168)


GABABR1
2445
92
−
GABA

Gamma-aminobutyric acid B








receptor, 1








(NT 493-2940 of SEQ ID NO: 171)








(AA 165-979 of SEQ ID NO: 172)


VIPR2
1254
48
−
VIP

Vasoactive intestinal peptide








receptor 2








(NT 493-1749 of SEQ ID NO: 175)








(AA 165-582 of SEQ ID NO: 176)





aProteins of human origin unless otherwise noted.


NT = nucleotide


AA = amino acid






Eighteen of twenty-two Mistic-L fusion constructs expressed in E. coli (as described in Example 1) were localized to the cell membrane (see Table 3). As known to those of ordinary skill in the art, expression levels were influenced by induction conditions and proteolytic susceptibility of the expressed protein (in particular, the cargo protein domain and the linker between the Mistic-L and cargo protein domains). Nonetheless, following thrombin-mediated cleavage from Mistic-L, yields of cargo proteins from the membrane fractions of recombinant bacteria often exceeded 1 mg per liter of culture (see for example, FIG. 5B). Such consistent, high-yield, heterologous production of structurally distinct eukaryotic IM proteins in E. coli is unprecedented (for review see Tate, FEBS Lett., 504:94-98, 2001).
For those Mistic-L fusion constructs that were not localized to the cell membrane (see Table 3), the expression of Mistic-L with no fusion partner domain (or very truncated snippets of the fusion partner domain) was observed. This result indicates that the cargo protein was likely separated from its Mistic-L domain, for example by endogenous proteases, before Mistic-L could traffic the cargo protein to the membrane. Accordingly, it is believed that such Mistic-L fusion constructs may be successfully produced in protease-deficient bacteria or under other circumstances that limit proteolysis of the Mistic-L fusion protein. Some eukaryotic proteins are thought to require post-translational modification to fold into their correct conformation. Such post-translational modifications do not naturally occur within a typical bacterial host cell and proteins requiring such modifications for proper folding are preferably avoided in those method embodiments involving bacterial expression.
FIG. 5B demonstrates the high-yield expression of several representative Mistic-L fusion constructs. Proteins in LDAO-solubilized membrane fractions were purified by Ni-NTA affinity chromatography. The indicated Mistic-L-fused protein (RAI3, CRFR2b, ActR IIb, BMPR II or aKv1.1) is shown by the open arrow in the respective lanes marked “−”. A portion of each affinity-purified fusion protein was digested with thrombin to yield an isolated cargo (i.e., fusion partner) protein. In FIG. 5B, isolated RAI3, CRFR2b, ActR IIb, BMPR II or aKv1.1 are shown by the solid arrows in the respective lanes marked “+”. N-terminal Edman degradation sequencing of at least 14 residues of the cargo protein after separation from Mistic-L was performed for select samples (including RAI3, BMPR II and aKv1.1) to confirm protein identity. Additional bands in the pre-digestion sample of aKv1.1 (FIG. 5B, aKv1.1, bracket) were determined to be truncated products containing fragments of the N-terminal domain (T1) of this channel. The region between T1 and the membrane-spanning domain of this channel is known to be flexible and proteolytically susceptible (Kobertz et al., Biochem., 39(34), 10347-10352, 2000).
Recombinant proteins produced by fusion to Mistic-L retained their native conformations and functions. For example, aKv1.1 was cleaved from a Mistic-L-aKv1.1 fusion protein using thrombin, solubilized in 3 mM LDAO, and purified by size exclusion chromatography on a Superose-6 column. As shown in FIG. 6, aKv1.1 eluted from the column (peak 1) was an expected 300 kDa tetrameric assembly (i.e., four 50 kDa monomers plus 100 kDa micelle). As shown in the FIG. 6 inset, aKv1.1 and Mistic-L present in a single sample (lane entitled “load”) were substantially separated by size exclusion chromatography (compare Kv1.1 monomer in lane 1 and Mistic-L in lane 2 with “load” lane). Thus, aKv1.1 (and other Mistic-L cargo proteins) were (and can be) purified to near homogeneity using a simple two-step purification as described herein.
Several of the cargo proteins described in this Example specifically bind known ligands. Selected ligand-binding cargo proteins were purified from their respective Mistic-L fusion proteins as described in this Example. As shown in Table 3, at least, Alk3, BMPRII, CRFR1, and CRFR2β retained their native ligand-binding affinity and specificity.
In a particular example, BMPRII, ALK3 and CRFR2β were incubated with 5×105 cpm 125I-BMP7 tracer in the presence or absence of 40 nM unlabeled BMP7 as competitor. BMPRII and ALK3 are type II and type I receptors of BMP7, respectively. CRFR2β is a G-protein coupled receptor with no known affinity for BMP7 and was included as a control. Samples were prepared in triplicate in 1.7 ml eppendorf tubes in a final volume of 1 ml binding buffer (150 mM NaCl, 50 mM Tris (pH 7.5), 0.1% BSA). Binding was performed for 150 minutes at room temperature. After an initial 60 minutes of binding, 20 μl of 50% Ni-NTA resin (Qiagen) was added to each sample and allowed to bind for the balance of the 90 minute incubation period. Complexes were precipitated by centrifugation and washed three times in 1 ml binding buffer. After the final wash, bound 125I-BMP7 tracer was quantified in a gamma counter. BMP7 was iodinated using a modified Cloramine-T procedure (see, e.g., Lawler et al., J. Neurosci. Meth., 49:141-53, 1993). As shown in FIG. 11, unlabeled BMP7 competitively inhibited 125I-BMP7 binding to BMPRII and ALK3, but had no significant effect on the amount of signal measured in the CRFR2β sample.
This and the foregoing examples demonstrate that Mistic-L functions to assist in the heterologous expression of a wide variety of IM proteins while displaying no specific affinity for any protein once proteolytically cleaved from its cargo. Structural and functional data from cleaved cargo proteins further demonstrate that Mistic-L fusions can be used to produce IM proteins fully folded and in their native conformations. Mistic-L lacks any lengthy spans of hydrophobic residues or any motif resembling a signal sequence, and yet is fully membrane integrated with a periplasmically exposed C-terminus. All of these unusual characteristics suggest that Mistic-L, in particular, and Mistic polypeptides, in general, may autonomously associate with bacterial membranes.
In one non-binding, proposed mechanism (see FIG. 15, Schematic I), a Mistic polypeptide (e.g., Mistic-L, M1, M2, M3 or M4) is produced in the cytoplasm as a soluble, hydrophilic polypeptide. It subsequently undergoes a conformational change, folding into a stable helical bundle that autonomously integrates into the membrane. Downstream cargo proteins are then positioned for facilitated, spontaneous folding and membrane integration, possibly in a co-translational manner. A Mistic polypeptide's ability to autonomously associate with the membrane may (but need not) account for its high efficiency in assisting the production and integration of downstream fusion proteins into the membrane. Another, non-binding proposed mechanism for the membrane-associating activity of a Mistic polypeptide is shown in FIG. 15, Schematic II. In this model, it is proposed that the Mistic polypeptide undergoes a conformation rearrangement upon association with a membrane, which rearrangement facilitates the insertion of a Mistic fusion partner (e.g., IM protein) in the membrane.
Example 5
Mistic-L Structural Mutations do not Affect Membrane-Targeting Function
This example describes the functional characterization of additional Mistic-L variants. Among other things, such variants specify particular mutations that do and do not affect the membrane-associating and protein-trafficking functions of Mistic-L.
To further demonstrate Mistic-L's direct role in assisting the production of recombinant IM proteins, mutations were introduced at three sites (residues 13, 36, and 75) within the core of the Mistic-L structure (see FIGS. 1A and 3D). In particular, W13A, Q36E, or M75A mutations were made using a QuickchangeTM kit (Stratagene) in conformance with manufacturer's instructions. The three-dimension structure of Mistic-L (see generally FIG. 3 and, specifically, FIG. 3D), shows these residue to be in the core of the Mistic-L protein. Mutation (particularly, non-conservative mutation) of a core residue may disrupt the structural integrity of the protein.
Wild type (Wt) and mutant Mistic-L proteins were expressed in E. coli (as described in Example 1) either alone or fused to aKv1.1. The N-terminal T1 domain of aKv1.1 was deleted from fusion protein expression constructs to minimize proteolytic degradation artifacts (as described in Example 4). As shown in FIG. 7, wild type Mistic-L and all three Mistic-L mutants localized to the cell membrane (lanes 5-8 from the left). No appreciable accumulation of the W13A or Q36E mutant in the cytoplasm was observed (lanes 1-3 from the left).
When expressed as a fusion protein with modified aKv1.1, the W13A and Q36E mutants retained the ability to traffic aKv1.1 to the cell membrane (FIG. 7, lanes 10 and 11 from the left). Mutation of Trp13 to Ala (W13A) reduced the amount of W13A-aKv1.1 fusion protein observed in the membrane fraction by 2-3 fold (compare FIG. 7, lanes 9 and 10 from the left). However, such a minor reduction indicates that Mistic-L residue 13 can be modified without substantial effect on protein function.
In comparison, the M75A-aKv1.1 fusion protein was not detected in the cell membrane fraction (right-most lane of FIG. 7). This result suggests that non-conservative mutations of the methionine at position 75 (such as to alanine) of wild-type Mistic-L sufficiently destabilized Mistic-L's structure such that it partitioned between the membrane and the cytoplasm, and failed to traffic fused aKv1.1 to the membrane.
This Example demonstrates that at least two core Mistic-L residues tolerate mutation without substantial adverse effects on protein function. However, non-conservative mutation of Met75 should be avoided to maintain Mistic-L's membrane-associating and protein-trafficking functions.
Example 6
Linker Length can be Varied to Optimize Mistic Fusion Protein Expression
This Example demonstrates that the number of amino acids between a Mistic polypeptide domain and a fusion partner domain in a Mistic fusion protein can be varied to optimize expression of the fusion protein.
As shown schematically in FIG. 8, wild-type Mistic-L was fused with a variable length linker (0, 5, 8, or 22 amino acids) to wild-type aKv1.1 (FIG. 8B) or to a truncated aKv1.1 (FIG. 8A), which has the N-terminal T1 domain removed. These Mistic-L fusion proteins were then expressed in E. coli as described in Example 1, purified by Ni-NTA affinity chromatography, and separated on SDS-PAGE gels.
As shown in FIG. 8C, a Mistic-L-wt aKv1.1 fusion protein was observed regardless of the length of the linker between the Mistic-L and wt aKv1.1 domains. Nonetheless, comparatively higher expression was observed for the Mistic-L-wt aKv1.1 fusion protein having a linker of 5 amino acids connecting Mistic-L to the aKv1.1 T1 domain. As further shown in FIG. 8C, Mistic-L and Mistic-L “+T1” side products were observed in each sample, which suggests that the domain linker and the amino acid sequence between the T1 domain and the TM pore-forming domain are protease sensitive.
Example 7
Exemplar Mistic Expression Vectors
This Example describes several representative expression vectors that are useful for the expression of Mistic fusion proteins (such as, Mistic-L-, M1-, M2-, M3-, and M4-IM protein fusions) in bacteria.
GatewayTM technology was selected for the construction of exemplary Mistic polypeptide expression vectors. As known to the ordinarily skilled artisan, GatewayTM-adapted vectors permit rapid and efficient transfer of DNA segments (such as IM protein coding sequences) between multiple different cloning vectors while maintaining orientation and reading frame of the transferred DNA segments (see, for example, Walthout et al., Meth. Enzymol., 328:575, 2000; and U.S. Pat. Nos. 6,720,140; 6,277,608; 6,270,969; 6,171,861; 6,143,557; and 5,888,732).
As shown in FIG. 9A, pMis2.1 and pMisT2.1 vectors can be used to express fusion proteins having an N-terminal Mistic polypeptide (e.g., Mistic-L, M1, M2, M3, or M4, or functional fragments or variants of any thereof) domain. pMis2.1 has a peptide tag (e.g., “6 His”) linked in frame to the C-terminus of the Mistic domain. pMisT2.1 has an exogenous helix domain cloned in-frame between the Mistic domain and the peptide tag; thus, the exogenous helix follows the C-terminus of the Mistic domain and precedes the N-terminus of a peptide tag. In each of these vectors, a protease-recognition sequence (e.g., “Thr” for thrombin recognition site) and a fusion partner domain (e.g., “IM protein”) are flanked by recombination sites (“attB1” and “attB2”), which permits the protease-recognition site and fusion partner domain (collectively, the “fusion partner cassette”) to be cloned in and out of the GatewayTM-based vector with ease. Given this vector configuration, any fusion partner cassette can be ready inserted into the vector and expressed with an N-terminal Mistic domain.
As shown in Example 4, many fusion partners are successfully expressed when linked to an N-terminal Mistic-L domain by a relatively short linker (e.g., 15-36 amino acids). Nonetheless, it is recognized that geometric restrictions may arise in connection with some IM fusion partners. The natural orientation of an IM fusion partner domain in the membrane may not correspond with the geometry permitted by the fusion protein. For example, the N-terminus of a fusion partner domain may not normally be located on the same side of the membrane as the C-terminus of the Mistic domain. pMisT2.1 was designed to relieve such constraints. The exogenous transmembrane helix (such as, a KvPae transmembrane helix, a S1 transmembrane domain from a Pseudomonas K+ channel, or a synthetic helix), which is inserted generally between the Mistic and fusion partner domains, traverses the membrane to permit a fusion partner domain to assume its natural orientation with respect to the membrane.
Another geometric consideration taken into account in vector design was the distance between the natural N-terminus of an IM fusion partner and the membrane. To accommodate this distance, pMis2. 1 and pMisT2. 1 were engineered to have up to 36 amino acids between the C-terminus of the Mistic domain or the C-terminus of the exogenous helix and the N-terminus of an IM fusion partner, respectively. As shown in FIG. 9A, the up to 36 amino acids can include sequences encoding other functions (such as, peptide tag or protease-recognition site).
FIG. 9B shows another representative Mistic fusion protein expression vector design, which is also based on GatewayTM technology. In this implementation, sequences encoding a variable-length linker, a protease-recognition site, and a fusion partner protein (e.g., “IM Protein”) are contained within the recombination elements (attB1 and attB2). As discussed above, the sequences flanked by the recombination elements (referred to as the fusion partner cassette) can be cloned in and out of the GatewayTM-based vector with particular ease. In the illustrated vector, the fusion partner cassette is flanked on the N-terminus by sequences encoding an N-tagged (e.g., His-tagged) Mistic domain followed by an optional exogenous helix (“TM helix”), and on the C-terminus by a second protease-recognition site (e.g., “TEV”) and a second peptide tag (e.g., “StrepTag” or “BioTag”). The second C-terminal peptide tag can be used to isolate full-length Mistic fusion proteins (or fusion partner domains) from truncated degradation products. Moreover, the C-terminal affinity tag can be subsequently (and optionally) removed using the second protease processing site.
The vector shown in FIG. 9B further illustrates a second promoter (“D”) driving the expression of another protein (“Protein 2”). In this optional configuration, the illustrated vector can be used to express membrane protein systems involving more than one unique protein chain. Di-cistronic vectors with dual Mistic domain fusions and single Mistic domain fusions are contemplated. In one specific example, the Kv channel, rKv4.2, will be expressed in association with an interacting IM protein, di-peptidyl peptidase (DPP) VI, or a cytoplasmic modulator, K+ channel interacting protein (KChIP).
Example 8
Mistic-L Stabilizes its Fusion Partners
This Example demonstrates that a representative Mistic polypeptide facilitates (for example, stabilizes) the expression of its fusion partner(s). Although not being bound by any one theory, it is believed that a Mistic polypeptide stabilizes its fusion partner by increasing its solubility and/or by preventing its aggregation.
FIG. 12 illustrates that Mistic-L can stabilize an IM protein fusion partner. The figure shows the gel filtration elution profile a Mistic-L-KvPae fusion protein and a KvPae fusion partner domain after thrombin removal of the Mistic-L domain by overnight digestion at room temperature. Gel filtration was performed using a Superdex 200TM column run at 1 ml per minute in a solution of 300 mM KCl, 50 mM Tris (pH 8.0), 3 mM LDAO, 1 mM DTT.
Mistic-L-KvPae and cleaved KvPae are represented by the leftmost and rightmost peaks, respectively, of the elution profile shown in FIG. 12. As indicated by the size of the Mistic-L-KvPae peak, a large amount of Mistic-L-KvPae is recovered in the absence of thrombin cleavage of the fusion partners. However, when the fusion partners were cleaved, more than 90% of the KvPae protein was lost, mostly due to protein aggregation (aggregates were removed from the sample by a pre-filter to avoid clogging the column) or precipitation (the sample became cloudy during the course of the thrombin digestion).
This Example indicates that Mistic polypeptides assist in maintaining a fusion partner (e.g., KvPae) in solution (for example, by increasing solubility), or preventing its aggregation (for example by sterically separating aggregation prone regions or domains of KvPae). This and other disclosed properties of Mistic polypeptides (such as Mistic-L, M1, M2, M3, and M4) permit the successful heterologous expression of fusion partner proteins (such as membrane proteins) that otherwise could not be expressed at all or in useful amounts.
Example 9
M1 is a Functional Fragment of Mistic-L
Sequence analysis revealed that the B. subtilis gene for Mistic-L may also have an internal, alternative translation start site that would yield an 84 amino acid protein. Thus, a truncated form of Mistic-L, which included amino acid residues 27 to 110, was produced. This Mistic-L variant, which lacks the N-terminus, was named “M1.” M1 was created by full vector PCR using oligos annealing directly upstream and downstream of, and directed away from, the 26 amino acids being deleted, followed by blunt ended ligation of the resulting PCR product.
M1 was fused to a variety of cargo proteins, including Alk3, BmpRII, or CRFR2β in a manner analogous to that described in Example 4 for the making of Mistic-L fusion proteins. As shown in Example 11, M1 retained the membrane-associating functions of Mistic-L, which proves M1 a functional fragment of Mistic-L and shows that the 26 N-terminal amino acids of Mistic-L are not necessary for Mistic polypeptides to associate with a membrane.
Example 10
Isolation of Several Mistic-L/M1 Orthologs
This Example demonstrates the cloning of several Mistic-L and Mistic M1 orthologs.
Genomic DNA, obtained from the Bacillus Genetic Stock Center (BGSC), from four Bacillus species (B. licheniformis, BGSCID 5A36; B. mojavensis, BGSCID 28A1; B. atrophaeus, BGSCID 11A1; and B. pumilus, BGSCID 8A3) was amplified using two “MisticSeeker” oligonucleotides:








(SEQ ID NO: 186)









ATGCTAATACGACTCACTATAGGGGCTCTTTACTTTAAATTGTGCCC;



and


 








(SEQ ID NO: 187)









ATGGCTAGTTATTGCTCAGCGGCCGACTGWNGANACNGTNABNABNGCCC



ACCADATNCC.






The MisticSeeker oligonucleotides were (are) complementary to conserved regions of the genes upstream (YugP) and downstream (YugO-b) of the Mistic-L gene in the Bacillus subtilis genome.

PCR was conducted for 30 cycles with one minute incubations between melting (94° C.), annealing (50° C.), and elongation (72° C.), temperatures using Vent DNA polymerase. The amplified product was sequenced using the same MisticSeeker oligos. For B. licheniformis (M2), B. mojavensis (M3), and B. atrophaeus (M4), 252 base pair open-reading-frames were found that translated to proteins homologous to the C-terminal 84 amino acids of the B. subtilis Mistic-L protein. In all cases, the Mistic homologue is located just upstream and partially overlapping a K+ channel gene (YugO-C). The identity conservation for these homologues varied from 93% to 62% (see FIG. 13).
The conservation pattern between the homologues was mapped to the Mistic-L structure (see FIG. 13). Non-conservative residues (numbered 1-9 in FIG. 13) mapped to the flexible loop regions of the protein. This pattern indicates that the overall structural fold of the protein, less the N-terminal helix, is common to Mistic polypeptides (e.g., Mistic-L (including M1), M2, M3 and M4). It further indicates that the flexible loop regions of a Mistic polypeptide can absorb non-conservative amino acid substitutions with no substantial adverse functional consequence.
Example 11
Proteins Fused to Other Mistic Polypeptide are Targeted to the Membrane
This Example demonstrates that M1, M2, M3, and M4 possess the same membrane-associating property as Mistic-L and are similarly capable of facilitating the association of a fusion partner protein with a membrane and further facilitating the isolation of relatively large amounts of otherwise difficult-to-isolate fusion partners (such as IM proteins).
Fusion constructs comprising M1, M2, M3, or M4 fused to ALK3, BMPRII, or CRFRIIβ were constructed in a manner similar to that described for Mistic-L fusion proteins in Example 4. The amino acid sequences and corresponding nucleic acid sequences for these fusion constructs are provided in SEQ ID NOs: 196-219 (with odd numbers being nucleic acid sequences and even numbers being amino acid sequences; see also Table 1) and amino acid sequences for these His-tagged M1-M4 fusion proteins were expressed in bacterial host cells, as described in Example 2. Briefly, freshly transformed colonies were cultured in TB and induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at an O.D. of 1.0. Growth was continued overnight at 10-18 ° C. Cells were harvested and resuspended in 50 mM Tris pH 8.0, 300 mM KCl, 10% glycerol, 10 mM imidazole with 1 mg/ml lysozyme. Cells were disrupted by sonication on ice and membranes were pelleted by high speed centrifugation. Membranes were solubilized by sonication in the above buffer with the addition of 20 mM LDAO. Insoluble material was removed by high speed centrifugation and the desired fusion protein was optionally purified from the resulting supernatant using Ni-NTA affinity chromatography (Qiagen). Membrane-associated proteins in the presence or absence of thrombin (which cleaved the cargo domain from the Mistic domain) were visualized on SDS gels.
As shown in FIG. 14, each of M1, M2, M3 and M4 fused to their respective cargoes (Alk3, BMPRII, and CRFR2β) were isolated in the absence of thrombin (lanes marked “−”). In the presence of thrombin (lanes marked “+”), the respective cargo proteins were released from the Mistic domains (see band at approximately 70 kD for Alk3 and approximately 55 kD for BMPRII, and bands at approximately 45 kD and 33 kD for CRFR2β). In all cases, the level of expression of M1, M2, M3 or M4 fusion proteins was largely comparable to that of Mistic-L fusion proteins.
While this invention has been described with an emphasis upon particular embodiments, it will be obvious to those of ordinary skill in the art that variations of the particular embodiments may be used and it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the claims following Table 4.







