Abstract:
Nuclear magnetic resonance methods for identifying sites in a DNA-binding and dimerization domain of a papillomavirus E2 protein are disclosed. Preferably the sites are ligand binding sites.

Description:
[0001]    This application claims the benefit of U.S. Provisional Application Ser. No. 60/197,459, filed Apr. 17, 2000, No. 60/211,055, filed Jun. 13, 2000, and No. 60/268,444 filed Feb. 13, 2001, which are incorporated herein by reference in their entireties. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    An important aspect in understanding the function of biochemical processes is the elucidation of the nature of the associations between various species including, for example, the associations between ligands and proteins. Such associations may be non-covalent, wherein juxtapositions are energetically favored by hydrogen bonding, van der Waals forces, or electrostatic interactions, or they may be covalent. When physical binding is being studied, a target molecule is typically exposed to one or more compounds suspected of being ligands, and assays are then performed to determine if complexes between the target molecule and one or more of those compounds are formed. Such assays, as are well known in the art, test for gross changes (e.g., size, charge, and mobility) in the target molecule that indicate complex formation.  
           [0003]    Where functional changes are measured, assay conditions are established that allow for measurement of biological or chemical events related to the target molecule (e.g., enzyme catalyzed reaction and receptor-mediated enzyme activation). To identify an alteration, the function of the target molecule is determined before and after exposure to the test compounds.  
           [0004]    Assays involving the use of nuclear magnetic resonance (NMR) techniques are also known. NMR techniques may be used, for example, in conjunction with other assay methods to assess hits identified from physical binding screens or functional assay screens. If  1 H,  13 C, and/or  15 N resonance assignments are known for the target as well as either a solution or X-ray crystallographic structure, then the binding site location of identified ligands can be determined using NMR techniques. As such, definitive resonance assignments of the target are required as a first step. A DNA-binding protein, E2, which is encoded by the papillomavirus and is involved in transcriptional regulation and viral replication, is one such target.  
         SUMMARY OF THE INVENTION  
         [0005]    In one aspect, the present invention provides a nuclear magnetic resonance method for identifying a site in a DNA-binding and dimerization domain of a papillomavirus E2 protein. In one embodiment, the method includes providing a first set of chemical shifts for atoms of a mixture including a ligand and the papillomavirus E2 protein, comparing the first set of chemical shifts to a second set of chemical shifts as listed in Table 1, and identifying at least a portion of the atoms that exhibit changes in chemical shifts, wherein the site includes the identified atoms. Preferably providing the first set of chemical shifts includes providing a mixture of the ligand and the papillomavirus E2 protein, allowing the ligand to interact with the papillomavirus E2 protein, obtaining a nuclear magnetic resonance spectrum of the mixture, and measuring chemical shifts of atoms from the spectrum. Preferably allowing the ligand to interact includes allowing the ligand and the protein to reach a binding equilibrium. Preferably the site is a ligand binding site. Preferably the papillomavirus E2 protein is encoded by the HPV-18 strain.  
           [0006]    In another embodiment, the method includes providing a first  1 H- 15 N heteronuclear single quantum correlation spectrum of a mixture including a ligand and the papillomavirus E2 protein, comparing the first  1 H- 15 N heteronuclear single quantum correlation spectrum to a second  1 H- 15 N heteronuclear single quantum correlation spectrum as illustrated in FIG. 2, and identifying at least a portion of the amino acids having atoms that exhibit changes in chemical shifts, wherein the site includes the identified amino acids. Preferably providing the first spectrum includes providing a mixture of the ligand and the papillomavirus E2 protein, allowing the ligand to interact with the papillomavirus E2 protein, and obtaining a  1 H- 15 N heteronuclear single quantum correlation spectrum of the mixture. Preferably allowing the ligand to interact includes allowing the ligand and the protein to reach a binding equilibrium. Preferably the site is a ligand binding site. Preferably the papillomavirus E2 protein is encoded by the HPV-18 strain.  
           [0007]    In another aspect, the present invention provides a machine-readable data storage medium including a data storage material encoded with nuclear magnetic resonance chemical shifts as listed in Table 1, wherein when a first set of chemical shifts is provided, the chemical shifts encoded on the data storage material are capable of being read by the machine to create a second set of chemical shifts, and the machine having programmed instructions that are capable of causing the machine to compare the first and second sets of chemical shifts to arrive at structural information.  
           [0008]    In another aspect, the present invention provides a computer-assisted method for identifying a ligand binding site in a DNA-binding and dimerization domain of a papillomavirus E2 protein. The method includes providing a first set of nuclear magnetic resonance chemical shifts for atoms of a mixture including the ligand and the papillomavirus E2 protein, causing the first set of chemical shifts to be entered into memory of a computer, causing the computer to read a second set of chemical shifts as listed in Table 1 from a machine-readable data storage medium, causing the computer to compare the first and second sets of chemical shifts, and causing the computer to identify at least a portion of the atoms that exhibit changes in chemical shifts, wherein the ligand binding site includes the identified atoms. Preferably the papillomavirus E2 protein is encoded by the HPV-18 strain. Preferably the method further includes causing the computer to visually display a spatial arrangement of atoms of the ligand binding site.  
           [0009]    Methods disclosed in the present invention for identifying sites offer advantages over other methods known in the art. For example, the present invention preferably provides methods for efficiently identifying binding sites for a wide range of chemically and physically diverse potential ligands.  
           [0010]    The term “binding” as used herein, refers to a condition of proximity between a chemical entity or compound, or portions thereof, and the target protein or portions thereof. The association may be non-covalent, wherein the juxtaposition is energetically favored by hydrogen bonding, van der Waals forces, or electrostatic interactions, or it may be covalent. The association may be a static interaction, or an equilibrium may be reached between associated and non-associated species. Preferably, a ligand that binds to a ligand binding site in a DNA-binding and dimerization domain of a papillomavirus E2 protein would also be expected to bind to or interfere with another ligand binding site whose structure defines a shape that falls within an acceptable