TABLE 4





Mistic-L Atomic Structure Coordinates
























ATOM
1
N
CYS
3
−14.858
−5.458
−2.728
1.00
0.00


ATOM
2
H
CYS
3
−14.461
−4.881
−3.442
1.00
0.00


ATOM
3
CA
CYS
3
−14.902
−4.856
−1.406
1.00
0.00


ATOM
4
HA
CYS
3
−14.452
−5.574
−0.720
1.00
0.00


ATOM
5
CB
CYS
3
−14.067
−3.575
−1.339
1.00
0.00


ATOM
6
2HB
CYS
3
−14.280
−2.948
−2.205
1.00
0.00


ATOM
7
QB
CYS
3
−14.280
−2.948
−2.205
1.00
0.00


ATOM
8
SG
CYS
3
−14.442
−2.664
0.202
1.00
0.00


ATOM
9
HG
CYS
3
−15.293
−1.803
−0.349
1.00
0.00


ATOM
10
C
CYS
3
−16.366
−4.601
−1.043
1.00
0.00


ATOM
11
O
CYS
3
−16.861
−5.123
−0.045
1.00
0.00


ATOM
12
N
THR
4
−17.018
−3.801
−1.873
1.00
0.00


ATOM
13
H
THR
4
−16.608
−3.381
−2.683
1.00
0.00


ATOM
14
CA
THR
4
−18.416
−3.472
−1.652
1.00
0.00


ATOM
15
HA
THR
4
−18.660
−3.691
−0.612
1.00
0.00


ATOM
16
CB
THR
4
−18.596
−1.974
−1.913
1.00
0.00


ATOM
17
HB
THR
4
−18.110
−1.682
−2.844
1.00
0.00


ATOM
18
QG2
THR
4
−20.416
−1.452
−1.906
1.00
0.00


ATOM
19
OG1
THR
4
−18.052
−1.352
−0.753
1.00
0.00


ATOM
20
1HG
THR
4
−17.357
−0.683
−1.019
1.00
0.00


ATOM
21
CG2
THR
4
−20.067
−1.553
−1.907
1.00
0.00


ATOM
22
1HG2
THR
4
−20.621
−2.174
−1.203
1.00
0.00


ATOM
23
2HG2
THR
4
−20.143
−0.508
−1.606
1.00
0.00


ATOM
24
3HG2
THR
4
−20.485
−1.674
−2.907
1.00
0.00


ATOM
25
C
THR
4
−19.316
−4.353
−2.520
1.00
0.00


ATOM
26
O
THR
4
−20.538
−4.325
−2.382
1.00
0.00


ATOM
27
N
PHE
5
−18.677
−5.114
−3.396
1.00
0.00


ATOM
28
H
PHE
5
−17.683
−5.131
−3.501
1.00
0.00


ATOM
29
CA
PHE
5
−19.404
−6.002
−4.286
1.00
0.00


ATOM
30
HA
PHE
5
−20.453
−5.709
−4.251
1.00
0.00


ATOM
31
CB
PHE
5
−18.782
−5.850
−5.675
1.00
0.00


ATOM
32
2HB
PHE
5
−18.522
−6.838
−6.057
1.00
0.00


ATOM
33
QB
PHE
5
−18.522
−6.838
−6.057
1.00
0.00


ATOM
34
QD
PHE
5
−19.768
−5.089
−6.780
1.00
0.00


ATOM
35
QE
PHE
5
−21.256
−3.941
−8.444
1.00
0.00


ATOM
36
QR
PHE
5
−20.806
−4.288
−7.941
1.00
0.00


ATOM
37
CG
PHE
5
−19.688
−5.151
−6.690
1.00
0.00


ATOM
38
CD1
PHE
5
−19.410
−3.881
−7.088
1.00
0.00


ATOM
39
1HD
PHE
5
−18.541
−3.361
−6.684
1.00
0.00


ATOM
40
CE1
PHE
5
−20.252
−3.231
−8.030
1.00
0.00


ATOM
41
1HE
PHE
5
−20.028
−2.213
−8.349
1.00
0.00


ATOM
42
CZ
PHE
5
−21.335
−3.879
−8.534
1.00
0.00


ATOM
43
HZ
PHE
5
−21.982
−3.381
−9.257
1.00
0.00


ATOM
44
CE2
PHE
5
−21.614
−5.149
−8.136
1.00
0.00


ATOM
45
2HE
PHE
5
−22.483
−5.670
−8.540
1.00
0.00


ATOM
46
CD2
PHE
5
−20.772
−5.799
−7.194
1.00
0.00


ATOM
47
2HD
PHE
5
−20.996
−6.818
−6.875
1.00
0.00


ATOM
48
C
PHE
5
−19.270
−7.460
−3.838
1.00
0.00


ATOM
49
O
PHE
5
−20.192
−8.016
−3.243
1.00
0.00


ATOM
50
N
PHE
6
−18.116
−8.035
−4.139
1.00
0.00


ATOM
51
H
PHE
6
−17.371
−7.574
−4.622
1.00
0.00


ATOM
52
CA
PHE
6
−17.849
−9.416
−3.773
1.00
0.00


ATOM
53
HA
PHE
6
−18.729
−9.787
−3.248
1.00
0.00


ATOM
54
CB
PHE
6
−17.559
−10.173
−5.070
1.00
0.00


ATOM
55
2HB
PHE
6
−16.702
−9.714
−5.564
1.00
0.00


ATOM
56
QB
PHE
6
−16.702
−9.714
−5.564
1.00
0.00


ATOM
57
QD
PHE
6
−17.252
−11.795
−4.853
1.00
0.00


ATOM
58
QE
PHE
6
−16.788
−14.240
−4.526
1.00
0.00


ATOM
59
QR
PHE
6
−16.928
−13.501
−4.625
1.00
0.00


ATOM
60
CG
PHE
6
−17.277
−11.664
−4.871
1.00
0.00


ATOM
61
CD1
PHE
6
−18.275
−12.573
−5.033
1.00
0.00


ATOM
62
1HD
PHE
6
−19.277
−12.239
−5.306
1.00
0.00


ATOM
63
CE1
PHE
6
−18.013
−13.955
−4.848
1.00
0.00


ATOM
64
1HE
PHE
6
−18.813
−14.685
−4.978
1.00
0.00


ATOM
65
CZ
PHE
6
−16.764
−14.371
−4.508
1.00
0.00


ATOM
66
HZ
PHE
6
−16.562
−15.433
−4.366
1.00
0.00


ATOM
67
CE2
PHE
6
−15.765
−13.463
−4.346
1.00
0.00


ATOM
68
2HE
PHE
6
−14.764
−13.795
−4.073
1.00
0.00


ATOM
69
CD2
PHE
6
−16.027
−12.079
−4.531
1.00
0.00


ATOM
70
2HD
PHE
6
−15.227
−11.351
−4.401
1.00
0.00


ATOM
71
C
PHE
6
−16.630
−9.514
−2.855
1.00
0.00


ATOM
72
O
PHE
6
−16.750
−9.918
−1.700
1.00
0.00


ATOM
73
N
GLU−
7
−15.484
−9.140
−3.404
1.00
0.00


ATOM
74
H
GLU−
7
−15.395
−8.814
−4.345
1.00
0.00


ATOM
75
CA
GLU−
7
−14.243
−9.182
−2.649
1.00
0.00


ATOM
76
HA
GLU−
7
−13.973
−10.237
−2.595
1.00
0.00


ATOM
77
CB
GLU−
7
−13.134
−8.417
−3.374
1.00
0.00


ATOM
78
2HB
GLU−
7
−12.239
−8.392
−2.753
1.00
0.00


ATOM
79
QB
GLU−
7
−12.239
−8.392
−2.753
1.00
0.00


ATOM
80
CG
GLU−
7
−12.809
−9.067
−4.721
1.00
0.00


ATOM
81
2HG
GLU−
7
−12.114
−8.436
−5.274
1.00
0.00


ATOM
82
QG
GLU−
7
−12.114
−8.436
−5.274
1.00
0.00


ATOM
83
CD
GLU−
7
−12.200
−10.457
−4.526
1.00
0.00


ATOM
84
OE1
GLU−
7
−12.995
−11.411
−4.389
1.00
0.00


ATOM
85
OE2
GLU−
7
−10.951
−10.533
−4.517
1.00
0.00


ATOM
86
C
GLU−
7
−14.460
−8.628
−1.240
1.00
0.00


ATOM
87
O
GLU−
7
−15.502
−8.039
−0.954
1.00
0.00


ATOM
88
N
LYS+
8
−13.461
−8.836
−0.396
1.00
0.00


ATOM
89
H
LYS+
8
−12.617
−9.317
−0.635
1.00
0.00


ATOM
90
CA
LYS+
8
−13.530
−8.365
0.977
1.00
0.00


ATOM
91
HA
LYS+
8
−14.464
−7.814
1.086
1.00
0.00


ATOM
92
CB
LYS+
8
−13.585
−9.547
1.947
1.00
0.00


ATOM
93
2HB
LYS+
8
−12.574
−9.883
2.175
1.00
0.00


ATOM
94
QB
LYS+
8
−12.574
−9.883
2.175
1.00
0.00


ATOM
95
CG
LYS+
8
−14.304
−9.161
3.242
1.00
0.00


ATOM
96
2HG
LYS+
8
−14.899
−8.263
3.077
1.00
0.00


ATOM
97
QG
LYS+
8
−14.899
−8.263
3.077
1.00
0.00


ATOM
98
CD
LYS+
8
−15.207
−10.296
3.728
1.00
0.00


ATOM
99
2HD
LYS+
8
−15.355
−10.212
4.804
1.00
0.00


ATOM
100
QD
LYS+
8
−15.355
−10.212
4.804
1.00
0.00


ATOM
101
CE
LYS+
8
−16.562
−10.260
3.017
1.00
0.00


ATOM
102
2HE
LYS+
8
−16.696
−11.169
2.431
1.00
0.00


ATOM
103
QE
LYS+
8
−16.696
−11.169
2.431
1.00
0.00


ATOM
104
NZ
LYS+
8
−17.660
−10.130
4.001
1.00
0.00


ATOM
105
1HZ
LYS+
8
−18.409
−10.744
3.749
1.00
0.00


ATOM
106
2HZ
LYS+
8
−17.323
−10.371
4.911
1.00
0.00


ATOM
107
3HZ
LYS+
8
−17.992
−9.186
4.007
1.00
0.00


ATOM
108
QZ
LYS+
8
−17.908
−10.100
4.222
1.00
0.00


ATOM
109
C
LYS+
8
−12.368
−7.407
1.241
1.00
0.00


ATOM
110
O
LYS+
8
−12.559
−6.193
1.298
1.00
0.00


ATOM
111
N
HIS+
9
−11.186
−7.988
1.398
1.00
0.00


ATOM
112
H
HIS+
9
−11.039
−8.976
1.351
1.00
0.00


ATOM
113
CA
HIS+
9
−9.993
−7.200
1.655
1.00
0.00


ATOM
114
HA
HIS+
9
−10.331
−6.231
2.022
1.00
0.00


ATOM
115
CB
HIS+
9
−9.143
−7.844
2.752
1.00
0.00


ATOM
116
2HB
HIS+
9
−9.503
−7.504
3.723
1.00
0.00


ATOM
117
QB
HIS+
9
−9.503
−7.504
3.723
1.00
0.00


ATOM
118
CG
HIS+
9
−9.153
−9.354
2.729
1.00
0.00


ATOM
119
ND1
HIS+
9
−9.771
−10.114
3.707
1.00
0.00


ATOM
120
CD2
HIS+
9
−8.615
−10.235
1.838
1.00
0.00


ATOM
121
1HD
HIS+
9
−10.260
−9.759
4.503
1.00
0.00


ATOM
122
CE1
HIS+
9
−9.605
−11.395
3.408
1.00
0.00


ATOM
123
NE2
HIS+
9
−8.888
−11.467
2.249
1.00
0.00


ATOM
124
2HD
HIS+
9
−8.056
−9.972
0.940
1.00
0.00


ATOM
125
1HE
HIS+
9
−9.976
−12.241
3.986
1.00
0.00


ATOM
126
C
HIS+
9
−9.221
−6.997
0.350
1.00
0.00


ATOM
127
O
HIS+
9
−8.445
−6.051
0.225
1.00
0.00


ATOM
128
N
HIS+
10
−9.462
−7.900
−0.590
1.00
0.00


ATOM
129
H
HIS+
10
−10.094
−8.666
−0.479
1.00
0.00


ATOM
130
CA
HIS+
10
−8.799
−7.832
−1.880
1.00
0.00


ATOM
131
HA
HIS+
10
−7.730
−7.921
−1.688
1.00
0.00


ATOM
132
CB
HIS+
10
−9.214
−9.006
−2.767
1.00
0.00


ATOM
133
2HB
HIS+
10
−8.555
−9.045
−3.635
1.00
0.00


ATOM
134
QB
HIS+
10
−8.555
−9.045
−3.635
1.00
0.00


ATOM
135
CG
HIS+
10
−9.181
−10.345
−2.069
1.00
0.00


ATOM
136
ND1
HIS+
10
−8.059
−10.822
−1.416
1.00
0.00


ATOM
137
CD2
HIS+
10
−10.145
−11.300
−1.928
1.00
0.00


ATOM
138
1HD
HIS+
10
−7.181
−10.349
−1.339
1.00
0.00


ATOM
139
CE1
HIS+
10
−8.344
−12.012
−0.908
1.00
0.00


ATOM
140
NE2
HIS+
10
−9.637
−12.307
−1.228
1.00
0.00


ATOM
141
2HD
HIS+
10
−11.158
−11.247
−2.324
1.00
0.00


ATOM
142
1HE
HIS+
10
−7.665
−12.645
−0.337
1.00
0.00


ATOM
143
C
HIS+
10
−9.075
−6.473
−2.528
1.00
0.00


ATOM
144
O
HIS+
10
−8.281
−5.992
−3.335
1.00
0.00


ATOM
145
N
ARG+
11
−10.205
−5.892
−2.150
1.00
0.00


ATOM
146
H
ARG+
11
−10.845
−6.289
−1.493
1.00
0.00


ATOM
147
CA
ARG+
11
−10.595
−4.599
−2.684
1.00
0.00


ATOM
148
HA
ARG+
11
−9.816
−4.346
−3.401
1.00
0.00


ATOM
149
CB
ARG+
11
−11.954
−4.680
−3.383
1.00
0.00


ATOM
150
2HB
ARG+
11
−12.477
−3.728
−3.282
1.00
0.00


ATOM
151
QB
ARG+
11
−12.477
−3.728
−3.282
1.00
0.00


ATOM
152
CG
ARG+
11
−11.789
−5.023
−4.864
1.00
0.00


ATOM
153
2HG
ARG+
11
−11.155
−5.903
−4.968
1.00
0.00


ATOM
154
QG
ARG+
11
−11.155
−5.903
−4.968
1.00
0.00


ATOM
155
CD
ARG+
11
−13.146
−5.283
−5.522
1.00
0.00


ATOM
156
2HD
ARG+
11
−13.740
−5.950
−4.897
1.00
0.00


ATOM
157
QD
ARG+
11
−13.740
−5.950
−4.897
1.00
0.00


ATOM
158
NE
ARG+
11
−13.864
−4.005
−5.723
1.00
0.00


ATOM
159
HE
ARG+
11
−13.500
−3.188
−5.277
1.00
0.00


ATOM
160
CZ
ARG+
11
−14.972
−3.875
−6.467
1.00
0.00


ATOM
161
NH1
ARG+
11
−15.555
−2.675
−6.594
1.00
0.00


ATOM
162
1HH1
ARG+
11
−16.382
−2.578
−7.148
1.00
0.00


ATOM
163
2HH1
ARG+
11
−15.166
−1.878
−6.133
1.00
0.00


ATOM
164
QH1
ARG+
11
−15.774
−2.228
−6.641
1.00
0.00


ATOM
165
NH2
ARG+
11
−15.494
−4.943
−7.084
1.00
0.00


ATOM
166
1HH2
ARG+
11
−16.319
−4.846
−7.638
1.00
0.00


ATOM
167
2HH2
ARG+
11
−15.057
−5.838
−6.989
1.00
0.00


ATOM
168
QH2
ARG+
11
−15.688
−5.342
−7.314
1.00
0.00


ATOM
169
C
ARG+
11
−10.669
−3.561
−1.562
1.00
0.00


ATOM
170
O
ARG+
11
−11.197
−2.468
−1.755
1.00
0.00


ATOM
171
N
LYS+
12
−10.131
−3.942
−0.411
1.00
0.00


ATOM
172
H
LYS+
12
−9.704
−4.833
−0.261
1.00
0.00


ATOM
173
CA
LYS+
12
−10.129
−3.058
0.742
1.00
0.00


ATOM
174
HA
LYS+
12
−10.930
−2.332
0.600
1.00
0.00


ATOM
175
CB
LYS+
12
−10.448
−3.841
2.017
1.00
0.00


ATOM
176
2HB
LYS+
12
−9.595
−4.459
2.293
1.00
0.00


ATOM
177
QB
LYS+
12
−9.595
−4.459
2.293
1.00
0.00


ATOM
178
CG
LYS+
12
−10.794
−2.894
3.169
1.00
0.00


ATOM
179
2HG
LYS+
12
−10.271
−1.947
3.035
1.00
0.00


ATOM
180
QG
LYS+
12
−10.271
−1.947
3.035
1.00
0.00


ATOM
181
CD
LYS+
12
−12.302
−2.646
3.241
1.00
0.00


ATOM
182
2HD
LYS+
12
−12.837
−3.557
2.976
1.00
0.00


ATOM
183
QD
LYS+
12
−12.837
−3.557
2.976
1.00
0.00


ATOM
184
CE
LYS+
12
−12.716
−2.194
4.643
1.00
0.00


ATOM
185
2HE
LYS+
12
−12.333
−1.192
4.837
1.00
0.00


ATOM
186
QE
LYS+
12
−12.333
−1.192
4.837
1.00
0.00


ATOM
187
NZ
LYS+
12
−14.191
−2.201
4.775
1.00
0.00


ATOM
188
1HZ
LYS+
12
−14.466
−1.536
5.469
1.00
0.00


ATOM
189
2HZ
LYS+
12
−14.605
−1.958
3.897
1.00
0.00


ATOM
190
3HZ
LYS+
12
−14.496
−3.113
5.047
1.00
0.00


ATOM
191
QZ
LYS+
12
−14.523
−2.202
4.805
1.00
0.00


ATOM
192
C
LYS+
12
−8.798
−2.306
0.800
1.00
0.00


ATOM
193
O
LYS+
12
−8.774
−1.093
1.003
1.00
0.00


ATOM
194
N
TRP
13
−7.722
−3.057
0.618
1.00
0.00


ATOM
195
H
TRP
13
−7.749
−4.043
0.453
1.00
0.00


ATOM
196
CA
TRP
13
−6.389
−2.477
0.647
1.00
0.00


ATOM
197
HA
TRP
13
−6.207
−2.112
1.657
1.00
0.00


ATOM
198
CB
TRP
13
−5.323
−3.535
0.356
1.00
0.00


ATOM
199
2HB
TRP
13
−5.118
−3.544
−0.715
1.00
0.00


ATOM
200
QB
TRP
13
−5.118
−3.544
−0.715
1.00
0.00


ATOM
201
CG
TRP
13
−4.011
−3.320
1.111
1.00
0.00


ATOM
202
CD1
TRP
13
−3.797
−2.561
2.194
1.00
0.00


ATOM
203
CD2
TRP
13
−2.730
−3.906
0.794
1.00
0.00


ATOM
204
CE3
TRP
13
−2.363
−4.786
−0.241
1.00
0.00


ATOM
205
CE2
TRP
13
−1.809
−3.458
1.717
1.00
0.00


ATOM
206
NE1
TRP
13
−2.477
−2.614
2.596
1.00
0.00


ATOM
207
HD
TRP
13
−4.568
−1.975
2.694
1.00
0.00


ATOM
208
3HE
TRP
13
−3.072
−5.153
−0.982
1.00
0.00


ATOM
209
CZ3
TRP
13
−1.013
−5.153
−0.243
1.00
0.00


ATOM
210
CZ2
TRP
13
−0.461
−3.835
1.701
1.00
0.00


ATOM
211
1HE
TRP
13
−2.041
−2.090
3.447
1.00
0.00


ATOM
212
3HZ
TRP
13
−0.674
−5.834
−1.025
1.00
0.00


ATOM
213
CH2
TRP
13
−0.072
−4.711
0.679
1.00
0.00


ATOM
214
2HZ
TRP
13
0.248
−3.467
2.443
1.00
0.00


ATOM
215
HH
TRP
13
0.963
−5.044
0.609
1.00
0.00


ATOM
216
C
TRP
13
−6.361
−1.311
−0.344
1.00
0.00


ATOM
217
O
TRP
13
−5.733
−0.286
−0.083
1.00
0.00


ATOM
218
N
ASP−
14
−7.047
−1.506
−1.459
1.00
0.00


ATOM
219
H
ASP−
14
−7.555
−2.343
−1.664
1.00
0.00


ATOM
220
CA
ASP−
14
−7.108
−0.484
−2.490
1.00
0.00


ATOM
221
HA
ASP−
14
−6.081
−0.140
−2.614
1.00
0.00


ATOM
222
CB
ASP−
14
−7.650
−1.056
−3.802
1.00
0.00


ATOM
223
2HB
ASP−
14
−7.294
−2.080
−3.909
1.00
0.00


ATOM
224
QB
ASP−
14
−7.294
−2.080
−3.909
1.00
0.00


ATOM
225
CG
ASP−
14
−9.175
−1.053
−3.926
1.00
0.00


ATOM
226
OD1
ASP−
14
−9.727
0.052
−4.115
1.00
0.00


ATOM
227
OD2
ASP−
14
−9.754
−2.157
−3.831
1.00
0.00


ATOM
228
C
ASP−
14
−8.049
0.635
−2.037
1.00
0.00


ATOM
229
O
ASP−
14
−7.917
1.776
−2.475
1.00
0.00


ATOM
230
N
ILE
15
−8.978
0.268
−1.166
1.00
0.00


ATOM
231
H
ILE
15
−9.078
−0.664
−0.815
1.00
0.00


ATOM
232
CA
ILE
15
−9.939
1.227
−0.650
1.00
0.00


ATOM
233
HA
ILE
15
−10.258
1.851
−1.484
1.00
0.00


ATOM
234
CB
ILE
15
−11.182
0.507
−0.123
1.00
0.00


ATOM
235
HB
ILE
15
−11.094
−0.551
−0.368
1.00
0.00


ATOM
236
QG2
ILE
15
−11.292
0.636
1.763
1.00
0.00


ATOM
237
CG2
ILE
15
−11.271
0.611
1.402
1.00
0.00


ATOM
238
1HG2
ILE
15
−12.198
0.152
1.744
1.00
0.00


ATOM
239
2HG2
ILE
15
−10.423
0.096
1.851
1.00
0.00


ATOM
240
3HG2
ILE
15
−11.255
1.660
1.696
1.00
0.00


ATOM
241
CG1
ILE
15
−12.448
1.026
−0.807
1.00
0.00


ATOM
242
2HG1
ILE
15
−12.532
2.103
−0.657
1.00
0.00


ATOM
243
QG1
ILE
15
−12.532
2.103
−0.657
1.00
0.00


ATOM
244
QD1
ILE
15
−12.424
0.640
−2.660
1.00
0.00


ATOM
245
CD1
ILE
15
−12.429
0.714
−2.304
1.00
0.00


ATOM
246
1HD1
ILE
15
−12.643
1.622
−2.867
1.00
0.00


ATOM
247
2HD1
ILE
15
−11.445
0.337
−2.585
1.00
0.00


ATOM
248
3HD1
ILE
15
−13.183
−0.040
−2.528
1.00
0.00


ATOM
249
C
ILE
15
−9.257
2.117
0.390
1.00
0.00


ATOM
250
O
ILE
15
−9.575
3.300
0.504
1.00
0.00


ATOM
251
N
LEU
16
−8.330
1.516
1.121
1.00
0.00


ATOM
252
H
LEU
16
−8.077
0.553
1.022
1.00
0.00


ATOM
253
CA
LEU
16
−7.600
2.240
2.148
1.00
0.00


ATOM
254
HA
LEU
16
−8.310
2.893
2.655
1.00
0.00


ATOM
255
CB
LEU
16
−7.047
1.273
3.196
1.00
0.00


ATOM
256
2HB
LEU
16
−6.402
0.553
2.693
1.00
0.00


ATOM
257
QB
LEU
16
−6.402
0.553
2.693
1.00
0.00


ATOM
258
CG
LEU
16
−6.259
1.906
4.344
1.00
0.00


ATOM
259
HG
LEU
16
−6.228
1.195
5.170
1.00
0.00


ATOM
260
QD1
LEU
16
−4.469
2.246
3.832
1.00
0.00


ATOM
261
QD2
LEU
16
−7.123
3.466
4.982
1.00
0.00


ATOM
262
CD1
LEU
16
−4.813
2.182
3.930
1.00
0.00


ATOM
263
1HD1
LEU
16
−4.135
1.745
4.665
1.00
0.00


ATOM
264
2HD1
LEU
16
−4.624
1.737
2.953
1.00
0.00


ATOM
265
3HD1
LEU
16
−4.648
3.257
3.879
1.00
0.00


ATOM
266
CD2
LEU
16
−6.958
3.166
4.859
1.00
0.00


ATOM
267
1HD2
LEU
16
−6.982
3.916
4.069
1.00
0.00


ATOM
268
2HD2
LEU
16
−7.976
2.921
5.158
1.00
0.00


ATOM
269
3HD2
LEU
16
−6.412
3.560
5.717
1.00
0.00


ATOM
270
QQD
LEU
16
−5.796
2.856
4.407
1.00
0.00


ATOM
271
C
LEU
16
−6.528
3.110
1.488
1.00
0.00


ATOM
272
O
LEU
16
−6.174
4.167
2.008
1.00
0.00


ATOM
273
N
LEU
17
−6.042
2.632
0.352
1.00
0.00


ATOM
274
H
LEU
17
−6.335
1.771
−0.065
1.00
0.00


ATOM
275
CA
LEU
17
−5.017
3.353
−0.385
1.00
0.00


ATOM
276
HA
LEU
17
−4.449
3.941
0.336
1.00
0.00


ATOM
277
CB
LEU
17
−4.041
2.375
−1.040
1.00
0.00


ATOM
278
2HB
LEU
17
−4.609
1.530
−1.430
1.00
0.00


ATOM
279
QB
LEU
17
−4.609
1.530
−1.430
1.00
0.00


ATOM
280
CG
LEU
17
−3.183
2.942
−2.174
1.00
0.00


ATOM
281
HG
LEU
17
−3.113
4.021
−2.043
1.00
0.00


ATOM
282
QD1
LEU
17
−1.422
2.252
−2.103
1.00
0.00


ATOM
283
QD2
LEU
17
−3.996
2.636
−3.857
1.00
0.00


ATOM
284
CD1
LEU
17
−1.760
2.385
−2.117
1.00
0.00


ATOM
285
1HD1
LEU
17
−1.167
2.976
−1.419
1.00
0.00


ATOM
286
2HD1
LEU
17
−1.788
1.348
−1.781
1.00
0.00


ATOM
287
3HD1
LEU
17
−1.310
2.433
−3.108
1.00
0.00


ATOM
288
CD2
LEU
17
−3.841
2.694
−3.535
1.00
0.00


ATOM
289
1HD2
LEU
17
−3.083
2.737
−4.317
1.00
0.00


ATOM
290
2HD2
LEU
17
−4.311
1.711
−3.537
1.00
0.00


ATOM
291
3HD2
LEU
17
−4.595
3.459
−3.717
1.00
0.00


ATOM
292
QQD
LEU
17
−2.709
2.444
−2.980
1.00
0.00


ATOM
293
C
LEU
17
−5.685
4.311
−1.373
1.00
0.00


ATOM
294
O
LEU
17
−5.043
5.226
−1.886
1.00
0.00


ATOM
295
N
GLU−
18
−6.965
4.067
−1.612
1.00
0.00


ATOM
296
H
GLU−
18
−7.480
3.320
−1.191
1.00
0.00


ATOM
297
CA
GLU−
18
−7.726
4.897
−2.530
1.00
0.00


ATOM
298
HA
GLU−
18
−6.985
5.381
−3.166
1.00
0.00


ATOM
299
CB
GLU−
18
−8.652
4.044
−3.400
1.00
0.00


ATOM
300
2HB
GLU−
18
−9.303
3.443
−2.765
1.00
0.00


ATOM
301
QB
GLU−
18
−9.303
3.443
−2.765
1.00
0.00


ATOM
302
CG
GLU−
18
−9.502
4.923
−4.321
1.00
0.00


ATOM
303
2HG
GLU−
18
−9.518
4.497
−5.324
1.00
0.00


ATOM
304
QG
GLU−
18
−9.518
4.497
−5.324
1.00
0.00


ATOM
305
CD
GLU−
18
−10.933
5.049
−3.792
1.00
0.00


ATOM
306
OE1
GLU−
18
−11.322
6.194
−3.475
1.00
0.00


ATOM
307
OE2
GLU−
18
−11.604
3.997
−3.716
1.00
0.00


ATOM
308
C
GLU−
18
−8.518
5.953
−1.758
1.00
0.00


ATOM
309
O
GLU−
18
−8.975
6.938
−2.336
1.00
0.00


ATOM
310
N
LYS+
19
−8.657
5.713
−0.463
1.00
0.00


ATOM
311
H
LYS+
19
−8.284
4.910
0.001
1.00
0.00


ATOM
312
CA
LYS+
19
−9.387
6.633
0.395
1.00
0.00


ATOM
313
HA
LYS+
19
−9.943
7.311
−0.251
1.00
0.00


ATOM
314
CB
LYS+
19
−10.410
5.876
1.244
1.00
0.00


ATOM
315
2HB
LYS+
19
−10.815
6.539
2.009
1.00
0.00


ATOM
316
QB
LYS+
19
−10.815
6.539
2.009
1.00
0.00


ATOM
317
CG
LYS+
19
−11.548
5.333
0.377
1.00
0.00


ATOM
318
2HG
LYS+
19
−11.161
4.574
−0.303
1.00
0.00


ATOM
319
QG
LYS+
19
−11.161
4.574
−0.303
1.00
0.00


ATOM
320
CD
LYS+
19
−12.657
4.732
1.243
1.00
0.00


ATOM
321
2HD
LYS+
19
−12.598
5.141
2.251
1.00
0.00


ATOM
322
QD
LYS+
19
−12.598
5.141
2.251
1.00
0.00


ATOM
323
CE
LYS+
19
−14.037
5.026
0.648
1.00
0.00


ATOM
324
2HE
LYS+
19
−14.024
4.833
−0.426
1.00
0.00


ATOM
325
QE
LYS+
19
−14.024
4.833
−0.426
1.00
0.00


ATOM
326
NZ
LYS+
19
−15.070
4.189
1.297
1.00
0.00


ATOM
327
1HZ
LYS+
19
−15.829
4.044
0.661
1.00
0.00


ATOM
328
2HZ
LYS+
19
−14.674
3.306
1.549
1.00
0.00


ATOM
329
3HZ
LYS+
19
−15.406
4.651
2.118
1.00
0.00


ATOM
330
QZ
LYS+
19
−15.303
4.000
1.443
1.00
0.00


ATOM
331
C
LYS+
19
−8.392
7.454
1.218
1.00
0.00


ATOM
332
O
LYS+
19
−8.620
8.634
1.475
1.00
0.00


ATOM
333
N
SER
20
−7.310
6.796
1.607
1.00
0.00


ATOM
334
H
SER
20
−7.132
5.835
1.394
1.00
0.00


ATOM
335
CA
SER
20
−6.280
7.450
2.395
1.00
0.00


ATOM
336
HA
SER
20
−6.796
7.854
3.267
1.00
0.00


ATOM
337
CB
SER
20
−5.216
6.449
2.852
1.00
0.00


ATOM
338
2HB
SER
20
−4.818
5.923
1.985
1.00
0.00


ATOM
339
QB
SER
20
−4.818
5.923
1.985
1.00
0.00


ATOM
340
OG
SER
20
−4.150
7.085
3.552
1.00
0.00


ATOM
341
HG
SER
20
−3.293
6.600
3.382
1.00
0.00


ATOM
342
C
SER
20
−5.635
8.576
1.583
1.00
0.00


ATOM
343
O
SER
20
−5.730
9.745
1.953
1.00
0.00


ATOM
344
N
THR
21
−4.994
8.183
0.493
1.00
0.00


ATOM
345
H
THR
21
−4.922
7.229
0.200
1.00
0.00


ATOM
346
CA
THR
21
−4.334
9.144
−0.375
1.00
0.00


ATOM
347
HA
THR
21
−4.555
10.146
−0.008
1.00
0.00


ATOM
348
CB
THR
21
−2.827
8.893
−0.298
1.00
0.00


ATOM
349
HB
THR
21
−2.269
9.797
−0.544
1.00
0.00


ATOM
350
QG2
THR
21
−2.292
8.203
1.382
1.00
0.00


ATOM
351
OG1
THR
21
−2.608
7.813
−1.201
1.00
0.00


ATOM
352
1HG
THR
21
−3.121
7.008
−0.904
1.00
0.00


ATOM
353
CG2
THR
21
−2.395
8.336
1.059
1.00
0.00


ATOM
354
1HG2
THR
21
−2.944
8.842
1.853
1.00
0.00


ATOM
355
2HG2
THR
21
−2.605
7.266
1.098
1.00
0.00


ATOM
356
3HG2
THR
21
−1.325
8.500
1.197
1.00
0.00


ATOM
357
C
THR
21
−4.894
9.052
−1.796
1.00
0.00


ATOM
358
O
THR
21
−4.146
9.138
−2.769
1.00
0.00


ATOM
359
N
GLY
22
−6.206
8.878
−1.870
1.00
0.00


ATOM
360
H
GLY
22
−6.807
8.809
−1.075
1.00
0.00


ATOM
361
CA
GLY
22
−6.874
8.773
−3.156
1.00
0.00


ATOM
362
1HA
GLY
22
−6.165
8.987
−3.957
1.00
0.00


ATOM
363
2HA
GLY
22
−7.226
7.753
−3.306
1.00
0.00


ATOM
364
QA
GLY
22
−6.695
8.370
−3.631
1.00
0.00


ATOM
365
C
GLY
22
−8.054
9.743
−3.241
1.00
0.00


ATOM
366
O
GLY
22
−8.724
9.822
−4.269
1.00
0.00


ATOM
367
N
VAL
23
−8.274
10.455
−2.146
1.00
0.00


ATOM
368
H
VAL
23
−7.724
10.384
−1.313
1.00
0.00


ATOM
369
CA
VAL
23
−9.362
11.416
−2.083
1.00
0.00


ATOM
370
HA
VAL
23
−9.794
11.489
−3.081
1.00
0.00


ATOM
371
CB
VAL
23
−10.450
10.913
−1.131
1.00
0.00


ATOM
372
HB
VAL
23
−11.159
11.726
−0.973
1.00
0.00


ATOM
373
QG1
VAL
23
−11.393
9.456
−1.889
1.00
0.00


ATOM
374
QG2
VAL
23
−9.716
10.446
0.551
1.00
0.00


ATOM
375
CG1
VAL
23
−11.212
9.736
−1.743
1.00
0.00


ATOM
376
1HG1
VAL
23
−10.951
9.643
−2.798
1.00
0.00


ATOM
377
2HG1
VAL
23
−10.944
8.818
−1.221
1.00
0.00


ATOM
378
3HG1
VAL
23
−12.284
9.908
−1.648
1.00
0.00


ATOM
379
CG2
VAL
23
−9.858
10.536
0.228
1.00
0.00


ATOM
380
1HG2
VAL
23
−8.785
10.371
0.124
1.00
0.00


ATOM
381
2HG2
VAL
23
−10.033
11.344
0.938
1.00
0.00


ATOM
382
3HG2
VAL
23
−10.331
9.623
0.591
1.00
0.00


ATOM
383
QQG
VAL
23
−10.555
9.951
−0.669
1.00
0.00


ATOM
384
C
VAL
23
−8.806
12.785
−1.685
1.00
0.00


ATOM
385
O
VAL
23
−9.177
13.802
−2.268
1.00
0.00


ATOM
386
N
MET
24
−7.926
12.766
−0.695
1.00
0.00


ATOM
387
H
MET
24
−7.629
11.933
−0.226
1.00
0.00


ATOM
388
CA
MET
24
−7.314
13.992
−0.213
1.00
0.00


ATOM
389
HA
MET
24
−6.440
13.679
0.359
1.00
0.00


ATOM
390
CB
MET
24
−6.917
14.868
−1.403
1.00
0.00


ATOM
391
2HB
MET
24
−7.806
15.332
−1.829
1.00
0.00


ATOM
392
QB
MET
24
−7.806
15.332
−1.829
1.00
0.00


ATOM
393
CG
MET
24
−5.922
15.949
−0.980
1.00
0.00


ATOM
394
2HG
MET
24
−4.907
15.641
−1.234
1.00
0.00


ATOM
395
QG
MET
24
−4.907
15.641
−1.234
1.00
0.00


ATOM
396
SD
MET
24
−6.311
17.490
−1.793
1.00
0.00


ATOM
397
QE
MET
24
−8.181
17.951
−0.840
1.00
0.00


ATOM
398
CE
MET
24
−7.865
17.873
−1.002
1.00
0.00


ATOM
399
1HE
MET
24
−8.561
17.048
−1.149
1.00
0.00


ATOM
400
2HE
MET
24
−7.701
18.025
0.066
1.00
0.00


ATOM
401
3HE
MET
24
−8.280
18.781
−1.438
1.00
0.00


ATOM
402
C
MET
24
−8.277
14.765
0.691
1.00
0.00


ATOM
403
O
MET
24
−8.234
15.993
0.743
1.00
0.00


ATOM
404
N
GLU−
25
−9.122
14.014
1.382
1.00
0.00


ATOM
405
H
GLU−
25
−9.150
13.016
1.334
1.00
0.00


ATOM
406
CA
GLU−
25
−10.094
14.613
2.280
1.00
0.00


ATOM
407
HA
GLU−
25
−9.511
15.026
3.104
1.00
0.00


ATOM
408
CB
GLU−
25
−10.855
15.746
1.589
1.00
0.00


ATOM
409
2HB
GLU−
25
−10.322
16.686
1.728
1.00
0.00


ATOM
410
QB
GLU−
25
−10.322
16.686
1.728
1.00
0.00


ATOM
411
CG
GLU−
25
−11.019
15.463
0.094
1.00
0.00


ATOM
412
2HG
GLU−
25
−10.867
14.402
−0.098
1.00
0.00


ATOM
413
QG
GLU−
25
−10.867
14.402
−0.098
1.00
0.00


ATOM
414
CD
GLU−
25
−12.409
15.880
−0.394
1.00
0.00


ATOM
415
OE1
GLU−
25
−12.522
17.031
−0.866
1.00
0.00


ATOM
416
OE2
GLU−
25
−13.326
15.038
−0.282
1.00
0.00


ATOM
417
C
GLU−
25
−11.056
13.548
2.809
1.00
0.00


ATOM
418
O
GLU−
25
−11.322
13.485
4.008
1.00
0.00


ATOM
419
N
ALA
26
−11.554
12.736
1.886
1.00
0.00


ATOM
420
H
ALA
26
−11.333
12.794
0.913
1.00
0.00


ATOM
421
CA
ALA
26
−12.481
11.676
2.245
1.00
0.00


ATOM
422
HA
ALA
26
−13.364
12.142
2.684
1.00
0.00


ATOM
423
QB
ALA
26
−13.000
10.738
0.684
1.00
0.00


ATOM
424
CB
ALA
26
−12.901
10.918
0.984
1.00
0.00


ATOM
425
1HB
ALA
26
−12.246
10.058
0.842
1.00
0.00


ATOM
426
2HB
ALA
26
−13.930
10.577
1.090
1.00
0.00


ATOM
427
3HB
ALA
26
−12.824
11.579
0.120
1.00
0.00


ATOM
428
C
ALA
26
−11.829
10.763
3.284
1.00
0.00


ATOM
429
O
ALA
26
−12.513
9.990
3.953
1.00
0.00


ATOM
430
N
MET
27
−10.514
10.882
3.387
1.00
0.00


ATOM
431
H
MET
27
−9.964
11.514
2.839
1.00
0.00


ATOM
432
CA
MET
27
−9.761
10.077
4.334
1.00
0.00


ATOM
433
HA
MET
27
−10.304
10.149
5.277
1.00
0.00


ATOM
434
CB
MET
27
−9.697
8.631
3.838
1.00
0.00


ATOM
435
2HB
MET
27
−8.664
8.363
3.620
1.00
0.00


ATOM
436
QB
MET
27
−8.664
8.363
3.620
1.00
0.00


ATOM
437
CG
MET
27
−10.270
7.668
4.879
1.00
0.00


ATOM
438
2HG
MET
27
−10.233
6.646
4.501
1.00
0.00


ATOM
439
QG
MET
27
−10.233
6.646
4.501
1.00
0.00


ATOM
440
SD
MET
27
−9.342
7.788
6.399
1.00
0.00


ATOM
441
QE
MET
27
−10.791
8.774
7.643
1.00
0.00


ATOM
442
CE
MET
27
−10.547
8.608
7.433
1.00
0.00


ATOM
443
1HE
MET
27
−10.541
9.676
7.219
1.00
0.00


ATOM
444
2HE
MET
27
−11.537
8.200
7.229
1.00
0.00


ATOM
445
3HE
MET
27
−10.296
8.445
8.480
1.00
0.00


ATOM
446
C
MET
27
−8.342
10.620
4.510
1.00
0.00


ATOM
447
O
MET
27
−7.439
9.889
4.913
1.00
0.00


ATOM
448
N
LYS+
28
−8.189
11.899
4.199
1.00
0.00


ATOM
449
H
LYS+
28
−8.928
12.487
3.872
1.00
0.00


ATOM
450
CA
LYS+
28
−6.895
12.549
4.318
1.00
0.00


ATOM
451
HA
LYS+
28
−6.201
11.821
4.739
1.00
0.00


ATOM
452
CB
LYS+
28
−6.361
12.934
2.937
1.00
0.00


ATOM
453
2HB
LYS+
28
−7.191
13.049
2.240
1.00
0.00


ATOM
454
QB
LYS+
28
−7.191
13.049
2.240
1.00
0.00


ATOM
455
CG
LYS+
28
−5.561
14.238
3.002
1.00
0.00


ATOM
456
2HG
LYS+
28
−4.947
14.337
2.108
1.00
0.00


ATOM
457
QG
LYS+
28
−4.947
14.337
2.108
1.00
0.00


ATOM
458
CD
LYS+
28
−6.491
15.446
3.127
1.00
0.00


ATOM
459
2HD
LYS+
28
−7.495
15.111
3.389
1.00
0.00


ATOM
460
QD
LYS+
28
−7.495
15.111
3.389
1.00
0.00


ATOM
461
CE
LYS+
28
−5.980
16.424
4.187
1.00
0.00


ATOM
462
2HE
LYS+
28
−5.031
16.071
4.590
1.00
0.00


ATOM
463
QE
LYS+
28
−5.031
16.071
4.590
1.00
0.00


ATOM
464
NZ
LYS+
28
−5.809
17.775
3.607
1.00
0.00


ATOM
465
1HZ
LYS+
28
−5.404
18.382
4.290
1.00
0.00


ATOM
466
2HZ
LYS+
28
−5.207
17.720
2.810
1.00
0.00


ATOM
467
3HZ
LYS+
28
−6.700
18.134
3.328
1.00
0.00


ATOM
468
QZ
LYS+
28
−5.770
18.079
3.476
1.00
0.00


ATOM
469
C
LYS+
28
−7.007
13.729
5.285
1.00
0.00


ATOM
470
O
LYS+
28
−6.000
14.322
5.667
1.00
0.00


ATOM
471
N
VAL
29
−8.243
14.034
5.654
1.00
0.00


ATOM
472
H
VAL
29
−9.057
13.547
5.339
1.00
0.00


ATOM
473
CA
VAL
29
−8.501
15.132
6.570
1.00
0.00


ATOM
474
HA
VAL
29
−7.537
15.538
6.876
1.00
0.00


ATOM
475
CB
VAL
29
−9.275
16.240
5.853
1.00
0.00


ATOM
476
HB
VAL
29
−8.856
16.349
4.853
1.00
0.00


ATOM
477
QG1
VAL
29
−11.103
15.782
5.670
1.00
0.00


ATOM
478
QG2
VAL
29
−9.077
17.895
6.751
1.00
0.00


ATOM
479
CG1
VAL
29
−10.752
15.870
5.706
1.00
0.00


ATOM
480
1HG1
VAL
29
−11.126
16.241
4.751
1.00
0.00


ATOM
481
2HG1
VAL
29
−10.861
14.785
5.741
1.00
0.00


ATOM
482
3HG1
VAL
29
−11.322
16.319
6.519
1.00
0.00


ATOM
483
CG2
VAL
29
−9.115
17.578
6.578
1.00
0.00


ATOM
484
1HG2
VAL
29
−9.103
17.408
7.655
1.00
0.00


ATOM
485
2HG2
VAL
29
−8.180
18.047
6.274
1.00
0.00


ATOM
486
3HG2
VAL
29
−9.950
18.232
6.324
1.00
0.00


ATOM
487
QQG
VAL
29
−10.090
16.839
6.211
1.00
0.00


ATOM
488
C
VAL
29
−9.227
14.600
7.807
1.00
0.00


ATOM
489
O
VAL
29
−10.295
15.093
8.165
1.00
0.00


ATOM
490
N
THR
30
−8.618
13.598
8.426
1.00
0.00


ATOM
491
H
THR
30
−7.750
13.202
8.128
1.00
0.00


ATOM
492
CA
THR
30
−9.194
12.993
9.615
1.00
0.00


ATOM
493
HA
THR
30
−10.204
13.379
9.743
1.00
0.00


ATOM
494
CB
THR
30
−9.249
11.480
9.396
1.00
0.00


ATOM
495
HB
THR
30
−8.637
11.188
8.543
1.00
0.00


ATOM
496
QG2
THR
30
−8.761
10.505
10.944
1.00
0.00


ATOM
497
OG1
THR
30
−10.637
11.203
9.235
1.00
0.00


ATOM
498
1HG
THR
30
−11.134
11.450
10.067
1.00
0.00


ATOM
499
CG2
THR
30
−8.854
10.692
10.646
1.00
0.00


ATOM
500
1HG2
THR
30
−7.809
10.890
10.886
1.00
0.00


ATOM
501
2HG2
THR
30
−9.482
10.999
11.483
1.00
0.00


ATOM
502
3HG2
THR
30
−8.990
9.626
10.463
1.00
0.00


ATOM
503
C
THR
30
−8.397
13.399
10.857
1.00
0.00


ATOM
504
O
THR
30
−8.735
13.001
11.972
1.00
0.00


ATOM
505
N
SER
31
−7.357
14.184
10.624
1.00
0.00


ATOM
506
H
SER
31
−7.089
14.503
9.715
1.00
0.00


ATOM
507
CA
SER
31
−6.510
14.647
11.711
1.00
0.00


ATOM
508
HA
SER
31
−6.890
15.636
11.970
1.00
0.00


ATOM
509
CB
SER
31
−6.619
13.724
12.926
1.00
0.00


ATOM
510
2HB
SER
31
−6.704
12.691
12.589
1.00
0.00


ATOM
511
QB
SER
31
−6.704
12.691
12.589
1.00
0.00


ATOM
512
OG
SER
31
−5.496
13.849
13.793
1.00
0.00


ATOM
513
HG
SER
31
−5.579
14.681
14.343
1.00
0.00


ATOM
514
C
SER
31
−5.057
14.737
11.240
1.00
0.00


ATOM
515
O
SER
31
−4.222
15.347
11.906
1.00
0.00


ATOM
516
N
GLU−
32
−4.799
14.120
10.096
1.00
0.00


ATOM
517
H
GLU−
32
−5.484
13.626
9.561
1.00
0.00


ATOM
518
CA
GLU−
32
−3.462
14.122
9.529
1.00
0.00


ATOM
519
HA
GLU−
32
−3.595
13.893
8.473
1.00
0.00


ATOM
520
CB
GLU−
32
−2.812
15.501
9.665
1.00
0.00


ATOM
521
2HB
GLU−
32
−2.072
15.481
10.465
1.00
0.00


ATOM
522
QB
GLU−
32
−2.072
15.481
10.465
1.00
0.00


ATOM
523
CG
GLU−
32
−2.142
15.922
8.356
1.00
0.00


ATOM
524
2HG
GLU−
32
−1.935
15.041
7.748
1.00
0.00


ATOM
525
QG
GLU−
32
−1.935
15.041
7.748
1.00
0.00


ATOM
526
CD
GLU−
32
−3.031
16.891
7.572
1.00
0.00


ATOM
527
OE1
GLU−
32
−3.837
16.388
6.759
1.00
0.00


ATOM
528
OE2
GLU−
32
−2.885
18.109
7.804
1.00
0.00


ATOM
529
C
GLU−
32
−2.603
13.043
10.191
1.00
0.00


ATOM
530
O
GLU−
32
−2.259
12.045
9.560
1.00
0.00


ATOM
531
N
GLU−
33
−2.281
13.280
11.455
1.00
0.00


ATOM
532
H
GLU−
33
−2.565
14.094
11.960
1.00
0.00


ATOM
533
CA
GLU−
33
−1.469
12.340
12.209
1.00
0.00


ATOM
534
HA
GLU−
33
−0.617
12.113
11.567
1.00
0.00


ATOM
535
CB
GLU−
33
−0.963
12.972
13.507
1.00
0.00


ATOM
536
2HB
GLU−
33
−0.966
12.228
14.303
1.00
0.00


ATOM
537
QB
GLU−
33
−0.966
12.228
14.303
1.00
0.00


ATOM
538
CG
GLU−
33
0.449
13.532
13.331
1.00
0.00


ATOM
539
2HG
GLU−
33
0.403
14.492
12.816
1.00
0.00


ATOM
540
QG
GLU−
33
0.403
14.492
12.816
1.00
0.00


ATOM
541
CD
GLU−
33
1.144
13.705
14.683
1.00
0.00


ATOM
542
OE1
GLU−
33
0.412
13.932
15.672
1.00
0.00


ATOM
543
OE2
GLU−
33
2.390
13.607
14.699
1.00
0.00


ATOM
544
C
GLU−
33
−2.260
11.063
12.492
1.00
0.00


ATOM
545
O
GLU−
33
−1.695
9.970
12.520
1.00
0.00


ATOM
546
N
LYS+
34
−3.558
11.241
12.697
1.00
0.00


ATOM
547
H
LYS+
34
−4.010
12.132
12.673
1.00
0.00


ATOM
548
CA
LYS+
34
−4.433
10.116
12.977
1.00
0.00


ATOM
549
HA
LYS+
34
−3.822
9.331
13.423
1.00
0.00


ATOM
550
CB
LYS+
34
−5.492
10.506
14.010
1.00
0.00


ATOM
551
2HB
LYS+
34
−6.443
10.039
13.755
1.00
0.00


ATOM
552
QB
LYS+
34
−6.443
10.039
13.755
1.00
0.00


ATOM
553
CG
LYS+
34
−5.069
10.078
15.417
1.00
0.00


ATOM
554
2HG
LYS+
34
−4.753
10.952
15.987
1.00
0.00


ATOM
555
QG
LYS+
34
−4.753
10.952
15.987
1.00
0.00


ATOM
556
CD
LYS+
34
−6.218
9.377
16.145
1.00
0.00


ATOM
557
2HD
LYS+
34
−6.400
8.403
15.691
1.00
0.00


ATOM
558
QD
LYS+
34
−6.400
8.403
15.691
1.00
0.00


ATOM
559
CE
LYS+
34
−5.898
9.201
17.631
1.00
0.00


ATOM
560
2HE
LYS+
34
−6.535
9.854
18.227
1.00
0.00


ATOM
561
QE
LYS+
34
−6.535
9.854
18.227
1.00
0.00


ATOM
562
NZ
LYS+
34
−6.101
7.793
18.042
1.00
0.00


ATOM
563
1HZ
LYS+
34
−5.213
7.347
18.151
1.00
0.00


ATOM
564
2HZ
LYS+
34
−6.597
7.766
18.909
1.00
0.00


ATOM
565
3HZ
LYS+
34
−6.630
7.314
17.341
1.00
0.00


ATOM
566
QZ
LYS+
34
−6.147
7.476
18.134
1.00
0.00


ATOM
567
C
LYS+
34
−5.018
9.590
11.665
1.00
0.00


ATOM
568
O
LYS+
34
−5.445
8.440
11.588
1.00
0.00


ATOM
569
N
GLU−
35
−5.019
10.460
10.665
1.00
0.00


ATOM
570
H
GLU−
35
−4.670
11.394
10.735
1.00
0.00


ATOM
571
CA
GLU−
35
−5.544
10.097
9.359
1.00
0.00


ATOM
572
HA
GLU−
35
−6.438
9.506
9.561
1.00
0.00


ATOM
573
CB
GLU−
35
−5.938
11.343
8.563
1.00
0.00


ATOM
574
2HB
GLU−
35
−5.140
12.084
8.621
1.00
0.00


ATOM
575
QB
GLU−
35
−5.140
12.084
8.621
1.00
0.00


ATOM
576
CG
GLU−
35
−6.209
10.993
7.098
1.00
0.00


ATOM
577
2HG
GLU−
35
−6.785
11.791
6.630
1.00
0.00


ATOM
578
QG
GLU−
35
−6.785
11.791
6.630
1.00
0.00


ATOM
579
CD
GLU−
35
−4.900
10.787
6.332
1.00
0.00


ATOM
580
OE1
GLU−
35
−3.870
11.306
6.817
1.00
0.00


ATOM
581
OE2
GLU−
35
−4.957
10.115
5.280
1.00
0.00


ATOM
582
C
GLU−
35
−4.524
9.255
8.592
1.00
0.00


ATOM
583
O
GLU−
35
−4.867
8.609
7.602
1.00
0.00


ATOM
584
N
GLN
36
−3.292
9.287
9.078
1.00
0.00


ATOM
585
H
GLN
36
−3.023
9.815
9.883
1.00
0.00


ATOM
586
CA
GLN
36
−2.221
8.534
8.449
1.00
0.00


ATOM
587
HA
GLN
36
−2.624
8.197
7.495
1.00
0.00


ATOM
588
CB
GLN
36
−1.002
9.423
8.192
1.00
0.00


ATOM
589
2HB
GLN
36
−0.286
9.311
9.006
1.00
0.00


ATOM
590
QB
GLN
36
−0.286
9.311
9.006
1.00
0.00


ATOM
591
CG
GLN
36
−0.333
9.066
6.864
1.00
0.00


ATOM
592
2HG
GLN
36
0.225
8.136
6.972
1.00
0.00


ATOM
593
QG
GLN
36
0.225
8.136
6.972
1.00
0.00


ATOM
594
CD
GLN
36
−1.371
8.919
5.749
1.00
0.00


ATOM
595
OE1
GLN
36
−1.469
7.898
5.088
1.00
0.00


ATOM
596
NE2
GLN
36
−2.139
9.991
5.579
1.00
0.00


ATOM
597
1HE2
GLN
36
−2.007
10.797
6.156
1.00
0.00


ATOM
598
2HE2
GLN
36
−2.848
9.992
4.874
1.00
0.00


ATOM
599
QE2
GLN
36
−2.428
10.394
5.515
1.00
0.00


ATOM
600
C
GLN
36
−1.845
7.326
9.311
1.00
0.00


ATOM
601
O
GLN
36
−1.531
6.258
8.786
1.00
0.00


ATOM
602
N
LEU
37
−1.888
7.535
10.618
1.00
0.00


ATOM
603
H
LEU
37
−2.145
8.406
11.036
1.00
0.00


ATOM
604
CA
LEU
37
−1.557
6.477
11.557
1.00
0.00


ATOM
605
HA
LEU
37
−0.727
5.912
11.132
1.00
0.00


ATOM
606
CB
LEU
37
−1.072
7.069
12.882
1.00
0.00


ATOM
607
2HB
LEU
37
−1.659
7.963
13.094
1.00
0.00


ATOM
608
QB
LEU
37
−1.659
7.963
13.094
1.00
0.00


ATOM
609
CG
LEU
37
−1.147
6.143
14.097
1.00
0.00


ATOM
610
HG
LEU
37
−2.178
5.805
14.207
1.00
0.00


ATOM
611
QD1
LEU
37
−0.078
4.601
13.843
1.00
0.00


ATOM
612
QD2
LEU
37
−0.690
7.068
15.685
1.00
0.00


ATOM
613
CD1
LEU
37
−0.283
4.897
13.891
1.00
0.00


ATOM
614
1HD1
LEU
37
0.665
5.021
14.415
1.00
0.00


ATOM
615
2HD1
LEU
37
−0.804
4.024
14.286
1.00
0.00


ATOM
616
3HD1
LEU
37
−0.095
4.756
12.828
1.00
0.00


ATOM
617
CD2
LEU
37
−0.778
6.891
15.381
1.00
0.00


ATOM
618
1HD2
LEU
37
−0.717
6.185
16.208
1.00
0.00


ATOM
619
2HD2
LEU
37
0.188
7.381
15.250
1.00
0.00


ATOM
620
3HD2
LEU
37
−1.540
7.640
15.596
1.00
0.00


ATOM
621
QQD
LEU
37
−0.384
5.835
14.764
1.00
0.00


ATOM
622
C
LEU
37
−2.756
5.539
11.705
1.00
0.00


ATOM
623
O
LEU
37
−2.644
4.338
11.460
1.00
0.00


ATOM
624
N
SER
38
−3.877
6.121
12.106
1.00
0.00


ATOM
625
H
SER
38
−3.960
7.097
12.304
1.00
0.00


ATOM
626
CA
SER
38
−5.096
5.351
12.290
1.00
0.00


ATOM
627
HA
SER
38
−4.878
4.654
13.099
1.00
0.00


ATOM
628
CB
SER
38
−6.261
6.255
12.695
1.00
0.00


ATOM
629
2HB
SER
38
−7.049
5.651
13.146
1.00
0.00


ATOM
630
QB
SER
38
−7.049
5.651
13.146
1.00
0.00


ATOM
631
OG
SER
38
−5.858
7.268
13.613
1.00
0.00


ATOM
632
HG
SER
38
−6.101
8.170
13.256
1.00
0.00


ATOM
633
C
SER
38
−5.435
4.593
11.005
1.00
0.00


ATOM
634
O
SER
38
−6.016
3.510
11.054
1.00
0.00


ATOM
635
N
THR
39
−5.056
5.191
9.885
1.00
0.00


ATOM
636
H
THR
39
−4.585
6.072
9.853
1.00
0.00


ATOM
637
CA
THR
39
−5.314
4.586
8.590
1.00
0.00


ATOM
638
HA
THR
39
−6.212
3.975
8.668
1.00
0.00


ATOM
639
CB
THR
39
−5.547
5.711
7.580
1.00
0.00


ATOM
640
HB
THR
39
−5.916
5.315
6.634
1.00
0.00


ATOM
641
QG2
THR
39
−6.701
7.055
8.246
1.00
0.00


ATOM
642
OG1
THR
39
−4.281
6.358
7.482
1.00
0.00


ATOM
643
1HG
THR
39
−4.140
6.955
8.272
1.00
0.00


ATOM
644
CG2
THR
39
−6.480
6.798
8.118
1.00
0.00


ATOM
645
1HG2
THR
39
−5.989
7.324
8.936
1.00
0.00


ATOM
646
2HG2
THR
39
−6.715
7.502
7.321
1.00
0.00


ATOM
647
3HG2
THR
39
−7.399
6.339
8.481
1.00
0.00


ATOM
648
C
THR
39
−4.167
3.649
8.203
1.00
0.00


ATOM
649
O
THR
39
−4.344
2.753
7.379
1.00
0.00


ATOM
650
N
ALA
40
−3.017
3.890
8.814
1.00
0.00


ATOM
651
H
ALA
40
−2.881
4.621
9.482
1.00
0.00


ATOM
652
CA
ALA
40
−1.841
3.079
8.544
1.00
0.00


ATOM
653
HA
ALA
40
−1.860
2.810
7.488
1.00
0.00


ATOM
654
QB
ALA
40
−0.281
4.095
8.887
1.00
0.00


ATOM
655
CB
ALA
40
−0.580
3.900
8.821
1.00
0.00


ATOM
656
1HB
ALA
40
−0.716
4.477
9.736
1.00
0.00


ATOM
657
2HB
ALA
40
0.271
3.229
8.937
1.00
0.00


ATOM
658
3HB
ALA
40
−0.398
4.578
7.987
1.00
0.00


ATOM
659
C
ALA
40
−1.903
1.803
9.384
1.00
0.00


ATOM
660
O
ALA
40
−1.138
0.868
9.156
1.00
0.00


ATOM
661
N
ILE
41
−2.822
1.805
10.339
1.00
0.00


ATOM
662
H
ILE
41
−3.441
2.570
10.518
1.00
0.00


ATOM
663
CA
ILE
41
−2.993
0.658
11.216
1.00
0.00


ATOM
664
HA
ILE
41
−2.317
−0.123
10.867
1.00
0.00


ATOM
665
CB
ILE
41
−2.578
1.010
12.646
1.00
0.00


ATOM
666
HB
ILE
41
−2.902
0.205
13.304
1.00
0.00


ATOM
667
QG2
ILE
41
−0.695
1.132
12.796
1.00
0.00


ATOM
668
CG2
ILE
41
−1.056
1.108
12.767
1.00
0.00


ATOM
669
1HG2
ILE
41
−0.697
0.344
13.456
1.00
0.00


ATOM
670
2HG2
ILE
41
−0.603
0.956
11.788
1.00
0.00


ATOM
671
3HG2
ILE
41
−0.784
2.094
13.144
1.00
0.00


ATOM
672
CG1
ILE
41
−3.274
2.288
13.120
1.00
0.00


ATOM
673
2HG1
ILE
41
−3.985
2.621
12.364
1.00
0.00


ATOM
674
QG1
ILE
41
−3.985
2.621
12.364
1.00
0.00


ATOM
675
QD1
ILE
41
−4.174
2.001
14.761
1.00
0.00


ATOM
676
CD1
ILE
41
−4.001
2.056
14.447
1.00
0.00


ATOM
677
1HD1
ILE
41
−4.806
2.784
14.552
1.00
0.00


ATOM
678
2HD1
ILE
41
−4.418
1.049
14.461
1.00
0.00


ATOM
679
3HD1
ILE
41
−3.298
2.170
15.271
1.00
0.00


ATOM
680
C
ILE
41
−4.430
0.145
11.101
1.00
0.00


ATOM
681
O
ILE
41
−4.678
−1.051
11.248
1.00
0.00


ATOM
682
N
ASP−
42
−5.337
1.074
10.841
1.00
0.00


ATOM
683
H
ASP−
42
−5.126
2.044
10.723
1.00
0.00


ATOM
684
CA
ASP−
42
−6.743
0.730
10.704
1.00
0.00


ATOM
685
HA
ASP−
42
−7.123
0.675
11.724
1.00
0.00


ATOM
686
CB
ASP−
42
−7.494
1.793
9.900
1.00
0.00


ATOM
687
2HB
ASP−
42
−8.109
1.294
9.152
1.00
0.00


ATOM
688
QB
ASP−
42
−8.109
1.294
9.152
1.00
0.00


ATOM
689
CG
ASP−
42
−8.388
2.718
10.730
1.00
0.00


ATOM
690
OD1
ASP−
42
−8.504
2.454
11.946
1.00
0.00


ATOM
691
OD2
ASP−
42
−8.934
3.668
10.128
1.00
0.00


ATOM
692
C
ASP−
42
−6.867
−0.601
9.959
1.00
0.00


ATOM
693
O
ASP−
42
−7.223
−1.618
10.554
1.00
0.00


ATOM
694
N
ARG+
43
−6.569
−0.552
8.670
1.00
0.00


ATOM
695
H
ARG+
43
−6.280
0.280
8.194
1.00
0.00


ATOM
696
CA
ARG+
43
−6.643
−1.740
7.839
1.00
0.00


ATOM
697
HA
ARG+
43
−7.145
−2.484
8.458
1.00
0.00


ATOM
698
CB
ARG+
43
−7.453
−1.476
6.568
1.00
0.00


ATOM
699
2HB
ARG+
43
−7.187
−2.204
5.803
1.00
0.00


ATOM
700
QB
ARG+
43
−7.187
−2.204
5.803
1.00
0.00


ATOM
701
CG
ARG+
43
−8.955
−1.549
6.851
1.00
0.00


ATOM
702
2HG
ARG+
43
−9.126
−1.578
7.926
1.00
0.00


ATOM
703
QG
ARG+
43
−9.126
−1.578
7.926
1.00
0.00


ATOM
704
CD
ARG+
43
−9.686
−0.349
6.245
1.00
0.00


ATOM
705
2HD
ARG+
43
−10.424
−0.691
5.519
1.00
0.00


ATOM
706
QD
ARG+
43
−10.424
−0.691
5.519
1.00
0.00


ATOM
707
NE
ARG+
43
−10.352
0.429
7.313
1.00
0.00


ATOM
708
HE
ARG+
43
−9.998
1.340
7.520
1.00
0.00


ATOM
709
CZ
ARG+
43
−11.401
−0.012
8.021
1.00
0.00


ATOM
710
NH1
ARG+
43
−11.942
0.763
8.972
1.00
0.00


ATOM
711
1HH1
ARG+
43
−12.724
0.434
9.500
1.00
0.00


ATOM
712
2HH1
ARG+
43
−11.562
1.670
9.153
1.00
0.00


ATOM
713
QH1
ARG+
43
−12.143
1.052
9.327
1.00
0.00


ATOM
714
NH2
ARG+
43
−11.910
−1.228
7.778
1.00
0.00


ATOM
715
1HH2
ARG+
43
−12.693
−1.557
8.307
1.00
0.00


ATOM
716
2HH2
ARG+
43
−11.507
−1.806
7.069
1.00
0.00


ATOM
717
QH2
ARG+
43
−12.100
−1.682
7.688
1.00
0.00


ATOM
718
C
ARG+
43
−5.237
−2.207
7.451
1.00
0.00


ATOM
719
O
ARG+
43
−5.004
−3.400
7.267
1.00
0.00


ATOM
720
N
MET
44
−4.339
−1.240
7.338
1.00
0.00


ATOM
721
H
MET
44
−4.537
−0.271
7.489
1.00
0.00


ATOM
722
CA
MET
44
−2.963
−1.536
6.975
1.00
0.00


ATOM
723
HA
MET
44
−3.014
−2.013
5.997
1.00
0.00


ATOM
724
CB
MET
44
−2.159
−0.234
6.919
1.00
0.00


ATOM
725
2HB
MET
44
−1.148
−0.412
7.286
1.00
0.00


ATOM
726
QB
MET
44
−1.148
−0.412
7.286
1.00
0.00


ATOM
727
CG
MET
44
−2.101
0.312
5.492
1.00
0.00


ATOM
728
2HG
MET
44
−2.040
1.400
5.513
1.00
0.00


ATOM
729
QG
MET
44
−2.040
1.400
5.513
1.00
0.00


ATOM
730
SD
MET
44
−0.684
−0.359
4.639
1.00
0.00


ATOM
731
QE
MET
44
−1.094
0.321
2.642
1.00
0.00


ATOM
732
CE
MET
44
−1.024
0.206
2.980
1.00
0.00


ATOM
733
1HE
MET
44
−0.153
0.732
2.590
1.00
0.00


ATOM
734
2HE
MET
44
−1.248
−0.649
2.343
1.00
0.00


ATOM
735
3HE
MET
44
−1.880
0.881
2.994
1.00
0.00


ATOM
736
C
MET
44
−2.323
−2.489
7.986
1.00
0.00


ATOM
737
O
MET
44
−1.369
−3.195
7.661
1.00
0.00


ATOM
738
N
ASN
45
−2.873
−2.478
9.191
1.00
0.00


ATOM
739
H
ASN
45
−3.648
−1.901
9.447
1.00
0.00


ATOM
740
CA
ASN
45
−2.367
−3.334
10.252
1.00
0.00


ATOM
741
HA
ASN
45
−1.339
−3.008
10.417
1.00
0.00


ATOM
742
CB
ASN
45
−3.196
−3.177
11.529
1.00
0.00


ATOM
743
2HB
ASN
45
−4.257
−3.179
11.280
1.00
0.00


ATOM
744
QB
ASN
45
−4.257
−3.179
11.280
1.00
0.00


ATOM
745
CG
ASN
45
−2.898
−4.306
12.517
1.00
0.00


ATOM
746
OD1
ASN
45
−1.785
−4.476
12.988
1.00
0.00


ATOM
747
ND2
ASN
45
−3.951
−5.066
12.805
1.00
0.00


ATOM
748
1HD2
ASN
45
−4.837
−4.872
12.384
1.00
0.00


ATOM
749
2HD2
ASN
45
−3.857
−5.830
13.443
1.00
0.00


ATOM
750
QD2
ASN
45
−4.347
−5.351
12.913
1.00
0.00


ATOM
751
C
ASN
45
−2.454
−4.795
9.808
1.00
0.00


ATOM
752
O
ASN
45
−1.497
−5.340
9.261
1.00
0.00


ATOM
753
N
GLU−
46
−3.612
−5.389
10.059
1.00
0.00


ATOM
754
H
GLU−
46
−4.385
−4.939
10.505
1.00
0.00


ATOM
755
CA
GLU−
46
−3.837
−6.777
9.692
1.00
0.00


ATOM
756
HA
GLU−
46
−2.883
−7.279
9.860
1.00
0.00


ATOM
757
CB
GLU−
46
−4.902
−7.415
10.586
1.00
0.00


ATOM
758
2HB
GLU−
46
−5.892
−7.084
10.270
1.00
0.00


ATOM
759
QB
GLU−
46
−5.892
−7.084
10.270
1.00
0.00


ATOM
760
CG
GLU−
46
−4.827
−8.942
10.525
1.00
0.00


ATOM
761
2HG
GLU−
46
−4.205
−9.245
9.682
1.00
0.00


ATOM
762
QG
GLU−
46
−4.205
−9.245
9.682
1.00
0.00


ATOM
763
CD
GLU−
46
−4.250
−9.514
11.822
1.00
0.00


ATOM
764
OE1
GLU−
46
−3.153
−10.109
11.742
1.00
0.00


ATOM
765
OE2
GLU−
46
−4.920
−9.344
12.864
1.00
0.00


ATOM
766
C
GLU−
46
−4.225
−6.879
8.217
1.00
0.00


ATOM
767
O
GLU−
46
−4.044
−7.925
7.594
1.00
0.00


ATOM
768
N
GLY
47
−4.752
−5.779
7.698
1.00
0.00


ATOM
769
H
GLY
47
−4.896
−4.933
8.211
1.00
0.00


ATOM
770
CA
GLY
47
−5.168
−5.732
6.307
1.00
0.00


ATOM
771
1HA
GLY
47
−5.875
−6.538
6.108
1.00
0.00


ATOM
772
2HA
GLY
47
−5.690
−4.795
6.111
1.00
0.00


ATOM
773
QA
GLY
47
−5.782
−5.666
6.110
1.00
0.00


ATOM
774
C
GLY
47
−3.965
−5.857
5.370
1.00
0.00


ATOM
775
O
GLY
47
−4.030
−6.556
4.360
1.00
0.00


ATOM
776
N
LEU
48
−2.894
−5.169
5.740
1.00
0.00


ATOM
777
H
LEU
48
−2.850
−4.602
6.562
1.00
0.00


ATOM
778
CA
LEU
48
−1.678
−5.194
4.945
1.00
0.00


ATOM
779
HA
LEU
48
−1.973
−5.150
3.895
1.00
0.00


ATOM
780
CB
LEU
48
−0.828
−3.954
5.227
1.00
0.00


ATOM
781
2HB
LEU
48
−1.468
−3.193
5.673
1.00
0.00


ATOM
782
QB
LEU
48
−1.468
−3.193
5.673
1.00
0.00


ATOM
783
CG
LEU
48
0.380
−4.163
6.142
1.00
0.00


ATOM
784
HG
LEU
48
0.049
−4.709
7.026
1.00
0.00


ATOM
785
QD1
LEU
48
1.699
−5.223
5.292
1.00
0.00


ATOM
786
QD2
LEU
48
1.077
−2.506
6.737
1.00
0.00


ATOM
787
CD1
LEU
48
1.446
−5.019
5.455
1.00
0.00


ATOM
788
1HD1
LEU
48
1.050
−5.409
4.517
1.00
0.00


ATOM
789
2HD1
LEU
48
2.327
−4.410
5.253
1.00
0.00


ATOM
790
3HD1
LEU
48
1.721
−5.850
6.106
1.00
0.00


ATOM
791
CD2
LEU
48
0.943
−2.824
6.622
1.00
0.00


ATOM
792
1HD2
LEU
48
1.262
−2.916
7.661
1.00
0.00


ATOM
793
2HD2
LEU
48
1.796
−2.546
6.004
1.00
0.00


ATOM
794
3HD2
LEU
48
0.172
−2.058
6.545
1.00
0.00


ATOM
795
QQD
LEU
48
1.388
−3.865
6.014
1.00
0.00


ATOM
796
C
LEU
48
−0.942
−6.513
5.188
1.00
0.00


ATOM
797
O
LEU
48
−0.572
−7.205
4.241
1.00
0.00


ATOM
798
N
ASP−
49
−0.753
−6.821
6.463
1.00
0.00


ATOM
799
H
ASP−
49
−1.057
−6.252
7.227
1.00
0.00


ATOM
800
CA
ASP−
49
−0.068
−8.045
6.843
1.00
0.00


ATOM
801
HA
ASP−
49
0.940
−7.946
6.440
1.00
0.00


ATOM
802
CB
ASP−
49
−0.031
−8.206
8.364
1.00
0.00


ATOM
803
2HB
ASP−
49
−0.820
−8.898
8.660
1.00
0.00


ATOM
804
QB
ASP−
49
−0.820
−8.898
8.660
1.00
0.00


ATOM
805
CG
ASP−
49
1.300
−8.709
8.928
1.00
0.00


ATOM
806
OD1
ASP−
49
2.214
−8.937
8.108
1.00
0.00


ATOM
807
OD2
ASP−
49
1.371
−8.853
10.168
1.00
0.00


ATOM
808
C
ASP−
49
−0.816
−9.246
6.258
1.00
0.00


ATOM
809
O
ASP−
49
−0.200
−10.246
5.891
1.00
0.00


ATOM
810
N
ALA
50
−2.131
−9.106
6.189
1.00
0.00


ATOM
811
H
ALA
50
−2.623
−8.290
6.489
1.00
0.00


ATOM
812
CA
ALA
50
−2.969
−10.166
5.655
1.00
0.00


ATOM
813
HA
ALA
50
−2.554
−11.118
5.990
1.00
0.00


ATOM
814
QB
ALA
50
−4.724
−9.991
6.343
1.00
0.00


ATOM
815
CB
ALA
50
−4.388
−10.025
6.211
1.00
0.00


ATOM
816
1HB
ALA
50
−5.008
−10.838
5.835
1.00
0.00


ATOM
817
2HB
ALA
50
−4.357
−10.065
7.300
1.00
0.00


ATOM
818
3HB
ALA
50
−4.808
−9.071
5.895
1.00
0.00


ATOM
819
C
ALA
50
−2.934
−10.118
4.127
1.00
0.00


ATOM
820
O
ALA
50
−2.354
−10.994
3.487
1.00
0.00


ATOM
821
N
PHE
51
−3.561
−9.084
3.585
1.00
0.00


ATOM
822
H
PHE
51
−4.030
−8.376
4.112
1.00
0.00


ATOM
823
CA
PHE
51
−3.609
−8.910
2.143
1.00
0.00


ATOM
824
HA
PHE
51
−4.331
−9.628
1.754
1.00
0.00


ATOM
825
CB
PHE
51
−4.005
−7.455
1.881
1.00
0.00


ATOM
826
2HB
PHE
51
−3.174
−6.807
2.160
1.00
0.00


ATOM
827
QB
PHE
51
−3.174
−6.807
2.160
1.00
0.00


ATOM
828
QD
PHE
51
−4.428
−7.142
0.302
1.00
0.00


ATOM
829
QE
PHE
51
−5.065
−6.670
−2.080
1.00
0.00


ATOM
830
QR
PHE
51
−4.873
−6.812
−1.360
1.00
0.00


ATOM
831
CG
PHE
51
−4.394
−7.167
0.430
1.00
0.00


ATOM
832
CD1
PHE
51
−3.493
−7.372
−0.568
1.00
0.00


ATOM
833
1HD
PHE
51
−2.495
−7.741
−0.334
1.00
0.00


ATOM
834
CE1
PHE
51
−3.853
−7.105
−1.916
1.00
0.00


ATOM
835
1HE
PHE
51
−3.132
−7.269
−2.715
1.00
0.00


ATOM
836
CZ
PHE
51
−5.099
−6.644
−2.208
1.00
0.00


ATOM
837
HZ
PHE
51
−5.376
−6.439
−3.242
1.00
0.00


ATOM
838
CE2
PHE
51
−6.000
−6.439
−1.209
1.00
0.00


ATOM
839
2HE
PHE
51
−6.999
−6.070
−1.444
1.00
0.00


ATOM
840
CD2
PHE
51
−5.640
−6.707
0.138
1.00
0.00


ATOM
841
2HD
PHE
51
−6.362
−6.543
0.937
1.00
0.00


ATOM
842
C
PHE
51
−2.241
−9.174
1.513
1.00
0.00


ATOM
843
O
PHE
51
−2.152
−9.775
0.443
1.00
0.00


ATOM
844
N
ILE
52
−1.208
−8.713
2.203
1.00
0.00


ATOM
845
H
ILE
52
−1.289
−8.226
3.072
1.00
0.00


ATOM
846
CA
ILE
52
0.152
−8.892
1.725
1.00
0.00


ATOM
847
HA
ILE
52
0.190
−8.518
0.702
1.00
0.00


ATOM
848
CB
ILE
52
1.126
−8.050
2.551
1.00
0.00


ATOM
849
HB
ILE
52
0.658
−7.089
2.756
1.00
0.00


ATOM
850
QG2
ILE
52
1.480
−8.868
4.222
1.00
0.00


ATOM
851
CG2
ILE
52
1.412
−8.711
3.902
1.00
0.00


ATOM
852
1HG2
ILE
52
0.487
−9.118
4.310
1.00
0.00


ATOM
853
2HG2
ILE
52
2.134
−9.516
3.767
1.00
0.00


ATOM
854
3HG2
ILE
52
1.818
−7.970
4.590
1.00
0.00


ATOM
855
CG1
ILE
52
2.410
−7.770
1.768
1.00
0.00


ATOM
856
2HG1
ILE
52
2.926
−6.914
2.202
1.00
0.00


ATOM
857
QG1
ILE
52
2.926
−6.914
2.202
1.00
0.00


ATOM
858
QD1
ILE
52
3.555
−9.278
1.779
1.00
0.00


ATOM
859
CD1
ILE
52
3.336
−8.989
1.776
1.00
0.00


ATOM
860
1HD1
ILE
52
3.981
−8.961
0.899
1.00
0.00


ATOM
861
2HD1
ILE
52
3.947
−8.974
2.679
1.00
0.00


ATOM
862
3HD1
ILE
52
2.737
−9.900
1.759
1.00
0.00


ATOM
863
C
ILE
52
0.489
−10.384
1.707
1.00
0.00


ATOM
864
O
ILE
52
1.066
−10.883
0.742
1.00
0.00


ATOM
865
N
GLN
53
0.116
−11.055
2.787
1.00
0.00


ATOM
866
H
GLN
53
−0.353
−10.642
3.568
1.00
0.00


ATOM
867
CA
GLN
53
0.371
−12.481
2.908
1.00
0.00


ATOM
868
HA
GLN
53
1.421
−12.610
2.644
1.00
0.00


ATOM
869
CB
GLN
53
0.154
−12.957
4.346
1.00
0.00


ATOM
870
2HB
GLN
53
0.308
−12.126
5.035
1.00
0.00


ATOM
871
QB
GLN
53
0.308
−12.126
5.035
1.00
0.00


ATOM
872
CG
GLN
53
−1.255
−13.524
4.529
1.00
0.00


ATOM
873
2HG
GLN
53
−1.928
−13.077
3.796
1.00
0.00


ATOM
874
QG
GLN
53
−1.928
−13.077
3.796
1.00
0.00


ATOM
875
CD
GLN
53
−1.258
−15.046
4.372
1.00
0.00


ATOM
876
OE1
GLN
53
−0.234
−15.676
4.163
1.00
0.00


ATOM
877
NE2
GLN
53
−2.463
−15.598
4.483
1.00
0.00


ATOM
878
1HE2
GLN
53
−3.263
−15.024
4.654
1.00
0.00


ATOM
879
2HE2
GLN
53
−2.568
−16.589
4.395
1.00
0.00


ATOM
880
QE2
GLN
53
−2.915
−15.806
4.525
1.00
0.00


ATOM
881
C
GLN
53
−0.510
−13.263
1.932
1.00
0.00


ATOM
882
O
GLN
53
−0.330
−14.467
1.756
1.00
0.00


ATOM
883
N
LEU
54
−1.445
−12.547
1.324
1.00
0.00


ATOM
884
H
LEU
54
−1.584
−11.569
1.473
1.00
0.00


ATOM
885
CA
LEU
54
−2.354
−13.159
0.371
1.00
0.00


ATOM
886
HA
LEU
54
−2.228
−14.239
0.446
1.00
0.00


ATOM
887
CB
LEU
54
−3.806
−12.848
0.741
1.00
0.00


ATOM
888
2HB
LEU
54
−4.367
−12.685
−0.180
1.00
0.00


ATOM
889
QB
LEU
54
−4.367
−12.685
−0.180
1.00
0.00


ATOM
890
CG
LEU
54
−4.532
−13.913
1.565
1.00
0.00


ATOM
891
HG
LEU
54
−3.796
−14.644
1.902
1.00
0.00


ATOM
892
QD1
LEU
54
−5.316
−13.155
3.112
1.00
0.00


ATOM
893
QD2
LEU
54
−5.799
−14.842
0.509
1.00
0.00


ATOM
894
CD1
LEU
54
−5.166
−13.301
2.816
1.00
0.00


ATOM
895
1HD1
LEU
54
−5.284
−14.071
3.577
1.00
0.00


ATOM
896
2HD1
LEU
54
−4.523
−12.508
3.198
1.00
0.00


ATOM
897
3HD1
LEU
54
−6.141
−12.887
2.563
1.00
0.00


ATOM
898
CD2
LEU
54
−5.556
−14.664
0.712
1.00
0.00


ATOM
899
1HD2
LEU
54
−5.088
−15.547
0.277
1.00
0.00


ATOM
900
2HD2
LEU
54
−6.396
−14.968
1.337
1.00
0.00


ATOM
901
3HD2
LEU
54
−5.912
−14.011
−0.085
1.00
0.00


ATOM
902
QQD
LEU
54
−5.558
−13.999
1.811
1.00
0.00


ATOM
903
C
LEU
54
−1.973
−12.722
−1.045
1.00
0.00


ATOM
904
O
LEU
54
−2.229
−13.441
−2.010
1.00
0.00


ATOM
905
N
TYR
55
−1.367
−11.547
−1.124
1.00
0.00


ATOM
906
H
TYR
55
−1.163
−10.969
−0.334
1.00
0.00


ATOM
907
CA
TYR
55
−0.948
−11.005
−2.405
1.00
0.00


ATOM
908
HA
TYR
55
−1.201
−11.732
−3.177
1.00
0.00


ATOM
909
CB
TYR
55
−1.674
−9.668
−2.557
1.00
0.00


ATOM
910
2HB
TYR
55
−1.096
−9.024
−3.219
1.00
0.00


ATOM
911
QB
TYR
55
−1.096
−9.024
−3.219
1.00
0.00


ATOM
912
QD
TYR
55
−3.233
−9.802
−3.155
1.00
0.00


ATOM
913
QE
TYR
55
−5.598
−10.007
−4.065
1.00
0.00


ATOM
914
QR
TYR
55
−4.415
−9.904
−3.610
1.00
0.00


ATOM
915
CG
TYR
55
−3.097
−9.790
−3.104
1.00
0.00


ATOM
916
CD1
TYR
55
−4.001
−10.635
−2.492
1.00
0.00


ATOM
917
1HD
TYR
55
−3.701
−11.216
−1.619
1.00
0.00


ATOM
918
CE1
TYR
55
−5.341
−10.751
−3.007
1.00
0.00


ATOM
919
CZ
TYR
55
−5.683
−10.014
−4.098
1.00
0.00


ATOM
920
1HE
TYR
55
−6.065
−11.415
−2.534
1.00
0.00


ATOM
921
CE2
TYR
55
−4.818
−9.173
−4.723
1.00
0.00


ATOM
922
2HE
TYR
55
−5.131
−8.598
−5.596
1.00
0.00


ATOM
923
CD2
TYR
55
−3.478
−9.057
−4.209
1.00
0.00


ATOM
924
2HD
TYR
55
−2.764
−8.389
−4.691
1.00
0.00


ATOM
925
OH
TYR
55
−6.949
−10.124
−4.583
1.00
0.00


ATOM
926
HH
TYR
55
−7.357
−10.984
−4.282
1.00
0.00


ATOM
927
C
TYR
55
0.562
−10.757
−2.429
1.00
0.00


ATOM
928
O
TYR
55
1.287
−11.395
−3.191
1.00
0.00


ATOM
929
N
ASN
55
0.991
−9.831
−1.584
1.00
0.00


ATOM
930
H
ASN
55
0.394
−9.317
−0.967
1.00
0.00


ATOM
931
CA
ASN
56
2.401
−9.491
−1.498
1.00
0.00


ATOM
932
HA
ASN
56
2.472
−8.770
−0.685
1.00
0.00


ATOM
933
CB
ASN
56
3.248
−10.730
−1.199
1.00
0.00


ATOM
934
2HB
ASN
56
3.119
−11.464
−1.994
1.00
0.00


ATOM
935
QB
ASN
56
3.119
−11.464
−1.994
1.00
0.00


ATOM
936
CG
ASN
56
4.728
−10.364
−1.072
1.00
0.00


ATOM
937
OD1
ASN
56
5.108
−9.204
−1.059
1.00
0.00


ATOM
938
ND2
ASN
56
5.539
−11.413
−0.979
1.00
0.00


ATOM
939
1HD2
ASN
56
5.162
−12.339
−0.995
1.00
0.00


ATOM
940
2HD2
ASN
56
6.526
−11.277
−0.892
1.00
0.00


ATOM
941
QD2
ASN
56
5.844
−11.808
−0.943
1.00
0.00


ATOM
942
C
ASN
56
2.864
−8.913
−2.837
1.00
0.00


ATOM
943
O
ASN
56
4.026
−9.064
−3.213
1.00
0.00


ATOM
944
N
GLU−
57
1.932
−8.264
−3.519
1.00
0.00


ATOM
945
H
GLU−
57
0.990
−8.146
−3.206
1.00
0.00


ATOM
946
CA
GLU−
57
2.231
−7.662
−4.807
1.00
0.00


ATOM
947
HA
GLU−
57
3.316
−7.703
−4.902
1.00
0.00


ATOM
948
CB
GLU−
57
1.598
−8.464
−5.946
1.00
0.00


ATOM
949
2HB
GLU−
57
0.565
−8.148
−6.089
1.00
0.00


ATOM
950
QB
GLU−
57
0.565
−8.148
−6.089
1.00
0.00


ATOM
951
CG
GLU−
57
2.378
−8.276
−7.249
1.00
0.00


ATOM
952
2HG
GLU−
57
2.419
−7.217
−7.503
1.00
0.00


ATOM
953
QG
GLU−
57
2.419
−7.217
−7.503
1.00
0.00


ATOM
954
CD
GLU−
57
3.798
−8.832
−7.123
1.00
0.00.


ATOM
955
OE1
GLU−
57
3.981
−9.734
−6.277
1.00
0.00


ATOM
956
OE2
GLU−
57
4.668
−8.341
−7.875
1.00
0.00


ATOM
957
C
GLU−
57
1.761
−6.207
−4.835
1.00
0.00


ATOM
958
O
GLU−
57
2.076
−5.467
−5.766
1.00
0.00


ATOM
959
N
SER
58
1.015
−5.839
−3.804
1.00
0.00


ATOM
960
H
SER
58
0.763
−6.447
−3.051
1.00
0.00


ATOM
961
CA
SER
58
0.499
−4.485
−3.698
1.00
0.00


ATOM
962
HA
SER
58
0.451
−4.277
−2.629
1.00
0.00


ATOM
963
CB
SER
58
1.440
−3.483
−4.370
1.00
0.00


ATOM
964
2HB
SER
58
1.217
−3.434
−5.436
1.00
0.00


ATOM
965
QB
SER
58
1.217
−3.434
−5.436
1.00
0.00


ATOM
966
OG
SER
58
1.327
−2.181
−3.802
1.00
0.00


ATOM
967
HG
SER
58
1.698
−2.179
−2.874
1.00
0.00


ATOM
968
C
SER
58
−0.895
−4.406
−4.324
1.00
0.00


ATOM
969
O
SER
58
−1.372
−3.320
−4.652
1.00
0.00


ATOM
970
N
GLU−
59
−1.509
−5.570
−4.472
1.00
0.00


ATOM
971
H
GLU−
59
−1.115
−6.449
−4.203
1.00
0.00


ATOM
972
CA
GLU−
59
−2.839
−5.647
−5.054
1.00
0.00


ATOM
973
HA
GLU−
59
−3.335
−6.467
−4.535
1.00
0.00


ATOM
974
CB
GLU−
59
−3.620
−4.354
−4.807
1.00
0.00


ATOM
975
2HB
GLU−
59
−3.349
−3.612
−5.558
1.00
0.00


ATOM
976
QB
GLU−
59
−3.349
−3.612
−5.558
1.00
0.00


ATOM
977
CG
GLU−
59
−5.128
−4.607
−4.854
1.00
0.00


ATOM
978
2HG
GLU−
59
−5.563
−4.416
−3.873
1.00
0.00


ATOM
979
QG
GLU−
59
−5.563
−4.416
−3.873
1.00
0.00


ATOM
980
CD
GLU−
59
−5.802
−3.716
−5.899
1.00
0.00


ATOM
981
OE1
GLU−
59
−5.128
−3.417
−6.908
1.00
0.00


ATOM
982
OE2
GLU−
59
−6.975
−3.354
−5.665
1.00
0.00


ATOM
983
C
GLU−
59
−2.747
−5.956
−6.549
1.00
0.00


ATOM
984
O
GLU−
59
−3.276
−5.211
−7.373
1.00
0.00


ATOM
985
N
ILE
60
−2.075
−7.056
−6.853
1.00
0.00


ATOM
986
H
ILE
60
−1.648
−7.656
−6.177
1.00
0.00


ATOM
987
CA
ILE
60
−1.907
−7.472
−8.235
1.00
0.00


ATOM
988
HA
ILE
60
−1.858
−6.570
−8.845
1.00
0.00


ATOM
989
CB
ILE
60
−0.580
−8.213
−8.414
1.00
0.00


ATOM
990
HB
ILE
60
0.198
−7.650
−7.900
1.00
0.00


ATOM
991
QG2
ILE
60
−0.649
−9.929
−7.617
1.00
0.00


ATOM
992
CG2
ILE
60
−0.636
−9.600
−7.770
1.00
0.00


ATOM
993
1HG2
ILE
60
−0.999
−9.511
−6.746
1.00
0.00


ATOM
994
2HG2
ILE
60
−1.310
−10.239
−8.341
1.00
0.00


ATOM
995
3HG2
ILE
60
0.362
−10.038
−7.766
1.00
0.00


ATOM
996
CG1
ILE
60
−0.186
−8.283
−9.891
1.00
0.00


ATOM
997
2HG1
ILE
60
0.430
−9.164
−10.066
1.00
0.00


ATOM
998
QG1
ILE
60
0.430
−9.164
−10.066
1.00
0.00


ATOM
999
QD1
ILE
60
−1.720
−8.346
−11.000
1.00
0.00


ATOM
1000
CD1
ILE
60
−1.425
−8.334
−10.787
1.00
0.00


ATOM
1001
1HD1
ILE
60
−2.019
−7.431
−10.639
1.00
0.00


ATOM
1002
2HD1
ILE
60
−1.117
−8.398
−11.831
1.00
0.00


ATOM
1003
3HD1
ILE
60
−2.024
−9.208
−10.531
1.00
0.00


ATOM
1004
C
ILE
60
−3.129
−8.284
−8.670
1.00
0.00


ATOM
1005
O
ILE
60
−3.765
−7.966
−9.673
1.00
0.00


ATOM
1006
N
ASP−
61
−3.420
−9.318
−7.892
1.00
0.00


ATOM
1007
H
ASP−
61
−2.897
−9.569
−7.078
1.00
0.00


ATOM
1008
CA
ASP−
61
−4.554
−10.177
−8.184
1.00
0.00


ATOM
1009
HA
ASP−
61
−4.181
−10.912
−8.899
1.00
0.00


ATOM
1010
CB
ASP−
61
−5.064
−10.866
−6.917
1.00
0.00


ATOM
1011
2HB
ASP−
61
−5.875
−10.270
−6.499
1.00
0.00


ATOM
1012
QB
ASP−
61
−5.875
−10.270
−6.499
1.00
0.00


ATOM
1013
CG
ASP−
61
−5.562
−12.299
−7.116
1.00
0.00


ATOM
1014
OD1
ASP−
61
−6.652
−12.444
−7.710
1.00
0.00


ATOM
1015
OD2
ASP−
61
−4.842
−13.218
−6.667
1.00
0.00


ATOM
1016
C
ASP−
61
−5.695
−9.333
−8.756
1.00
0.00


ATOM
1017
O
ASP−
61
−6.346
−9.734
−9.720
1.00
0.00


ATOM
1018
N
GLU−
62
−5.903
−8.181
−8.136
1.00
0.00


ATOM
1019
H
GLU−
62
−5.369
−7.863
−7.353
1.00
0.00


ATOM
1020
CA
GLU−
62
−6.954
−7.277
−8.571
1.00
0.00


ATOM
1021
HA
GLU−
62
−7.863
−7.878
−8.594
1.00
0.00


ATOM
1022
CB
GLU−
62
−7.138
−6.129
−7.575
1.00
0.00


ATOM
1023
2HB
GLU−
62
−6.867
−5.186
−8.048
1.00
0.00


ATOM
1024
QB
GLU−
62
−6.867
−5.186
−8.048
1.00
0.00


ATOM
1025
CG
GLU−
62
−8.583
−6.061
−7.076
1.00
0.00


ATOM
1026
2HG
GLU−
62
−8.601
−5.680
−6.056
1.00
0.00


ATOM
1027
QG
GLU−
62
−8.601
−5.680
−6.056
1.00
0.00


ATOM
1028
CD
GLU−
62
−9.431
−5.163
−7.979
1.00
0.00


ATOM
1029
OE1
GLU−
62
−9.130
−3.951
−8.017
1.00
0.00


ATOM
1030
OE2
GLU−
62
−10.362
−5.709
−8.610
1.00
0.00


ATOM
1031
C
GLU−
62
−6.650
−6.742
−9.971
1.00
0.00


ATOM
1032
O
GLU−
62
−5.638
−6.075
−10.179
1.00
0.00


ATOM
1033
N
PRO
63
−7.568
−7.065
−10.922
1.00
0.00


ATOM
1034
CD
PRO
63
−8.779
−7.853
−10.713
1.00
0.00


ATOM
1035
CA
PRO
63
−7.407
−6.625
−12.297
1.00
0.00


ATOM
1036
HA
PRO
63
−6.454
−6.725
−12.585
1.00
0.00


ATOM
1037
CB
PRO
63
−8.336
−7.515
−13.106
1.00
0.00


ATOM
1038
2HB
PRO
63
−7.779
−8.303
−13.613
1.00
0.00


ATOM
1039
QB
PRO
63
−7.779
−8.303
−13.613
1.00
0.00


ATOM
1040
CG
PRO
63
−9.323
−8.102
−12.110
1.00
0.00


ATOM
1041
2HG
PRO
63
−9.453
−9.170
−12.285
1.00
0.00


ATOM
1042
QG
PRO
63
−9.453
−9.170
−12.285
1.00
0.00


ATOM
1043
2HD
PRO
63
−8.557
−8.790
−10.202
1.00
0.00


ATOM
1044
QD
PRO
63
−8.557
−8.790
−10.202
1.00
0.00


ATOM
1045
C
PRO
63
−7.732
−5.137
−12.439
1.00
0.00


ATOM
1046
O
PRO
63
−8.894
−4.764
−12.599
1.00
0.00


ATOM
1047
N
LEU
64
−6.686
−4.327
−12.375
1.00
0.00


ATOM
1048
H
LEU
64
−5.745
−4.638
−12.244
1.00
0.00


ATOM
1049
CA
LEU
64
−6.846
−2.887
−12.493
1.00
0.00


ATOM
1050
HA
LEU
64
−7.882
−2.695
−12.772
1.00
0.00


ATOM
1051
CB
LEU
64
−6.607
−2.208
−11.143
1.00
0.00


ATOM
1052
2HB
LEU
64
−5.913
−1.381
−11.292
1.00
0.00


ATOM
1053
QB
LEU
64
−5.913
−1.381
−11.292
1.00
0.00


ATOM
1054
CG
LEU
64
−7.852
−1.671
−10.434
1.00
0.00


ATOM
1055
HG
LEU
64
−7.652
−1.652
−9.362
1.00
0.00


ATOM
1056
QD1
LEU
64
−8.221
0.108
−10.965
1.00
0.00


ATOM
1057
QD2
LEU
64
−9.335
−2.815
−10.706
1.00
0.00


ATOM
1058
CD1
LEU
64
−8.150
−0.233
−10.863
1.00
0.00


ATOM
1059
1HD1
LEU
64
−9.219
−0.124
−11.049
1.00
0.00


ATOM
1060
2HD1
LEU
64
−7.847
0.453
−10.073
1.00
0.00


ATOM
1061
3HD1
LEU
64
−7.598
−0.004
−11.774
1.00
0.00


ATOM
1062
CD2
LEU
64
−9.050
−2.596
−10.653
1.00
0.00


ATOM
1063
1HD2
LEU
64
−9.552
−2.325
−11.582
1.00
0.00


ATOM
1064
2HD2
LEU
64
−8.707
−3.628
−10.714
1.00
0.00


ATOM
1065
3HD2
LEU
64
−9.747
−2.493
−9.821
1.00
0.00


ATOM
1066
QQD
LEU
64
−8.778
−1.354
−10.835
1.00
0.00


ATOM
1067
C
LEU
64
−5.939
−2.368
−13.611
1.00
0.00


ATOM
1068
O
LEU
64
−6.422
−1.955
−14.665
1.00
0.00


ATOM
1069
N
ILE
65
−4.643
−2.406
−13.344
1.00
0.00


ATOM
1070
H
ILE
65
−4.258
−2.742
−12.484
1.00
0.00


ATOM
1071
CA
ILE
65
−3.664
−1.944
−14.314
1.00
0.00


ATOM
1072
HA
ILE
65
−4.130
−1.996
−15.297
1.00
0.00


ATOM
1073
CB
ILE
65
−3.307
−0.478
−14.058
1.00
0.00


ATOM
1074
HB
ILE
65
−4.148
−0.005
−13.551
1.00
0.00


ATOM
1075
QG2
ILE
65
−1.808
−0.334
−12.911
1.00
0.00


ATOM
1076
CG2
ILE
65
−2.096
−0.361
−13.132
1.00
0.00


ATOM
1077
1HG2
ILE
65
−1.182
−0.506
−13.707
1.00
0.00


ATOM
1078
2HG2
ILE
65
−2.083
0.626
−12.671
1.00
0.00


ATOM
1079
3HG2
ILE
65
−2.159
−1.124
−12.354
1.00
0.00


ATOM
1080
CG1
ILE
65
−3.095
0.272
−15.376
1.00
0.00


ATOM
1081
2HG1
ILE
65
−2.651
1.247
−15.175
1.00
0.00


ATOM
1082
QG1
ILE
65
−2.651
1.247
−15.175
1.00
0.00


ATOM
1083
QD1
ILE
65
−1.974
−0.712
−16.543
1.00
0.00


ATOM
1084
CD1
ILE
65
−2.189
−0.523
−16.319
1.00
0.00


ATOM
1085
1HD1
ILE
65
−1.224
−0.691
−15.841
1.00
0.00


ATOM
1086
2HD1
ILE
65
−2.653
−1.483
−16.545
1.00
0.00


ATOM
1087
3HD1
ILE
65
−2.045
0.038
−17.242
1.00
0.00


ATOM
1088
C
ILE
65
−2.456
−2.883
−14.299
1.00
0.00


ATOM
1089
O
ILE
65
−1.878
−3.172
−15.345
1.00
0.00


ATOM
1090
N
GLN
66
−2.110
−3.332
−13.101
1.00
0.00


ATOM
1091
H
GLN
66
−2.586
−3.091
−12.255
1.00
0.00


ATOM
1092
CA
GLN
66
−0.981
−4.232
−12.937
1.00
0.00


ATOM
1093
HA
GLN
66
−1.263
−5.148
−13.457
1.00
0.00


ATOM
1094
CB
GLN
66
0.279
−3.655
−13.584
1.00
0.00


ATOM
1095
2HB
GLN
66
0.886
−3.156
−12.828
1.00
0.00


ATOM
1096
QB
GLN
66
0.886
−3.156
−12.828
1.00
0.00


ATOM
1097
CG
GLN
66
1.100
−4.753
−14.262
1.00
0.00


ATOM
1098
2HG
GLN
66
0.442
−5.395
−14.848
1.00
0.00


ATOM
1099
QG
GLN
66
0.442
−5.395
−14.848
1.00
0.00


ATOM
1100
CD
GLN
66
2.177
−4.154
−15.169
1.00
0.00


ATOM
1101
OE1
GLN
66
3.355
−4.140
−14.851
1.00
0.00


ATOM
1102
NE2
GLN
66
1.709
−3.660
−16.310
1.00
0.00


ATOM
1103
1HE2
GLN
66
0.730
−3.702
−16.510
1.00
0.00


ATOM
1104
2HE2
GLN
66
2.336
−3.245
−16.970
1.00
0.00


ATOM
1105
QE2
GLN
66
1.533
−3.473
−16.740
1.00
0.00


ATOM
1106
C
GLN
66
−0.750
−4.527
−11.453
1.00
0.00


ATOM
1107
O
GLN
66
−0.998
−5.639
−10.992
1.00
0.00


ATOM
1108
N
LEU
67
−0.276
−3.510
−10.748
1.00
0.00


ATOM
1109
H
LEU
67
−0.076
−2.609
−11.132
1.00
0.00


ATOM
1110
CA
LEU
67
−0.007
−3.647
−9.326
1.00
0.00


ATOM
1111
HA
LEU
67
−0.957
−3.537
−8.802
1.00
0.00


ATOM
1112
CB
LEU
67
0.522
−5.048
−9.013
1.00
0.00


ATOM
1113
2HB
LEU
67
−0.209
−5.777
−9.361
1.00
0.00


ATOM
1114
QB
LEU
67
−0.209
−5.777
−9.361
1.00
0.00


ATOM
1115
CG
LEU
67
1.882
−5.402
−9.619
1.00
0.00


ATOM
1116
HG
LEU
67
2.099
−6.445
−9.385
1.00
0.00


ATOM
1117
QD1
LEU
67
1.845
−5.248
−11.505
1.00
0.00


ATOM
1118
QD2
LEU
67
3.260
−4.358
−8.847
1.00
0.00


ATOM
1119
CD1
LEU
67
1.853
−5.278
−11.143
1.00
0.00


ATOM
1120
1HD1
LEU
67
2.827
−5.548
−11.548
1.00
0.00


ATOM
1121
2HD1
LEU
67
1.093
−5.946
−11.548
1.00
0.00


ATOM
1122
3HD1
LEU
67
1.616
−4.249
−11.419
1.00
0.00


ATOM
1123
CD2
LEU
67
2.996
−4.558
−8.995
1.00
0.00


ATOM
1124
1HD2
LEU
67
3.442
−3.925
−9.761
1.00
0.00


ATOM
1125
2HD2
LEU
67
2.580
−3.935
−8.204
1.00
0.00


ATOM
1126
3HD2
LEU
67
3.758
−5.216
−8.578
1.00
0.00


ATOM
1127
QQD
LEU
67
2.553
−4.803
−10.176
1.00
0.00


ATOM
1128
C
LEU
67
0.928
−2.522
−8.878
1.00
0.00


ATOM
1129
O
LEU
67
0.712
−1.911
−7.832
1.00
0.00


ATOM
1130
N
ASP−
68
1.946
−2.283
−9.691
1.00
0.00


ATOM
1131
H
ASP−
68
2.114
−2.785
−10.540
1.00
0.00


ATOM
1132
CA
ASP−
68
2.915
−1.242
−9.391
1.00
0.00


ATOM
1133
HA
ASP−
68
3.331
−1.510
−8.420
1.00
0.00


ATOM
1134
CB
ASP−
68
4.013
−1.189
−10.454
1.00
0.00


ATOM
1135
2HB
ASP−
68
3.792
−0.374
−11.144
1.00
0.00


ATOM
1136
QB
ASP−
68
3.792
−0.374
−11.144
1.00
0.00


ATOM
1137
CG
ASP−
68
5.431
−0.998
−9.911
1.00
0.00


ATOM
1138
OD1
ASP−
68
5.855
0.175
−9.831
1.00
0.00


ATOM
1139
OD2
ASP−
68
6.058
−2.030
−9.588
1.00
0.00


ATOM
1140
C
ASP−
68
2.210
0.115
−9.372
1.00
0.00


ATOM
1141
O
ASP−
68
2.146
0.771
−8.334
1.00
0.00


ATOM
1142
N
ASP−
69
1.698
0.497
−10.533
1.00
0.00


ATOM
1143
H
ASP−
69
1.755
−0.042
−11.373
1.00
0.00


ATOM
1144
CA
ASP−
69
0.999
1.764
−10.663
1.00
0.00


ATOM
1145
HA
ASP−
69
1.673
2.507
−10.236
1.00
0.00


ATOM
1146
CB
ASP−
69
0.702
2.081
−12.129
1.00
0.00


ATOM
1147
2HB
ASP−
69
−0.025
2.892
−12.170
1.00
0.00


ATOM
1148
QB
ASP−
69
−0.025
2.892
−12.170
1.00
0.00


ATOM
1149
CG
ASP−
69
1.921
2.476
−12.966
1.00
0.00


ATOM
1150
OD1
ASP−
69
1.778
2.491
−14.207
1.00
0.00


ATOM
1151
OD2
ASP−
69
2.970
2.753
−12.344
1.00
0.00


ATOM
1152
C
ASP−
69
−0.334
1.685
−9.917
1.00
0.00


ATOM
1153
O
ASP−
69
−0.834
2.695
−9.422
1.00
0.00


ATOM
1154
N
ASP−
70
−0.873
0.476
−9.859
1.00
0.00


ATOM
1155
H
ASP−
70
−0.460
−0.340
−10.263
1.00
0.00


ATOM
1156
CA
ASP−
70
−2.139
0.253
−9.181
1.00
0.00


ATOM
1157
HA
ASP−
70
−2.893
0.700
−9.829
1.00
0.00


ATOM
1158
CB
ASP−
70
−2.405
−1.242
−8.991
1.00
0.00


ATOM
1159
2HB
ASP−
70
−1.932
−1.565
−8.064
1.00
0.00


ATOM
1160
QB
ASP−
70
−1.932
−1.565
−8.064
1.00
0.00


ATOM
1161
CG
ASP−
70
−3.882
−1.635
−8.948
1.00
0.00


ATOM
1162
OD1
ASP−
70
−4.162
−2.825
−9.210
1.00
0.00


ATOM
1163
OD2
ASP−
70
−4.700
−0.737
−8.653
1.00
0.00


ATOM
1164
C
ASP−
70
−2.094
0.905
−7.798
1.00
0.00


ATOM
1165
O
ASP−
70
−3.091
1.457
−7.335
1.00
0.00


ATOM
1166
N
THR
71
−0.927
0.820
−7.176
1.00
0.00


ATOM
1167
H
THR
71
−0.120
0.369
−7.559
1.00
0.00


ATOM
1168
CA
THR
71
−0.739
1.395
−5.855
1.00
0.00


ATOM
1169
HA
THR
71
−1.644
1.938
−5.584
1.00
0.00


ATOM
1170
CB
THR
71
−0.518
0.247
−4.867
1.00
0.00


ATOM
1171
HB
THR
71
0.189
0.535
−4.089
1.00
0.00


ATOM
1172
QG2
THR
71
−2.137
−0.381
−4.113
1.00
0.00


ATOM
1173
OG1
THR
71
−0.073
−0.832
−5.683
1.00
0.00


ATOM
1174
1HG
THR
71
0.189
−1.609
−5.110
1.00
0.00


ATOM
1175
CG2
THR
71
−1.827
−0.261
−4.257
1.00
0.00


ATOM
1176
1HG2
THR
71
−1.621
−1.126
−3.627
1.00
0.00


ATOM
1177
2HG2
THR
71
−2.277
0.529
−3.656
1.00
0.00


ATOM
1178
3HG2
THR
71
−2.511
−0.546
−5.056
1.00
0.00


ATOM
1179
C
THR
71
0.405
2.409
−5.873
1.00
0.00


ATOM
1180
O
THR
71
0.230
3.556
−5.462
1.00
0.00


ATOM
1181
N
ALA
72
1.553
1.953
−6.353
1.00
0.00


ATOM
1182
H
ALA
72
1.687
1.018
−6.685
1.00
0.00


ATOM
1183
CA
ALA
72
2.726
2.806
−6.430
1.00
0.00


ATOM
1184
HA
ALA
72
3.092
2.958
−5.414
1.00
0.00


ATOM
1185
QB
ALA
72
4.072
1.941
−7.441
1.00
0.00


ATOM
1186
CB
ALA
72
3.814
2.106
−7.247
1.00
0.00


ATOM
1187
1HB
ALA
72
3.632
2.269
−8.309
1.00
0.00


ATOM
1188
2HB
ALA
72
4.788
2.515
−6.979
1.00
0.00


ATOM
1189
3HB
ALA
72
3.796
1.038
−7.034
1.00
0.00


ATOM
1190
C
ALA
72
2.329
4.158
−7.026
1.00
0.00


ATOM
1191
O
ALA
72
2.774
5.203
−6.554
1.00
0.00


ATOM
1192
N
GLU−
73
1.494
4.095
−8.053
1.00
0.00


ATOM
1193
H
GLU−
73
1.135
3.240
−8.430
1.00
0.00


ATOM
1194
CA
GLU−
73
1.032
5.301
−8.717
1.00
0.00


ATOM
1195
HA
GLU−
73
1.933
5.792
−9.085
1.00
0.00


ATOM
1196
CB
GLU−
73
0.125
4.962
−9.901
1.00
0.00


ATOM
1197
2HB
GLU−
73
−0.891
4.789
−9.549
1.00
0.00


ATOM
1198
QB
GLU−
73
−0.891
4.789
−9.549
1.00
0.00


ATOM
1199
CG
GLU−
73
0.128
6.090
−10.935
1.00
0.00


ATOM
1200
2HG
GLU−
73
0.852
5.868
−11.719
1.00
0.00


ATOM
1201
QG
GLU−
73
0.852
5.868
−11.719
1.00
0.00


ATOM
1202
CD
GLU−
73
−1.260
6.270
−11.553
1.00
0.00


ATOM
1203
OE1
GLU−
73
−1.307
6.680
−12.733
1.00
0.00


ATOM
1204
OE2
GLU−
73
−2.243
5.995
−10.831
1.00
0.00


ATOM
1205
C
GLU−
73
0.314
6.214
−7.721
1.00
0.00


ATOM
1206
O
GLU−
73
0.282
7.431
−7.899
1.00
0.00


ATOM
1207
N
LEU
74
−0.246
5.592
−6.694
1.00
0.00


ATOM
1208
H
LEU
74
−0.216
4.601
−6.557
1.00
0.00


ATOM
1209
CA
LEU
74
−0.962
6.333
−5.669
1.00
0.00


ATOM
1210
HA
LEU
74
−1.516
7.127
−6.168
1.00
0.00


ATOM
1211
CB
LEU
74
−1.988
5.433
−4.976
1.00
0.00


ATOM
1212
2HB
LEU
74
−1.786
5.446
−3.905
1.00
0.00


ATOM
1213
QB
LEU
74
−1.786
5.446
−3.905
1.00
0.00


ATOM
1214
CG
LEU
74
−3.457
5.799
−5.196
1.00
0.00


ATOM
1215
HG
LEU
74
−4.056
5.268
−4.455
1.00
0.00


ATOM
1216
QD1
LEU
74
−3.744
7.650
−4.922
1.00
0.00


ATOM
1217
QD2
LEU
74
−4.050
5.233
−6.903
1.00
0.00


ATOM
1218
CD1
LEU
74
−3.689
7.295
−4.974
1.00
0.00


ATOM
1219
1HD1
LEU
74
−4.747
7.474
−4.781
1.00
0.00


ATOM
1220
2HD1
LEU
74
−3.101
7.631
−4.121
1.00
0.00


ATOM
1221
3HD1
LEU
74
−3.385
7.845
−5.865
1.00
0.00


ATOM
1222
CD2
LEU
74
−3.936
5.342
−6.575
1.00
0.00


ATOM
1223
1HD2
LEU
74
−4.396
6.181
−7.096
1.00
0.00


ATOM
1224
2HD2
LEU
74
−3.086
4.978
−7.153
1.00
0.00


ATOM
1225
3HD2
LEU
74
−4.666
4.541
−6.460
1.00
0.00


ATOM
1226
QQD
LEU
74
−3.897
6.442
−5.912
1.00
0.00


ATOM
1227
C
LEU
74
0.045
6.970
−4.710
1.00
0.00


ATOM
1228
O
LEU
74
0.024
8.182
−4.500
1.00
0.00


ATOM
1229
N
MET
75
0.902
6.126
−4.155
1.00
0.00


ATOM
1230
H
MET
75
0.911
5.143
−4.332
1.00
0.00


ATOM
1231
CA
MET
75
1.914
6.593
−3.223
1.00
0.00


ATOM
1232
HA
MET
75
1.387
7.228
−2.511
1.00
0.00


ATOM
1233
CB
MET
75
2.559
5.392
−2.527
1.00
0.00


ATOM
1234
2HB
MET
75
2.951
5.699
−1.557
1.00
0.00


ATOM
1235
QB
MET
75
2.951
5.699
−1.557
1.00
0.00


ATOM
1236
CG
MET
75
1.549
4.259
−2.340
1.00
0.00


ATOM
1237
2HG
MET
75
1.702
3.496
−3.105
1.00
0.00


ATOM
1238
QG
MET
75
1.702
3.496
−3.105
1.00
0.00


ATOM
1239
SD
MET
75
1.731
3.540
−0.717
1.00
0.00


ATOM
1240
QE
MET
75
1.852
1.460
−1.241
1.00
0.00


ATOM
1241
CE
MET
75
1.831
1.812
−1.153
1.00
0.00


ATOM
1242
1HE
MET
75
2.869
1.482
−1.096
1.00
0.00


ATOM
1243
2HE
MET
75
1.226
1.227
−0.460
1.00
0.00


ATOM
1244
3HE
MET
75
1.461
1.671
−2.168
1.00
0.00


ATOM
1245
C
MET
75
2.991
7.405
−3.945
1.00
0.00


ATOM
1246
O
MET
75
3.775
8.107
−3.308
1.00
0.00


ATOM
1247
N
LYS+
76
2.995
7.281
−5.264
1.00
0.00


ATOM
1248
H
LYS+
76
2.355
6.708
−5.774
1.00
0.00


ATOM
1249
CA
LYS+
76
3.964
7.994
−6.080
1.00
0.00


ATOM
1250
HA
LYS+
76
4.890
8.056
−5.509
1.00
0.00


ATOM
1251
CB
LYS+
76
4.271
7.209
−7.356
1.00
0.00


ATOM
1252
2HB
LYS+
76
3.347
7.016
−7.901
1.00
0.00


ATOM
1253
QB
LYS+
76
3.347
7.016
−7.901
1.00
0.00


ATOM
1254
CG
LYS+
76
5.245
7.978
−8.251
1.00
0.00


ATOM
1255
2HG
LYS+
76
6.270
7.719
−7.986
1.00
0.00


ATOM
1256
QG
LYS+
76
6.270
7.719
−7.986
1.00
0.00


ATOM
1257
CD
LYS+
76
4.999
7.660
−9.728
1.00
0.00


ATOM
1258
2HD
LYS+
76
3.949
7.827
−9.970
1.00
0.00


ATOM
1259
QD
LYS+
76
3.949
7.827
−9.970
1.00
0.00


ATOM
1260
CE
LYS+
76
5.880
8.530
−10.629
1.00
0.00


ATOM
1261
2HE
LYS+
76
6.783
8.821
−10.093
1.00
0.00


ATOM
1262
QE
LYS+
76
6.783
8.821
−10.093
1.00
0.00


ATOM
1263
NZ
LYS+
76
6.243
7.796
−11.862
1.00
0.00


ATOM
1264
1HZ
LYS+
76
7.186
7.472
−11.792
1.00
0.00


ATOM
1265
2HZ
LYS+
76
5.630
7.014
−11.977
1.00
0.00


ATOM
1266
3HZ
LYS+
76
6.157
8.406
−12.650
1.00
0.00


ATOM
1267
QZ
LYS+
76
6.324
7.631
−12.140
1.00
0.00


ATOM
1268
C
LYS+
76
3.458
9.415
−6.338
1.00
0.00


ATOM
1269
O
LYS+
76
4.175
10.386
−6.101
1.00
0.00


ATOM
1270
N
GLN
77
2.226
9.492
−6.820
1.00
0.00


ATOM
1271
H
GLN
77
1.649
8.697
−7.011
1.00
0.00


ATOM
1272
CA
GLN
77
1.616
10.778
−7.114
1.00
0.00


ATOM
1273
HA
GLN
77
2.267
11.245
−7.854
1.00
0.00


ATOM
1274
CB
GLN
77
0.221
10.599
−7.713
1.00
0.00


ATOM
1275
2HB
GLN
77
−0.525
11.028
−7.044
1.00
0.00


ATOM
1276
QB
GLN
77
−0.525
11.028
−7.044
1.00
0.00


ATOM
1277
CG
GLN
77
0.124
11.266
−9.087
1.00
0.00


ATOM
1278
2HG
GLN
77
−0.732
10.866
−9.631
1.00
0.00


ATOM
1279
QG
GLN
77
−0.732
10.866
−9.631
1.00
0.00


ATOM
1280
CD
GLN
77
−0.014
12.783
−8.951
1.00
0.00


ATOM
1281
OE1
GLN
77
0.924
13.492
−8.625
1.00
0.00


ATOM
1282
NE2
GLN
77
−1.234
13.241
−9.219
1.00
0.00


ATOM
1283
1HE2
GLN
77
−1.960
12.606
−9.481
1.00
0.00


ATOM
1284
2HE2
GLN
77
−1.425
14.221
−9.157
1.00
0.00


ATOM
1285
QE2
GLN
77
−1.693
13.413
−9.319
1.00
0.00


ATOM
1286
C
GLN
77
1.564
11.640
−5.852
1.00
0.00


ATOM
1287
O
GLN
77
1.732
12.857
−5.919
1.00
0.00


ATOM
1288
N
ALA
78
1.330
10.977
−4.729
1.00
0.00


ATOM
1289
H
ALA
78
1.196
9.987
−4.682
1.00
0.00


ATOM
1290
CA
ALA
78
1.254
11.667
−3.453
1.00
0.00


ATOM
1291
HA
ALA
78
0.696
12.591
−3.608
1.00
0.00


ATOM
1292
QB
ALA
78
0.319
10.592
−2.207
1.00
0.00


ATOM
1293
CB
ALA
78
0.498
10.798
−2.446
1.00
0.00


ATOM
1294
1HB
ALA
78
1.038
9.863
−2.296
1.00
0.00


ATOM
1295
2HB
ALA
78
0.419
11.328
−1.497
1.00
0.00


ATOM
1296
3HB
ALA
78
−0.500
10.585
−2.828
1.00
0.00


ATOM
1297
C
ALA
78
2.667
12.014
−2.980
1.00
0.00


ATOM
1298
O
ALA
78
2.954
13.167
−2.660
1.00
0.00


ATOM
1299
N
ARG+
79
3.513
10.994
−2.949
1.00
0.00


ATOM
1300
H
ARG+
79
3.272
10.060
−3.211
1.00
0.00


ATOM
1301
CA
ARG+
79
4.890
11.176
−2.521
1.00
0.00


ATOM
1302
HA
ARG+
79
4.817
11.702
−1.568
1.00
0.00


ATOM
1303
CB
ARG+
79
5.590
9.829
−2.331
1.00
0.00


ATOM
1304
2HB
ARG+
79
4.912
9.128
−1.845
1.00
0.00


ATOM
1305
QB
ARG+
79
4.912
9.128
−1.845
1.00
0.00


ATOM
1306
CG
ARG+
79
6.048
9.256
−3.674
1.00
0.00


ATOM
1307
2HG
ARG+
79
5.426
9.655
−4.475
1.00
0.00


ATOM
1308
QG
ARG+
79
5.426
9.655
−4.475
1.00
0.00


ATOM
1309
CD
ARG+
79
7.516
9.595
−3.942
1.00
0.00


ATOM
1310
2HD
ARG+
79
7.853
10.363
−3.247
1.00
0.00


ATOM
1311
QD
ARG+
79
7.853
10.363
−3.247
1.00
0.00


ATOM
1312
NE
ARG+
79
8.351
8.381
−3.797
1.00
0.00


ATOM
1313
HE
ARG+
79
8.977
8.333
−3.020
1.00
0.00


ATOM
1314
CZ
ARG+
79
8.318
7.342
−4.643
1.00
0.00


ATOM
1315
NH1
ARG+
79
9.111
6.282
−4.433
1.00
0.00


ATOM
1316
1HH1
ARG+
79
9.087
5.507
−5.064
1.00
0.00


ATOM
1317
2HH1
ARG+
79
9.727
6.267
−3.644
1.00
0.00


ATOM
1318
QH1
ARG+
79
9.407
5.887
−4.354
1.00
0.00


ATOM
1319
NH2
ARG+
79
7.493
7.362
−5.698
1.00
0.00


ATOM
1320
1HH2
ARG+
79
7.469
6.587
−6.330
1.00
0.00


ATOM
1321
2HH2
ARG+
79
6.902
8.153
−5.856
1.00
0.00


ATOM
1322
QH2
ARG+
79
7.186
7.370
−6.093
1.00
0.00


ATOM
1323
C
ARG+
79
5.661
12.004
−3.550
1.00
0.00


ATOM
1324
O
ARG+
79
6.830
12.326
−3.343
1.00
0.00


ATOM
1325
N
ASP−
80
4.976
12.325
−4.637
1.00
0.00


ATOM
1326
H
ASP−
80
4.026
12.059
−4.798
1.00
0.00


ATOM
1327
CA
ASP−
80
5.583
13.110
−5.699
1.00
0.00


ATOM
1328
HA
ASP−
80
6.635
13.197
−5.426
1.00
0.00


ATOM
1329
CB
ASP−
80
5.431
12.414
−7.054
1.00
0.00


ATOM
1330
2HB
ASP−
80
4.371
12.247
−7.243
1.00
0.00


ATOM
1331
QB
ASP−
80
4.371
12.247
−7.243
1.00
0.00


ATOM
1332
CG
ASP−
80
6.025
13.174
−8.242
1.00
0.00


ATOM
1333
OD1
ASP−
80
7.154
12.814
−8.639
1.00
0.00


ATOM
1334
OD2
ASP−
80
5.336
14.098
−8.725
1.00
0.00


ATOM
1335
C
ASP−
80
4.886
14.469
−5.786
1.00
0.00


ATOM
1336
O
ASP−
80
5.437
15.419
−6.339
1.00
0.00


ATOM
1337
N
MET
81
3.684
14.518
−5.229
1.00
0.00


ATOM
1338
H
MET
81
3.243
13.741
−4.781
1.00
0.00


ATOM
1339
CA
MET
81
2.907
15.745
−5.237
1.00
0.00


ATOM
1340
HA
MET
81
3.321
16.350
−6.043
1.00
0.00


ATOM
1341
CB
MET
81
1.434
15.415
−5.488
1.00
0.00


ATOM
1342
2HB
MET
81
1.109
14.636
−4.799
1.00
0.00


ATOM
1343
QB
MET
81
1.109
14.636
−4.799
1.00
0.00


ATOM
1344
CG
MET
81
0.556
16.656
−5.313
1.00
0.00


ATOM
1345
2HG
MET
81
1.141
17.467
−4.880
1.00
0.00


ATOM
1346
QG
MET
81
1.141
17.467
−4.880
1.00
0.00


ATOM
1347
SD
MET
81
−0.111
17.160
−6.891
1.00
0.00


ATOM
1348
QE
MET
81
−2.050
16.243
−6.787
1.00
0.00


ATOM
1349
CE
MET
81
−1.722
16.398
−6.804
1.00
0.00


ATOM
1350
1HE
MET
81
−2.046
16.351
−5.765
1.00
0.00


ATOM
1351
2HE
MET
81
−2.435
16.988
−7.381
1.00
0.00


ATOM
1352
3HE
MET
81
−1.670
15.389
−7.214
1.00
0.00


ATOM
1353
C
MET
81
3.043
16.489
−3.906
1.00
0.00


ATOM
1354
O
MET
81
3.275
17.696
−3.887
1.00
0.00


ATOM
1355
N
TYR
82
2.893
15.736
−2.826
1.00
0.00


ATOM
1356
H
TYR
82
2.705
14.754
−2.851
1.00
0.00


ATOM
1357
CA
TYR
82
2.997
16.308
−1.495
1.00
0.00


ATOM
1358
HA
TYR
82
3.381
17.324
−1.594
1.00
0.00


ATOM
1359
CB
TYR
82
1.592
16.236
−0.894
1.00
0.00


ATOM
1360
2HB
TYR
82
1.663
15.856
0.125
1.00
0.00


ATOM
1361
QB
TYR
82
1.663
15.856
0.125
1.00
0.00


ATOM
1362
QD
TYR
82
0.784
17.703
−0.871
1.00
0.00


ATOM
1363
QE
TYR
82
−0.443
19.929
−0.836
1.00
0.00


ATOM
1364
QR
TYR
82
0.170
18.816
−0.854
1.00
0.00


ATOM
1365
CG
TYR
82
0.854
17.575
−0.873
1.00
0.00


ATOM
1366
CD1
TYR
82
−0.487
17.630
−1.197
1.00
0.00


ATOM
1367
1HD
TYR
82
−1.019
16.719
−1.467
1.00
0.00


ATOM
1368
CE1
TYR
82
−1.182
18.891
−1.176
1.00
0.00


ATOM
1369
1HE
TYR
82
−2.240
18.948
−1.430
1.00
0.00


ATOM
1370
CZ
TYR
82
−0.487
20.009
−0.835
1.00
0.00


ATOM
1371
CE2
TYR
82
0.834
19.991
−0.510
1.00
0.00


ATOM
1372
2HE
TYR
82
1.354
20.910
−0.242
1.00
0.00


ATOM
1373
CD2
TYR
82
1.528
18.729
−0.531
1.00
0.00


ATOM
1374
2HD
TYR
82
2.587
18.686
−0.274
1.00
0.00


ATOM
1375
OH
TYR
82
−1.143
21.201
−0.816
1.00
0.00


ATOM
1376
HH
TYR
82
−0.627
21.883
−1.333
1.00
0.00


ATOM
1377
C
TYR
82
3.955
15.495
−0.621
1.00
0.00


ATOM
1378
O
TYR
82
4.802
16.060
0.069
1.00
0.00


ATOM
1379
N
GLY
83
3.790
14.182
−0.682
1.00
0.00


ATOM
1380
H
GLY
83
3.100
13.731
−1.247
1.00
0.00


ATOM
1381
CA
GLY
83
4.630
13.286
0.095
1.00
0.00


ATOM
1382
1HA
GLY
83
5.577
13.129
−0.423
1.00
0.00


ATOM
1383
2HA
GLY
83
4.864
13.743
1.056
1.00
0.00


ATOM
1384
QA
GLY
83
5.220
13.436
0.317
1.00
0.00


ATOM
1385
C
GLY
83
3.936
11.941
0.318
1.00
0.00


ATOM
1386
O
GLY
83
4.594
10.933
0.567
1.00
0.00


ATOM
1387
N
GLN
84
2.614
11.970
0.222
1.00
0.00


ATOM
1388
H
GLN
84
2.087
12.794
0.020
1.00
0.00


ATOM
1389
CA
GLN
84
1.824
10.766
0.412
1.00
0.00


ATOM
1390
HA
GLN
84
1.783
10.290
−0.568
1.00
0.00


ATOM
1391
CB
GLN
84
2.501
9.815
1.401
1.00
0.00


ATOM
1392
2HB
GLN
84
2.863
10.378
2.262
1.00
0.00


ATOM
1393
QB
GLN
84
2.863
10.378
2.262
1.00
0.00


ATOM
1394
CG
GLN
84
1.531
8.728
1.868
1.00
0.00


ATOM
1395
2HG
GLN
84
0.637
9.188
2.289
1.00
0.00


ATOM
1396
QG
GLN
84
0.637
9.188
2.289
1.00
0.00


ATOM
1397
CD
GLN
84
1.140
7.807
0.710
1.00
0.00


ATOM
1398
OE1
GLN
84
1.605
7.945
−0.410
1.00
0.00


ATOM
1399
NE2
GLN
84
0.263
6.863
1.041
1.00
0.00


ATOM
1400
1HE2
GLN
84
−0.078
6.805
1.979
1.00
0.00


ATOM
1401
2HE2
GLN
84
−0.054
6.213
0.351
1.00
0.00


ATOM
1402
QE2
GLN
84
−0.066
6.509
1.165
1.00
0.00


ATOM
1403
C
GLN
84
0.411
11.125
0.876
1.00
0.00


ATOM
1404
O
GLN
84
−0.545
10.412
0.575
1.00
0.00


ATOM
1405
N
GLU−
85
0.325
12.231
1.600
1.00
0.00


ATOM
1406
H
GLU−
85
1.107
12.805
1.840
1.00
0.00


ATOM
1407
CA
GLU−
85
−0.956
12.694
2.109
1.00
0.00


ATOM
1408
HA
GLU−
85
−1.617
12.723
1.244
1.00
0.00


ATOM
1409
CB
GLU−
85
−1.523
11.716
3.140
1.00
0.00


ATOM
1410
2HB
GLU−
85
−0.810
11.589
3.956
1.00
0.00


ATOM
1411
QB
GLU−
85
−0.810
11.589
3.956
1.00
0.00


ATOM
1412
CG
GLU−
85
−2.858
12.216
3.695
1.00
0.00


ATOM
1413
2HG
GLU−
85
−3.573
11.394
3.726
1.00
0.00


ATOM
1414
QG
GLU−
85
−3.573
11.394
3.726
1.00
0.00


ATOM
1415
CD
GLU−
85
−2.684
12.804
5.097
1.00
0.00


ATOM
1416
OE1
GLU−
85
−1.696
12.415
5.756
1.00
0.00


ATOM
1417
OE2
GLU−
85
−3.543
13.628
5.477
1.00
0.00


ATOM
1418
C
GLU−
85
−0.815
14.096
2.703
1.00
0.00


ATOM
1419
O
GLU−
85
−1.619
14.504
3.539
1.00
0.00


ATOM
1420
N
LYS+
86
0.213
14.797
2.247
1.00
0.00


ATOM
1421
H
LYS+
86
0.863
14.459
1.566
1.00
0.00


ATOM
1422
CA
LYS+
86
0.470
16.146
2.723
1.00
0.00


ATOM
1423
HA
LYS+
86
1.228
16.583
2.073
1.00
0.00


ATOM
1424
CB
LYS+
86
−0.788
17.008
2.594
1.00
0.00


ATOM
1425
2HB
LYS+
86
−1.384
16.926
3.503
1.00
0.00


ATOM
1426
QB
LYS+
86
−1.384
16.926
3.503
1.00
0.00


ATOM
1427
CG
LYS+
86
−0.425
18.474
2.346
1.00
0.00


ATOM
1428
2HG
LYS+
86
−0.435
18.678
1.276
1.00
0.00


ATOM
1429
QG
LYS+
86
−0.435
18.678
1.276
1.00
0.00


ATOM
1430
CD
LYS+
86
−1.404
19.408
3.059
1.00
0.00


ATOM
1431
2HD
LYS+
86
−2.265
18.840
3.409
1.00
0.00


ATOM
1432
QD
LYS+
86
−2.265
18.840
3.409
1.00
0.00


ATOM
1433
CE
LYS+
86
−0.732
20.108
4.242
1.00
0.00


ATOM
1434
2HE
LYS+
86
0.352
20.040
4.144
1.00
0.00


ATOM
1435
QE
LYS+
86
0.352
20.040
4.144
1.00
0.00


ATOM
1436
NZ
LYS+
86
−1.141
21.529
4.305
1.00
0.00


ATOM
1437
1HZ
LYS+
86
−2.111
21.606
4.074
1.00
0.00


ATOM
1438
2HZ
LYS+
86
−0.989
21.879
5.230
1.00
0.00


ATOM
1439
3HZ
LYS+
86
−0.600
22.061
3.654
1.00
0.00


ATOM
1440
QZ
LYS+
86
−1.233
21.849
4.319
1.00
0.00


ATOM
1441
C
LYS+
86
1.029
16.083
4.146
1.00
0.00


ATOM
1442
O
LYS+
86
1.010
17.077
4.868
1.00
0.00


ATOM
1443
N
LEU
87
1.512
14.903
4.505
1.00
0.00


ATOM
1444
H
LEU
87
1.524
14.098
3.911
1.00
0.00


ATOM
1445
CA
LEU
87
2.075
14.696
5.828
1.00
0.00


ATOM
1446
HA
LEU
87
2.127
15.669
6.317
1.00
0.00


ATOM
1447
CB
LEU
87
1.152
13.816
6.672
1.00
0.00


ATOM
1448
2HB
LEU
87
1.130
12.819
6.231
1.00
0.00


ATOM
1449
QB
LEU
87
1.130
12.819
6.231
1.00
0.00


ATOM
1450
CG
LEU
87
1.520
13.680
8.151
1.00
0.00


ATOM
1451
HG
LEU
87
2.608
13.678
8.234
1.00
0.00


ATOM
1452
QD1
LEU
87
0.887
15.161
9.147
1.00
0.00


ATOM
1453
QD2
LEU
87
0.904
12.035
8.857
1.00
0.00


ATOM
1454
CD1
LEU
87
1.008
14.876
8.955
1.00
0.00


ATOM
1455
1HD1
LEU
87
1.809
15.256
9.592
1.00
0.00


ATOM
1456
2HD1
LEU
87
0.684
15.662
8.273
1.00
0.00


ATOM
1457
3HD1
LEU
87
0.168
14.565
9.575
1.00
0.00


ATOM
1458
CD2
LEU
87
1.023
12.351
8.721
1.00
0.00


ATOM
1459
1HD2
LEU
87
−0.067
12.327
8.689
1.00
0.00


ATOM
1460
2HD2
LEU
87
1.421
11.528
8.127
1.00
0.00


ATOM
1461
3HD2
LEU
87
1.357
12.249
9.754
1.00
0.00


ATOM
1462
QQD
LEU
87
0.895
13.598
9.002
1.00
0.00


ATOM
1463
C
LEU
87
3.496
14.145
5.693
1.00
0.00


ATOM
1464
O
LEU
87
4.093
13.708
6.677
1.00
0.00


ATOM
1465
N
ASN
88
3.997
14.184
4.468
1.00
0.00


ATOM
1466
H
ASN
88
3.506
14.541
3.673
1.00
0.00


ATOM
1467
CA
ASN
88
5.337
13.694
4.192
1.00
0.00


ATOM
1468
HA
ASN
88
5.210
12.642
3.935
1.00
0.00


ATOM
1469
CB
ASN
88
5.976
14.461
3.031
1.00
0.00


ATOM
1470
2HB
ASN
88
6.212
13.771
2.221
1.00
0.00


ATOM
1471
QB
ASN
88
6.212
13.771
2.221
1.00
0.00


ATOM
1472
CG
ASN
88
5.040
15.557
2.517
1.00
0.00


ATOM
1473
OD1
ASN
88
3.877
15.330
2.226
1.00
0.00


ATOM
1474
ND2
ASN
88
5.611
16.754
2.423
1.00
0.00


ATOM
1475
1HD2
ASN
88
6.571
16.872
2.678
1.00
0.00


ATOM
1476
2HD2
ASN
88
5.081
17.537
2.097
1.00
0.00


ATOM
1477
QD2
ASN
88
5.826
17.204
2.387
1.00
0.00


ATOM
1478
C
ASN
88
6.215
13.893
5.429
1.00
0.00


ATOM
1479
O
ASN
88
6.956
12.994
5.820
1.00
0.00


ATOM
1480
N
GLU−
89
6.100
15.078
6.011
1.00
0.00


ATOM
1481
H
GLU−
89
5.494
15.804
5.686
1.00
0.00


ATOM
1482
CA
GLU−
89
6.873
15.407
7.196
1.00
0.00


ATOM
1483
HA
GLU−
89
7.869
15.660
6.832
1.00
0.00


ATOM
1484
CB
GLU−
89
6.281
16.620
7.918
1.00
0.00


ATOM
1485
2HB
GLU−
89
6.337
16.469
8.996
1.00
0.00


ATOM
1486
QB
GLU−
89
6.337
16.469
8.996
1.00
0.00


ATOM
1487
CG
GLU−
89
7.025
17.901
7.537
1.00
0.00


ATOM
1488
2HG
GLU−
89
6.307
18.684
7.291
1.00
0.00


ATOM
1489
QG
GLU−
89
6.307
18.684
7.291
1.00
0.00


ATOM
1490
CD
GLU−
89
7.929
18.371
8.680
1.00
0.00


ATOM
1491
OE1
GLU−
89
7.430
18.389
9.826
1.00
0.00


ATOM
1492
OE2
GLU−
89
9.096
18.702
8.381
1.00
0.00


ATOM
1493
C
GLU−
89
6.953
14.199
8.131
1.00
0.00


ATOM
1494
O
GLU−
89
8.026
13.867
8.633
1.00
0.00


ATOM
1495
N
LYS+
90
5.804
13.571
8.335
1.00
0.00


ATOM
1496
H
LYS+
90
4.935
13.847
7.922
1.00
0.00


ATOM
1497
CA
LYS+
90
5.730
12.406
9.200
1.00
0.00


ATOM
1498
HA
LYS+
90
6.722
12.251
9.623
1.00
0.00


ATOM
1499
CB
LYS+
90
4.775
12.666
10.367
1.00
0.00


ATOM
1500
2HB
LYS+
90
3.924
11.986
10.303
1.00
0.00


ATOM
1501
QB
LYS+
90
3.924
11.986
10.303
1.00
0.00


ATOM
1502
CG
LYS+
90
5.485
12.477
11.709
1.00
0.00


ATOM
1503
2HG
LYS+
90
6.030
11.532
11.706
1.00
0.00


ATOM
1504
QG
LYS+
90
6.030
11.532
11.706
1.00
0.00


ATOM
1505
CD
LYS+
90
6.453
13.629
11.985
1.00
0.00


ATOM
1506
2HD
LYS+
90
5.892
14.519
12.270
1.00
0.00


ATOM
1507
QD
LYS+
90
5.892
14.519
12.270
1.00
0.00


ATOM
1508
CE
LYS+
90
7.439
13.263
13.096
1.00
0.00


ATOM
1509
2HE
LYS+
90
7.270
12.235
13.415
1.00
0.00


ATOM
1510
QE
LYS+
90
7.270
12.235
13.415
1.00
0.00


ATOM
1511
NZ
LYS+
90
8.833
13.421
12.624
1.00
0.00


ATOM
1512
1HZ
LYS+
90
8.915
13.052
11.699
1.00
0.00


ATOM
1513
2HZ
LYS+
90
9.075
14.392
12.618
1.00
0.00


ATOM
1514
3HZ
LYS+
90
9.450
12.928
13.237
1.00
0.00


ATOM
1515
QZ
LYS+
90
9.147
13.457
12.518
1.00
0.00


ATOM
1516
C
LYS+
90
5.361
11.179
8.364
1.00
0.00


ATOM
1517
O
LYS+
90
5.516
10.046
8.817
1.00
0.00


ATOM
1518
N
LEU
91
4.880
11.446
7.159
1.00
0.00


ATOM
1519
H
LEU
91
4.758
12.371
6.798
1.00
0.00


ATOM
1520
CA
LEU
91
4.488
10.377
6.256
1.00
0.00


ATOM
1521
HA
LEU
91
3.794
9.733
6.795
1.00
0.00


ATOM
1522
CB
LEU
91
3.738
10.943
5.050
1.00
0.00


ATOM
1523
2HB
LEU
91
4.191
10.543
4.143
1.00
0.00


ATOM
1524
QB
LEU
91
4.191
10.543
4.143
1.00
0.00


ATOM
1525
CG
LEU
91
2.234
10.661
5.002
1.00
0.00


ATOM
1526
HG
LEU
91
1.800
10.961
5.956
1.00
0.00


ATOM
1527
QD1
LEU
91
1.392
11.691
3.656
1.00
0.00


ATOM
1528
QD2
LEU
91
1.898
8.809
4.793
1.00
0.00


ATOM
1529
CD1
LEU
91
1.553
11.494
3.914
1.00
0.00


ATOM
1530
1HD1
LEU
91
0.959
12.281
4.377
1.00
0.00


ATOM
1531
2HD1
LEU
91
2.312
11.941
3.272
1.00
0.00


ATOM
1532
3HD1
LEU
91
0.905
10.852
3.317
1.00
0.00


ATOM
1533
CD2
LEU
91
1.962
9.165
4.833
1.00
0.00


ATOM
1534
1HD2
LEU
91
2.827
8.688
4.370
1.00
0.00


ATOM
1535
2HD2
LEU
91
1.780
8.716
5.809
1.00
0.00


ATOM
1536
3HD2
LEU
91
1.087
9.024
4.199
1.00
0.00


ATOM
1537
QQD
LEU
91
1.645
10.250
4.224
1.00
0.00


ATOM
1538
C
LEU
91
5.723
9.554
5.881
1.00
0.00


ATOM
1539
O
LEU
91
5.681
8.324
5.895
1.00
0.00


ATOM
1540
N
ASN
92
6.792
10.265
5.556
1.00
0.00


ATOM
1541
H
ASN
92
6.818
11.265
5.547
1.00
0.00


ATOM
1542
CA
ASN
92
8.036
9.616
5.177
1.00
0.00


ATOM
1543
HA
ASN
92
7.933
9.395
4.115
1.00
0.00


ATOM
1544
CB
ASN
92
9.234
10.534
5.426
1.00
0.00


ATOM
1545
2HB
ASN
92
9.660
10.324
6.407
1.00
0.00


ATOM
1546
QB
ASN
92
9.660
10.324
6.407
1.00
0.00


ATOM
1547
CG
ASN
92
10.303
10.343
4.348
1.00
0.00


ATOM
1548
OD1
ASN
92
10.321
11.015
3.330
1.00
0.00


ATOM
1549
ND2
ASN
92
11.190
9.393
4.628
1.00
0.00


ATOM
1550
1HD2
ASN
92
11.118
8.878
5.482
1.00
0.00


ATOM
1551
2HD2
ASN
92
11.929
9.193
3.984
1.00
0.00


ATOM
1552
QD2
ASN
92
11.524
9.036
4.733
1.00
0.00


ATOM
1553
C
ASN
92
8.224
8.353
6.021
1.00
0.00


ATOM
1554
O
ASN
92
8.761
7.357
5.542
1.00
0.00


ATOM
1555
N
THR
93
7.772
8.438
7.264
1.00
0.00


ATOM
1556
H
THR
93
7.336
9.253
7.646
1.00
0.00


ATOM
1557
CA
THR
93
7.883
7.314
8.179
1.00
0.00


ATOM
1558
HA
THR
93
8.741
6.712
7.880
1.00
0.00


ATOM
1559
CB
THR
93
8.115
7.872
9.585
1.00
0.00


ATOM
1560
HB
THR
93
7.347
8.599
9.847
1.00
0.00


ATOM
1561
QG2
THR
93
8.231
6.509
10.894
1.00
0.00


ATOM
1562
OG1
THR
93
9.432
8.414
9.530
1.00
0.00


ATOM
1563
1HG
THR
93
10.069
7.731
9.172
1.00
0.00


ATOM
1564
CG2
THR
93
8.209
6.771
10.643
1.00
0.00


ATOM
1565
1HG2
THR
93
9.252
6.624
10.923
1.00
0.00


ATOM
1566
2HG2
THR
93
7.634
7.062
11.522
1.00
0.00


ATOM
1567
3HG2
THR
93
7.808
5.842
10.238
1.00
0.00


ATOM
1568
C
THR
93
6.647
6.419
8.077
1.00
0.00


ATOM
1569
O
THR
93
6.755
5.196
8.153
1.00
0.00


ATOM
1570
N
ILE
94
5.502
7.062
7.907
1.00
0.00


ATOM
1571
H
ILE
94
5.422
8.057
7.845
1.00
0.00


ATOM
1572
CA
ILE
94
4.247
6.338
7.793
1.00
0.00


ATOM
1573
HA
ILE
94
4.186
5.656
8.641
1.00
0.00


ATOM
1574
CB
ILE
94
3.062
7.300
7.900
1.00
0.00


ATOM
1575
HB
ILE
94
3.069
7.950
7.025
1.00
0.00


ATOM
1576
QG2
ILE
94
1.420
6.359
7.883
1.00
0.00


ATOM
1577
CG2
ILE
94
1.735
6.539
7.886
1.00
0.00


ATOM
1578
1HG2
ILE
94
1.365
6.472
6.863
1.00
0.00


ATOM
1579
2HG2
ILE
94
1.887
5.536
8.284
1.00
0.00


ATOM
1580
3HG2
ILE
94
1.007
7.067
8.501
1.00
0.00


ATOM
1581
CG1
ILE
94
3.195
8.199
9.131
1.00
0.00


ATOM
1582
2HG1
ILE
94
2.774
9.182
8.916
1.00
0.00


ATOM
1583
QG1
ILE
94
2.774
9.182
8.916
1.00
0.00


ATOM
1584
QD1
ILE
94
2.312
7.440
10.624
1.00
0.00


ATOM
1585
CD1
ILE
94
2.481
7.586
10.338
1.00
0.00


ATOM
1586
1HD1
ILE
94
1.424
7.847
10.306
1.00
0.00


ATOM
1587
2HD1
ILE
94
2.590
6.501
10.311
1.00
0.00


ATOM
1588
3HD1
ILE
94
2.923
7.972
11.256
1.00
0.00


ATOM
1589
C
ILE
94
4.254
5.513
6.505
1.00
0.00


ATOM
1590
O
ILE
94
3.806
4.366
6.496
1.00
0.00


ATOM
1591
N
ILE
95
4.766
6.127
5.449
1.00
0.00


ATOM
1592
H
ILE
95
5.129
7.058
5.465
1.00
0.00


ATOM
1593
CA
ILE
95
4.838
5.462
4.158
1.00
0.00


ATOM
1594
HA
ILE
95
3.911
4.904
4.027
1.00
0.00


ATOM
1595
CB
ILE
95
4.910
6.491
3.029
1.00
0.00


ATOM
1596
HB
ILE
95
3.928
6.952
2.924
1.00
0.00


ATOM
1597
QG2
ILE
95
6.135
7.873
3.448
1.00
0.00


ATOM
1598
CG2
ILE
95
5.899
7.608
3.368
1.00
0.00


ATOM
1599
1HG2
ILE
95
6.557
7.280
4.174
1.00
0.00


ATOM
1600
2HG2
ILE
95
6.496
7.845
2.486
1.00
0.00


ATOM
1601
3HG2
ILE
95
5.351
8.495
3.685
1.00
0.00


ATOM
1602
CG1
ILE
95
5.239
5.819
1.695
1.00
0.00


ATOM
1603
2HG1
ILE
95
5.741
4.868
1.876
1.00
0.00


ATOM
1604
QG1
ILE
95
5.741
4.868
1.876
1.00
0.00


ATOM
1605
QD1
ILE
95
3.670
5.528
0.676
1.00
0.00


ATOM
1606
CD1
ILE
95
3.971
5.584
0.872
1.00
0.00


ATOM
1607
1HD1
ILE
95
3.525
6.543
0.610
1.00
0.00


ATOM
1608
2HD1
ILE
95
4.224
5.040
−0.039
1.00
0.00


ATOM
1609
3HD1
ILE
95
3.261
5.000
1.458
1.00
0.00


ATOM
1610
C
ILE
95
6.004
4.471
4.164
1.00
0.00


ATOM
1611
O
ILE
95
5.882
3.361
3.648
1.00
0.00


ATOM
1612
N
LYS+
96
7.108
4.909
4.752
1.00
0.00


ATOM
1613
H
LYS+
96
7.198
5.813
5.169
1.00
0.00


ATOM
1614
CA
LYS+
96
8.295
4.074
4.830
1.00
0.00


ATOM
1615
HA
LYS+
96
8.530
3.744
3.818
1.00
0.00


ATOM
1616
CB
LYS+
96
9.489
4.888
5.331
1.00
0.00


ATOM
1617
2HB
LYS+
96
9.234
5.377
6.271
1.00
0.00


ATOM
1618
QB
LYS+
96
9.234
5.377
6.271
1.00
0.00


ATOM
1619
CG
LYS+
96
10.715
3.995
5.531
1.00
0.00


ATOM
1620
2HG
LYS+
96
10.966
3.499
4.593
1.00
0.00


ATOM
1621
QG
LYS+
96
10.966
3.499
4.593
1.00
0.00


ATOM
1622
CD
LYS+
96
11.915
4.812
6.018
1.00
0.00


ATOM
1623
2HD
LYS+
96
11.784
5.064
7.070
1.00
0.00


ATOM
1624
QD
LYS+
96
11.784
5.064
7.070
1.00
0.00


ATOM
1625
CE
LYS+
96
13.219
4.034
5.831
1.00
0.00


ATOM
1626
2HE
LYS+
96
13.016
3.087
5.332
1.00
0.00


ATOM
1627
QE
LYS+
96
13.016
3.087
5.332
1.00
0.00


ATOM
1628
NZ
LYS+
96
14.185
4.825
5.035
1.00
0.00


ATOM
1629
1HZ
LYS+
96
14.871
5.224
5.644
1.00
0.00


ATOM
1630
2HZ
LYS+
96
14.635
4.229
4.371
1.00
0.00


ATOM
1631
3HZ
LYS+
96
13.700
5.556
4.554
1.00
0.00


ATOM
1632
QZ
LYS+
96
14.402
5.003
4.857
1.00
0.00


ATOM
1633
C
LYS+
96
7.990
2.841
5.684
1.00
0.00


ATOM
1634
O
LYS+
96
8.608
1.792
5.508
1.00
0.00


ATOM
1635
N
GLN
97
7.038
3.009
6.590
1.00
0.00


ATOM
1636
H
GLN
97
6.540
3.865
6.727
1.00
0.00


ATOM
1637
CA
GLN
97
6.645
1.922
7.472
1.00
0.00


ATOM
1638
HA
GLN
97
7.558
1.362
7.667
1.00
0.00


ATOM
1639
CB
GLN
97
6.095
2.461
8.794
1.00
0.00


ATOM
1640
2HB
GLN
97
5.503
1.689
9.287
1.00
0.00


ATOM
1641
QB
GLN
97
5.503
1.689
9.287
1.00
0.00


ATOM
1642
CG
GLN
97
7.228
2.910
9.717
1.00
0.00


ATOM
1643
2HG
GLN
97
6.845
3.619
10.451
1.00
0.00


ATOM
1644
QG
GLN
97
6.845
3.619
10.451
1.00
0.00


ATOM
1645
CD
GLN
97
7.858
1.715
10.435
1.00
0.00


ATOM
1646
OE1
GLN
97
8.842
1.142
9.997
1.00
0.00


ATOM
1647
NE2
GLN
97
7.237
1.371
11.560
1.00
0.00


ATOM
1648
1HE2
GLN
97
6.433
1.883
11.864
1.00
0.00


ATOM
1649
2HE2
GLN
97
7.573
0.600
12.102
1.00
0.00


ATOM
1650
QE2
GLN
97
7.003
1.242
11.983
1.00
0.00


ATOM
1651
C
GLN
97
5.621
1.021
6.778
1.00
0.00


ATOM
1652
O
GLN
97
5.713
−0.204
6.855
1.00
0.00


ATOM
1653
N
ILE
98
4.668
1.661
6.116
1.00
0.00


ATOM
1654
H
ILE
98
4.600
2.658
6.059
1.00
0.00


ATOM
1655
CA
ILE
98
3.628
0.933
5.410
1.00
0.00


ATOM
1656
HA
ILE
98
3.396
0.043
5.997
1.00
0.00


ATOM
1657
CB
ILE
98
2.348
1.768
5.331
1.00
0.00


ATOM
1658
HB
ILE
98
2.612
2.813
5.488
1.00
0.00


ATOM
1659
QG2
ILE
98
1.561
1.641
3.614
1.00
0.00


ATOM
1660
CG2
ILE
98
1.711
1.665
3.943
1.00
0.00


ATOM
1661
1HG2
ILE
98
0.790
2.246
3.923
1.00
0.00


ATOM
1662
2HG2
ILE
98
2.404
2.055
3.197
1.00
0.00


ATOM
1663
3HG2
ILE
98
1.489
0.621
3.723
1.00
0.00


ATOM
1664
CG1
ILE
98
1.370
1.377
6.441
1.00
0.00


ATOM
1665
2HG1
ILE
98
1.122
0.319
6.357
1.00
0.00


ATOM
1666
QG1
ILE
98
1.122
0.319
6.357
1.00
0.00


ATOM
1667
QD1
ILE
98
2.106
1.728
8.149
1.00
0.00


ATOM
1668
CD1
ILE
98
1.964
1.661
7.821
1.00
0.00


ATOM
1669
1HD1
ILE
98
2.241
2.713
7.889
1.00
0.00


ATOM
1670
2HD1
ILE
98
1.226
1.429
8.590
1.00
0.00


ATOM
1671
3HD1
ILE
98
2.850
1.042
7.969
1.00
0.00


ATOM
1672
C
ILE
98
4.158
0.490
4.045
1.00
0.00


ATOM
1673
O
ILE
98
3.437
−0.133
3.267
1.00
0.00


ATOM
1674
N
LEU
99
5.415
0.828
3.797
1.00
0.00


ATOM
1675
H
LEU
99
5.995
1.335
4.435
1.00
0.00


ATOM
1676
CA
LEU
99
6.051
0.473
2.539
1.00
0.00


ATOM
1677
HA
LEU
99
5.324
−0.088
1.952
1.00
0.00


ATOM
1678
CB
LEU
99
6.400
1.731
1.742
1.00
0.00


ATOM
1679
2HB
LEU
99
7.193
2.263
2.267
1.00
0.00


ATOM
1680
QB
LEU
99
7.193
2.263
2.267
1.00
0.00


ATOM
1681
CG
LEU
99
6.848
1.507
0.296
1.00
0.00


ATOM
1682
HG
LEU
99
7.142
0.463
0.188
1.00
0.00


ATOM
1683
QD1
LEU
99
5.422
1.813
−0.911
1.00
0.00


ATOM
1684
QD2
LEU
99
8.365
2.561
−0.116
1.00
0.00


ATOM
1685
CD1
LEU
99
5.696
1.754
−0.680
1.00
0.00


ATOM
1686
1HD1
LEU
99
5.567
0.881
−1.320
1.00
0.00


ATOM
1687
2HD1
LEU
99
4.777
1.932
−0.120
1.00
0.00


ATOM
1688
3HD1
LEU
99
5.921
2.625
−1.294
1.00
0.00


ATOM
1689
CD2
LEU
99
8.074
2.358
−0.038
1.00
0.00


ATOM
1690
1HD2
LEU
99
8.689
2.475
0.855
1.00
0.00


ATOM
1691
2HD2
LEU
99
8.656
1.869
−0.818
1.00
0.00


ATOM
1692
3HD2
LEU
99
7.751
3.339
−0.387
1.00
0.00


ATOM
1693
QQD
LEU
99
6.893
2.187
−0.514
1.00
0.00


ATOM
1694
C
LEU
99
7.252
−0.432
2.818
1.00
0.00


ATOM
1695
O
LEU
99
7.678
−1.189
1.947
1.00
0.00


ATOM
1696
N
SER
100
7.764
−0.324
4.035
1.00
0.00


ATOM
1697
H
SER
100
7.412
0.295
4.737
1.00
0.00


ATOM
1698
CA
SER
100
8.909
−1.122
4.439
1.00
0.00


ATOM
1699
HA
SER
100
9.380
−1.442
3.509
1.00
0.00


ATOM
1700
CB
SER
100
9.902
−0.291
5.254
1.00
0.00


ATOM
1701
2HB
SER
100
9.441
0.002
6.197
1.00
0.00


ATOM
1702
QB
SER
100
9.441
0.002
6.197
1.00
0.00


ATOM
1703
OG
SER
100
11.107
−1.004
5.516
1.00
0.00


ATOM
1704
HG
SER
100
11.337
−0.940
6.487
1.00
0.00


ATOM
1705
C
SER
100
8.441
−2.334
5.248
1.00
0.00


ATOM
1706
O
SER
100
9.215
−3.257
5.493
1.00
0.00


ATOM
1707
N
ILE
101
7.177
−2.290
5.642
1.00
0.00


ATOM
1708
H
ILE
101
6.554
−1.535
5.439
1.00
0.00


ATOM
1709
CA
ILE
101
6.597
−3.372
6.420
1.00
0.00


ATOM
1710
HA
ILE
101
7.257
−3.556
7.267
1.00
0.00


ATOM
1711
CB
ILE
101
5.237
−2.957
6.985
1.00
0.00


ATOM
1712
HB
ILE
101
4.879
−2.098
6.417
1.00
0.00


ATOM
1713
QG2
ILE
101
3.963
−4.340
6.774
1.00
0.00


ATOM
1714
CG2
ILE
101
4.208
−4.075
6.815
1.00
0.00


ATOM
1715
1HG2
ILE
101
4.455
−4.902
7.480
1.00
0.00


ATOM
1716
2HG2
ILE
101
3.215
−3.696
7.060
1.00
0.00


ATOM
1717
3HG2
ILE
101
4.218
−4.423
5.782
1.00
0.00


ATOM
1718
CG1
ILE
101
5.364
−2.513
8.445
1.00
0.00


ATOM
1719
2HG1
ILE
101
5.288
−3.380
9.101
1.00
0.00


ATOM
1720
QG1
ILE
101
5.288
−3.380
9.101
1.00
0.00


ATOM
1721
QD1
ILE
101
7.012
−1.629
8.744
1.00
0.00


ATOM
1722
CD1
ILE
101
6.696
−1.798
8.686
1.00
0.00


ATOM
1723
1HD1
ILE
101
7.136
−1.515
7.730
1.00
0.00


ATOM
1724
2HD1
ILE
101
6.524
−0.904
9.286
1.00
0.00


ATOM
1725
3HD1
ILE
101
7.376
−2.466
9.216
1.00
0.00


ATOM
1726
C
ILE
101
6.545
−4.638
5.563
1.00
0.00


ATOM
1727
O
ILE
101
6.205
−5.713
6.056
1.00
0.00


ATOM
1728
N
SER
102
6.886
−4.470
4.293
1.00
0.00


ATOM
1729
H
SER
102
7.161
−3.593
3.900
1.00
0.00


ATOM
1730
CA
SER
102
6.884
−5.587
3.362
1.00
0.00


ATOM
1731
HA
SER
102
7.710
−5.395
2.679
1.00
0.00


ATOM
1732
CB
SER
102
7.121
−6.911
4.091
1.00
0.00


ATOM
1733
2HB
SER
102
7.391
−7.679
3.366
1.00
0.00


ATOM
1734
QB
SER
102
7.391
−7.679
3.366
1.00
0.00


ATOM
1735
OG
SER
102
8.148
−6.804
5.072
1.00
0.00


ATOM
1736
HG
SER
102
7.837
−7.198
5.937
1.00
0.00


ATOM
1737
C
SER
102
5.556
−5.629
2.603
1.00
0.00


ATOM
1738
O
SER
102
5.015
−6.706
2.352
1.00
0.00


ATOM
1739
N
VAL
103
5.070
−4.447
2.257
1.00
0.00


ATOM
1740
H
VAL
103
5.517
−3.577
2.464
1.00
0.00


ATOM
1741
CA
VAL
103
3.817
−4.336
1.529
1.00
0.00


ATOM
1742
HA
VAL
103
3.385
−5.334
1.465
1.00
0.00


ATOM
1743
CB
VAL
103
2.841
−3.447
2.303
1.00
0.00


ATOM
1744
HB
VAL
103
2.088
−4.092
2.757
1.00
0.00


ATOM
1745
QG1
VAL
103
3.726
−2.516
3.693
1.00
0.00


ATOM
1746
QG2
VAL
103
1.951
−2.246
1.141
1.00
0.00


ATOM
1747
CG1
VAL
103
3.556
−2.695
3.425
1.00
0.00


ATOM
1748
1HG1
VAL
103
4.001
−3.411
4.117
1.00
0.00


ATOM
1749
2HG1
VAL
103
4.339
−2.065
3.001
1.00
0.00


ATOM
1750
3HG1
VAL
103
2.839
−2.072
3.960
1.00
0.00


ATOM
1751
CG2
VAL
103
2.122
−2.476
1.364
1.00
0.00


ATOM
1752
1HG2
VAL
103
1.426
−1.863
1.938
1.00
0.00


ATOM
1753
2HG2
VAL
103
2.855
−1.834
0.875
1.00
0.00


ATOM
1754
3HG2
VAL
103
1.572
−3.040
0.609
1.00
0.00


ATOM
1755
QQG
VAL
103
2.839
−2.381
2.417
1.00
0.00


ATOM
1756
C
VAL
103
4.096
−3.827
0.114
1.00
0.00


ATOM
1757
O
VAL
103
3.389
−4.180
−0.827
1.00
0.00


ATOM
1758
N
SER
104
5.130
−3.005
0.008
1.00
0.00


ATOM
1759
H
SER
104
5.701
−2.722
0.779
1.00
0.00


ATOM
1760
CA
SER
104
5.512
−2.444
−1.276
1.00
0.00


ATOM
1761
HA
SER
104
4.758
−1.688
−1.496
1.00
0.00


ATOM
1762
CB
SER
104
6.890
−1.783
−1.202
1.00
0.00


ATOM
1763
2HB
SER
104
7.654
−2.506
−1.487
1.00
0.00


ATOM
1764
QB
SER
104
7.654
−2.506
−1.487
1.00
0.00


ATOM
1765
OG
SER
104
6.980
−0.638
−2.045
1.00
0.00


ATOM
1766
HG
SER
104
7.685
−0.017
−1.704
1.00
0.00


ATOM
1767
C
SER
104
5.505
−3.536
−2.347
1.00
0.00


ATOM
1768
O
SER
104
4.876
−3.382
−3.393
1.00
0.00


ATOM
1769
N
GLU−
105
6.212
−4.616
−2.050
1.00
0.00


ATOM
1770
H
GLU−
105
6.721
−4.735
−1.197
1.00
0.00


ATOM
1771
CA
GLU−
105
6.295
−5.735
−2.974
1.00
0.00


ATOM
1772
HA
GLU−
105
5.305
−6.190
−2.967
1.00
0.00


ATOM
1773
CB
GLU−
105
6.615
−5.252
−4.391
1.00
0.00


ATOM
1774
2HB
GLU−
105
7.595
−5.622
−4.693
1.00
0.00


ATOM
1775
QB
GLU−
105
7.595
−5.622
−4.693
1.00
0.00


ATOM
1776
CG
GLU−
105
5.555
−5.731
−5.385
1.00
0.00


ATOM
1777
2HG
GLU−
105
4.563
−5.452
−5.027
1.00
0.00


ATOM
1778
QG
GLU−
105
4.563
−5.452
−5.027
1.00
0.00


ATOM
1779
CD
GLU−
105
5.791
−5.127
−6.771
1.00
0.00


ATOM
1780
OE1
GLU−
105
5.357
−3.972
−6.968
1.00
0.00


ATOM
1781
OE2
GLU−
105
6.400
−5.835
−7.601
1.00
0.00


ATOM
1782
C
GLU−
105
7.337
−6.747
−2.491
1.00
0.00


ATOM
1783
O
GLU−
105
7.985
−7.409
−3.301
1.00
0.00


ATOM
1784
N
GLU−
106
7.464
−6.833
−1.175
1.00
0.00


ATOM
1785
H
GLU−
106
6.933
−6.291
−0.525
1.00
0.00


ATOM
1786
CA
GLU−
106
8.417
−7.753
−0.576
1.00
0.00


ATOM
1787
HA
GLU−
106
8.749
−7.268
0.342
1.00
0.00


ATOM
1788
CB
GLU−
106
7.747
−9.083
−0.225
1.00
0.00


ATOM
1789
2HB
GLU−
106
7.505
−9.625
−1.139
1.00
0.00


ATOM
1790
QB
GLU−
106
7.505
−9.625
−1.139
1.00
0.00


ATOM
1791
CG
GLU−
106
8.657
−9.939
0.658
1.00
0.00


ATOM
1792
2HG
GLU−
106
9.700
−9.762
0.391
1.00
0.00


ATOM
1793
QG
GLU−
106
9.700
−9.762
0.391
1.00
0.00


ATOM
1794
CD
GLU−
106
8.443
−9.622
2.139
1.00
0.00


ATOM
1795
OE1
GLU−
106
9.177
−8.745
2.644
1.00
0.00


ATOM
1796
OE2
GLU−
106
7.549
−10.262
2.733
1.00
0.00


ATOM
1797
C
GLU−
106
9.607
−7.969
−1.511
1.00
0.00


ATOM
1798
O
GLU−
106
10.097
−9.089
−1.650
1.00
0.00


ATOM
1799
N
GLY
107
10.040
−6.879
−2.129
1.00
0.00


ATOM
1800
H
GLY
107
9.637
−5.972
−2.009
1.00
0.00


ATOM
1801
CA
GLY
107
11.165
−6.936
−3.047
1.00
0.00


ATOM
1802
1HA
GLY
107
11.323
−7.963
−3.371
1.00
0.00


ATOM
1803
2HA
GLY
107
10.941
−6.350
−3.938
1.00
0.00


ATOM
1804
QA
GLY
107
11.132
−7.157
−3.655
1.00
0.00


ATOM
1805
C
GLY
107
12.438
−6.402
−2.387
1.00
0.00


ATOM
1806
O
GLY
107
13.094
−5.512
−2.924
1.00
0.00


ATOM
1807
N
GLU−
108
12.751
−6.971
−1.231
1.00
0.00


ATOM
1808
H
GLU−
108
12.212
−7.696
−0.801
1.00
0.00


ATOM
1809
CA
GLU−
108
13.935
−6.565
−0.493
1.00
0.00


ATOM
1810
HA
GLU−
108
13.967
−7.215
0.382
1.00
0.00


ATOM
1811
CB
GLU−
108
15.200
−6.779
−1.325
1.00
0.00


ATOM
1812
2HB
GLU−
108
15.710
−5.827
−1.469
1.00
0.00


ATOM
1813
QB
GLU−
108
15.710
−5.827
−1.469
1.00
0.00


ATOM
1814
CG
GLU−
108
16.144
−7.771
−0.642
1.00
0.00


ATOM
1815
2HG
GLU−
108
15.921
−8.782
−0.980
1.00
0.00


ATOM
1816
QG
GLU−
108
15.921
−8.782
−0.980
1.00
0.00


ATOM
1817
CD
GLU−
108
17.606
−7.437
−0.946
1.00
0.00


ATOM
1818
OE1
GLU−
108
18.056
−7.814
−2.050
1.00
0.00


ATOM
1819
OE2
GLU−
108
18.239
−6.811
−0.069
1.00
0.00


ATOM
1820
C
GLU−
108
13.809
−5.106
−0.051
1.00
0.00


ATOM
1821
O
GLU−
108
14.799
−4.377
−0.016
1.00
0.00


ATOM
1822
N
LYS+
109
12.583
−4.723
0.275
1.00
0.00


ATOM
1823
H
LYS+
109
11.783
−5.322
0.244
1.00
0.00


ATOM
1824
CA
LYS+
109
12.316
−3.364
0.714
1.00
0.00


ATOM
1825
HA
LYS+
109
11.447
−3.398
1.372
1.00
0.00


ATOM
1826
CB
LYS+
109
13.487
−2.826
1.537
1.00
0.00


ATOM
1827
2HB
LYS+
109
14.228
−2.379
0.874
1.00
0.00


ATOM
1828
QB
LYS+
109
14.228
−2.379
0.874
1.00
0.00


ATOM
1829
CG
LYS+
109
13.012
−1.786
2.553
1.00
0.00


ATOM
1830
2HG
LYS+
109
11.991
−2.013
2.862
1.00
0.00


ATOM
1831
QG
LYS+
109
11.991
−2.013
2.862
1.00
0.00


ATOM
1832
CD
LYS+
109
13.926
−1.758
3.779
1.00
0.00


ATOM
1833
2HD
LYS+
109
14.389
−2.735
3.914
1.00
0.00


ATOM
1834
QD
LYS+
109
14.389
−2.735
3.914
1.00
0.00


ATOM
1835
CE
LYS+
109
15.012
−0.690
3.629
1.00
0.00


ATOM
1836
2HE
LYS+
109
14.904
−0.186
2.669
1.00
0.00


ATOM
1837
QE
LYS+
109
14.904
−0.186
2.669
1.00
0.00


ATOM
1838
NZ
LYS+
109
14.924
0.297
4.727
1.00
0.00


ATOM
1839
1HZ
LYS+
109
15.235
1.190
4.399
1.00
0.00


ATOM
1840
2HZ
LYS+
109
13.975
0.369
5.037
1.00
0.00


ATOM
1841
3HZ
LYS+
109
15.502
0.002
5.489
1.00
0.00


ATOM
1842
QZ
LYS+
109
14.904
0.520
4.975
1.00
0.00


ATOM
1843
C
LYS+
109
11.969
−2.500
−0.500
1.00
0.00


ATOM
1844
O
LYS+
109
11.079
−1.653
−0.431
1.00
0.00


ATOM
1845
N
GLU−
110
12.691
−2.743
−1.585
1.00
0.00


ATOM
1846
H
GLU−
110
13.412
−3.434
−1.633
1.00
0.00


ATOM
1847
CA
GLU−
110
12.471
−1.998
−2.813
1.00
0.00


ATOM
1848
HA
GLU−
110
11.563
−2.416
−3.247
1.00
0.00


ATOM
1849
CB
GLU−
110
12.248
−0.512
−2.520
1.00
0.00


ATOM
1850
2HB
GLU−
110
12.944
0.086
−3.107
1.00
0.00


ATOM
1851
QB
GLU−
110
12.944
0.086
−3.107
1.00
0.00


ATOM
1852
CG
GLU−
110
10.811
−0.100
−2.847
1.00
0.00


ATOM
1853
2HG
GLU−
110
10.166
−0.306
−1.993
1.00
0.00


ATOM
1854
QG
GLU−
110
10.166
−0.306
−1.993
1.00
0.00


ATOM
1855
CD
GLU−
110
10.736
1.387
−3.203
1.00
0.00


ATOM
1856
OE1
GLU−
110
11.163
2.197
−2.351
1.00
0.00


ATOM
1857
OE2
GLU−
110
10.253
1.680
−4.318
1.00
0.00


ATOM
1858
C
GLU−
110
13.646
−2.197
−3.772
1.00
0.00


ATOM
1859
O
GLU−
110
14.492
−1.317
−3.914
1.00
0.00


ATOM
1860
N
LEU
111
13.660
−3.361
−4.405
1.00
0.00


ATOM
1861
H
LEU
111
12.968
−4.072
−4.284
1.00
0.00


ATOM
1862
CA
LEU
111
14.718
−3.687
−5.347
1.00
0.00


ATOM
1863
HA
LEU
111
14.534
−4.700
−5.706
1.00
0.00


ATOM
1864
CB
LEU
111
14.658
−2.759
−6.562
1.00
0.00


ATOM
1865
2HB
LEU
111
15.655
−2.357
−6.737
1.00
0.00


ATOM
1866
QB
LEU
111
15.655
−2.357
−6.737
1.00
0.00


ATOM
1867
CG
LEU
111
14.157
−3.390
−7.864
1.00
0.00


ATOM
1868
HG
LEU
111
14.217
−4.474
−7.764
1.00
0.00


ATOM
1869
QD1
LEU
111
12.340
−2.956
−8.172
1.00
0.00


ATOM
1870
QD2
LEU
111
15.260
−2.901
−9.323
1.00
0.00


ATOM
1871
CD1
LEU
111
12.689
−3.040
−8.113
1.00
0.00


ATOM
1872
1HD1
LEU
111
12.624
−2.274
−8.886
1.00
0.00


ATOM
1873
2HD1
LEU
111
12.153
−3.931
−8.439
1.00
0.00


ATOM
1874
3HD1
LEU
111
12.243
−2.665
−7.192
1.00
0.00


ATOM
1875
CD2
LEU
111
15.048
−2.995
−9.043
1.00
0.00


ATOM
1876
1HD2
LEU
111
14.985
−1.918
−9.201
1.00
0.00


ATOM
1877
2HD2
LEU
111
16.081
−3.270
−8.826
1.00
0.00


ATOM
1878
3HD2
LEU
111
14.715
−3.514
−9.941
1.00
0.00


ATOM
1879
QQD
LEU
111
13.800
−2.929
−8.748
1.00
0.00


ATOM
1880
C
LEU
111
16.064
−3.666
−4.623
1.00
0.00


ATOM
1881
O
LEU
111
16.538
−4.700
−4.155
1.00
0.00


ATOM
1882
N
VAL
112
16.643
−2.476
−4.552
1.00
0.00


ATOM
1883
H
VAL
112
16.251
−1.639
−4.935
1.00
0.00


ATOM
1884
CA
VAL
112
17.927
−2.306
−3.892
1.00
0.00


ATOM
1885
HA
VAL
112
18.228
−3.280
−3.504
1.00
0.00


ATOM
1886
CB
VAL
112
18.980
−1.853
−4.906
1.00
0.00


ATOM
1887
HB
VAL
112
18.556
−1.032
−5.485
1.00
0.00


ATOM
1888
QG1
VAL
112
20.530
−1.208
−4.030
1.00
0.00


ATOM
1889
QG2
VAL
112
19.412
−3.251
−6.107
1.00
0.00


ATOM
1890
CG1
VAL
112
20.232
−1.332
−4.198
1.00
0.00


ATOM
1891
1HG1
VAL
112
20.119
−1.454
−3.121
1.00
0.00


ATOM
1892
2HG1
VAL
112
21.102
−1.894
−4.537
1.00
0.00


ATOM
1893
3HG1
VAL
112
20.369
−0.276
−4.431
1.00
0.00


ATOM
1894
CG2
VAL
112
19.329
−2.983
−5.876
1.00
0.00


ATOM
1895
1HG2
VAL
112
20.278
−2.762
−6.365
1.00
0.00


ATOM
1896
2HG2
VAL
112
19.413
−3.920
−5.327
1.00
0.00


ATOM
1897
3HG2
VAL
112
18.545
−3.070
−6.629
1.00
0.00


ATOM
1898
QQG
VAL
112
19.971
−2.229
−5.068
1.00
0.00


ATOM
1899
C
VAL
112
17.768
−1.336
−2.720
1.00
0.00


ATOM
1900
O
VAL
112
17.167
−0.273
−2.867
1.00
0.00


ATOM
1901
N
PRO
113
18.333
−1.747
−1.553
1.00
0.00


ATOM
1902
CD
PRO
113
19.053
−3.001
−1.343
1.00
0.00


ATOM
1903
CA
PRO
113
18.260
−0.927
−0.356
1.00
0.00


ATOM
1904
HA
PRO
113
17.347
−0.530
−0.262
1.00
0.00


ATOM
1905
CB
PRO
113
18.584
−1.872
0.789
1.00
0.00


ATOM
1906
2HB
PRO
113
17.679
−2.177
1.312
1.00
0.00


ATOM
1907
QB
PRO
113
17.679
−2.177
1.312
1.00
0.00


ATOM
1908
CG
PRO
113
19.279
−3.068
0.158
1.00
0.00


ATOM
1909
2HG
PRO
113
18.880
−3.998
0.564
1.00
0.00


ATOM
1910
QG
PRO
113
18.880
−3.998
0.564
1.00
0.00


ATOM
1911
2HD
PRO
113
18.474
−3.854
−1.695
1.00
0.00


ATOM
1912
QD
PRO
113
18.474
−3.854
−1.695
1.00
0.00


ATOM
1913
C
PRO
113
19.223
0.259
−0.445
1.00
0.00


ATOM
1914
O
PRO
113
18.821
1.405
−0.255
1.00
0.00


ATOM
1915
N
ARG+
114
20.477
−0.059
−0.734
1.00
0.00


ATOM
1916
H
ARG+
114
20.795
−0.994
−0.887
1.00
0.00


ATOM
1917
CA
ARG+
114
21.501
0.966
−0.850
1.00
0.00


ATOM
1918
HA
ARG+
114
21.073
1.843
−0.365
1.00
0.00


ATOM
1919
CB
ARG+
114
22.788
0.541
−0.139
1.00
0.00


ATOM
1920
2HB
ARG+
114
23.386
−0.081
−0.806
1.00
0.00


ATOM
1921
QB
ARG+
114
23.386
−0.081
−0.806
1.00
0.00


ATOM
1922
CG
ARG+
114
23.601
1.761
0.297
1.00
0.00


ATOM
1923
2HG
ARG+
114
22.941
2.496
0.759
1.00
0.00


ATOM
1924
QG
ARG+
114
22.941
2.496
0.759
1.00
0.00


ATOM
1925
CD
ARG+
114
24.700
1.362
1.284
1.00
0.00


ATOM
1926
2HD
ARG+
114
24.344
0.564
1.935
1.00
0.00


ATOM
1927
QD
ARG+
114
24.344
0.564
1.935
1.00
0.00


ATOM
1928
NE
ARG+
114
25.904
0.915
0.549
1.00
0.00


ATOM
1929
HE
ARG+
114
26.619
1.589
0.365
1.00
0.00


ATOM
1930
CZ
ARG+
114
26.095
−0.338
0.112
1.00
0.00


ATOM
1931
NH1
ARG+
114
27.217
−0.654
−0.548
1.00
0.00


ATOM
1932
1HH1
ARG+
114
27.359
−1.588
−0.873
1.00
0.00


ATOM
1933
2HH1
ARG+
114
27.913
0.045
−0.713
1.00
0.00


ATOM
1934
QH1
ARG+
114
27.636
−0.771
−0.793
1.00
0.00


ATOM
1935
NH2
ARG+
114
25.162
−1.274
0.335
1.00
0.00


ATOM
1936
1HH2
ARG+
114
25.305
−2.209
0.009
1.00
0.00


ATOM
1937
2HH2
ARG+
114
24.324
−1.038
0.826
1.00
0.00


ATOM
1938
QH2
ARG+
114
24.815
−1.623
0.417
1.00
0.00


ATOM
1939
C
ARG+
114
21.807
1.245
−2.323
1.00
0.00


ATOM
1940
O
ARG+
114
22.177
0.338
−3.066
1.00
0.00

## Claims

1. An isolated nucleic acid molecule encoding a polypeptide of no more than 125 amino acids in length comprising:
(a) an amino acid sequence as set forth in SEQ ID NO: 2;
(b) an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, and which is capable of associating with a membrane;
(c) an amino acid sequence that differs from SEQ ID NO: 2 by no more than about 10 conserved amino acid substitutions, and which is capable of associating with a membrane; or
(d) an amino acid sequence comprising amino acids 27-110 of SEQ ID NO: 2 which is capable of associating with a membrane.

2. The isolated nucleic acid of claim 1, wherein the polypeptide has no more than about 35% hydrophobic residues.

3. The nucleic acid molecule of claim 1 comprising a nucleic acid sequence as set forth in SEQ ID NO: 1.

4. An isolated nucleic acid encoding a polypeptide, wherein the polypeptide has the tertiary structure of the atomic structure coordinates set forth in PDB Accession No. 1YGM as disclosed in Table 4.

5. An isolated nucleic acid molecule encoding a fusion protein comprising a cargo protein domain and a Mistic domain; wherein the cargo protein domain comprises an integral membrane protein, and wherein the Mistic domain comprises
(a) an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 and is capable of associating with a membrane; or
(b) an amino acid sequence comprising amino acids 27-110 of SEQ ID NO: 2 which is capable of associating with a membrane.

6. The isolated nucleic acid molecule of claim 5, wherein the Mistic domain comprises the amino acid sequence set forth in SEQ ID NO: 2.

7. A method of producing a recombinant fusion protein, comprising expressing the isolated nucleic acid molecule of claim 5 in an expression system, which comprises a membrane or membrane-like structure, wherein at least a portion of the fusion protein is associated with the membrane or membrane-like structure.

8. A vector comprising a promoter sequence operably linked to an isolated nucleic acid molecule encoding a fusion protein comprising a cargo protein domain and a Mistic domain; wherein the Mistic domain comprises
(a) an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 and is capable of associating with a membrane; or
(b) an amino acid sequence comprising amino acids 27-110 of SEQ ID NO: 2 which is capable of associating with a membrane.

9. A cell transformed with the vector of claim 8.

10. The method of claim 7, wherein the expression system is a cell.

11. The vector of claim 8, wherein the cargo protein domain comprises an integral membrane protein.

12. The method of claim 7, further comprising isolating from the expression system a membrane fraction containing the fusion protein.

13. The method of claim 12, further comprising isolating the fusion protein from the membrane fraction.

14. The method of claim 7, wherein the fusion protein further comprises a protease-recognition site between the Mistic domain and the cargo protein domain.

15. A method of stabilizing expression of a recombinant protein comprising co-expressing the recombinant protein with a polypeptide encoded by the isolated nucleic acid molecule according to claim 1.

16. The method of claim 15, wherein stabilizing the expression of the recombinant protein comprises increasing the solubility of the recombinant protein or preventing the aggregation of the recombinant protein.

17. The method of claim 15, wherein co-expression comprising expressing the recombinant protein and the polypeptide as a fusion protein.

18. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprises at least 80% sequence identity to SEQ ID NO: 1.

19. The isolated nucleic acid molecule of claim 5, wherein the fusion protein further comprises a linker between the cargo protein domain and the Mistic domain, and one or more of:
at least one exogenous helix domain, and
a peptide tag.

20. A method for the increased expression of a recombinant fusion protein, comprising:
transfecting a cell with vector of claim 8; and
expressing the fusion protein in a cell, wherein the amount of the cargo protein domain expressed in the cell is greater than the amount expressed in a second cell transfected with an expression vector encoding the cargo protein domain alone.

21. The method of claim 20, wherein the amount of cargo protein domain expressed in the cell is at least 50-fold greater than the amount of cargo protein domain expressed in the second cell.

22. The method of claim 7, wherein 50% or more of the cargo protein domain is incorporated into the membrane.

23. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 which is capable of associating with a membrane.

24. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2 which is capable of associating with a membrane.

25. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprises at least 90% sequence identity to SEQ ID NO: 1.

26. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprises at least 95% sequence identity to SEQ ID NO: 1.

27. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprises at least 98% sequence identity to SEQ ID NO: 1.

28. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes an amino acid sequence comprising amino acids 27-110 of SEQ ID NO: 2 which is capable of associating with a membrane.

29. The isolated nucleic acid molecule of claim 5, wherein the Mistic domain comprises a sequence having at least 95% sequence identity to SEQ ID NO: 2 which is capable of associating with a membrane.

30. The nucleic acid molecule of claim 5, wherein the Mistic domain comprises a sequence having at least 98% sequence identity to SEQ ID NO: 2 which is capable of associating with a membrane.

31. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes an amino acid sequence comprising SEQ ID NO: 189 which is capable of associating with a membrane.

32. The nucleic acid molecule of claim 5, wherein the Mistic domain comprises SEQ ID NO: 189 and is capable of associating with a membrane.

33. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprising at least 90% sequence identity to SEQ ID NO: 2 does not include a substitution at position 75 of SEQ ID NO: 2.

34. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprising at least 90% sequence identity to SEQ ID NO: 2 includes a Met75Ile substitution in SEQ ID NO: 2.

35. The isolated nucleic acid molecule of claim 5, wherein the integral membrane protein comprises a potassium channel protein, G-protein coupled receptor protein, or a TGF-beta family receptor protein.

36. The isolated nucleic acid molecule of claim 5, wherein the Mistic domain of the fusion protein is N-terminal of the cargo protein domain.

37. The isolated nucleic acid molecule of claim 5, wherein the Mistic domain of the fusion protein is C-terminal of the cargo protein domain.

38. The isolated nucleic acid molecule of claim 5, further comprising a linker between the cargo protein domain and the Mistic domain.

39. The isolated nucleic acid molecule of claim 38, wherein the linker comprises from 1 to 100 amino acids.

40. The isolated nucleic acid molecule of claim 39, wherein the linker comprises an amino acid sequence as set forth in SEQ ID NO: 40, 42, 44, 46, 48, or 50.

41. The isolated nucleic acid molecule of claim 5, further comprising a protease-recognition site between the cargo protein domain and the Mistic domain.

42. The isolated nucleic acid molecule of claim 41, wherein the protease-recognition site is capable of being cleaved by thrombin, chymotrypsin, trypsin, plasmin, papain, pepsin, subtilisin, enterokinase or TEV protease.

43. The isolated nucleic acid molecule of claim 38, further comprising a protease-recognition site located in the linker.

44. The isolated nucleic acid molecule of claim 5, further comprising a peptide tag.

45. The isolated nucleic acid molecule of claim 44, wherein the peptide tag is located at the N-terminus of the Mistic domain, the C-terminus of the Mistic domain, the N-terminus of the cargo protein domain, or the C-terminus of the cargo protein domain.

46. The isolated nucleic acid molecule of claim 44, wherein the peptide tag comprises a FLAG tag, a His tag, a HA tag, a streptactin tag, or a biotinylation peptide.

47. The isolated nucleic acid molecule of claim 5, further comprising at least one exogenous helix domain.

48. The isolated nucleic acid molecule of claim 47, wherein the at least one exogenous helix domain is located between the Mistic domain and the cargo protein domain.

49. The isolated nucleic acid molecule of claim 5, further comprising one or more of a peptide tag, a linker, and a protease-recognition site, each of which is located between the Mistic domain and the cargo protein domain.

50. The cell of claim 9, wherein the cell is a prokaryotic cell.

51. The cell of claim 50, wherein the prokaryotic cell is a protease-deficient bacterial strain.

52. The method of claim 10, wherein the cell is a prokaryotic cell.

53. The method of claim 52, wherein the prokaryotic cell is a bacteria.

54. The method of claim 53, wherein the bacteria is protease-deficient bacteria.

55. The method of claim 53, wherein the prokaryotic cell is an E. coli strain selected from the group consisting of B1-21, B1-21 (DE3), B1-21 (DE3) pLysS, Origami B, OmpT-defective CD41, CD43 (DE3), and phosphatidylenthanolamine (PE)-deficient AD93.

56. A method of isolating a recombinant fusion protein or domain thereof, comprising:
expressing the isolated nucleic acid molecule of claim 5 in an expression system, which comprises a membrane or membrane-like structure, wherein at least a portion of the fusion protein is associated with the membrane or membrane-like structure;
isolating from the expression system a membrane fraction comprising the membrane or membrane-like structure; and
isolating the fusion protein or the cargo protein domain from the membrane fraction.

57. The method of claim 56, wherein the expression system is a cell-free expression system.

58. The method of claim 56, wherein the expression system comprises a cell.

59. The method of claim 58, wherein the cell is a prokaryotic cell.

60. The method of claim 56, wherein at least a portion of the fusion protein is incorporated into the membrane or membrane-like structure.

61. The method of claim 58, wherein a yield of isolated fusion protein or isolated cargo protein domain from the cell is no less than 0.1 mg/L of cells.

62. The method of claim 58, wherein a yield of isolated fusion protein or isolated cargo protein domain from the cell is no less than 1 mg/L of cells.

63. The method of claim 7, wherein the integral membrane protein comprises a potassium channel protein, G-protein coupled receptor protein, or a TGF-beta family receptor protein.

64. The vector of claim 8, wherein the cargo protein domain comprises a soluble protein.

65. A method of producing a recombinant fusion protein, comprising expressing the vector of claim 8 in an expression system, which comprises a membrane or membrane-like structure, wherein at least a portion of the fusion protein is associated with the membrane or membrane-like structure.

66. The isolated nucleic acid molecule of claim 5, wherein the Mistic domain is no more than 125 amino acids in length.

67. An isolated nucleic acid molecule encoding a polypeptide of no more than 125 amino acids in length wherein the nucleic acid molecule encodes an amino acid sequence comprising SEQ ID NO: 189 which is capable of associating with a membrane.

68. The nucleic acid molecule of claim 67, wherein the nucleic acid molecule comprises SEQ ID NO: 188.

69. An isolated nucleic acid molecule encoding a fusion protein comprising a cargo protein domain and a Mistic domain; wherein the cargo protein domain comprises a soluble protein, and wherein the Mistic domain comprises
(a) an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 and is capable of associating with a membrane; or
(b) an amino acid sequence comprising amino acids 27-110 of SEQ ID NO: 2 which is capable of associating with a membrane.

70. A method of producing a recombinant fusion protein, comprising expressing the isolated nucleic acid molecule of claim 69 in an expression system, which comprises a membrane or membrane-like structure, wherein at least a portion of the fusion protein is associated with the membrane or membrane-like structure.

71. The method of claim 70, wherein the fusion protein further comprises a protease-recognition site between the Mistic domain and the cargo protein domain.

72. The method of claim 71, wherein the cargo protein domain is not incorporated into the membrane and is tethered to the membrane by a portion of the fusion protein that is incorporated into the membrane.

73. The method of claim 72, further comprising digesting the protease-recognition site to release the cargo protein domain, and isolating the released cargo protein domain.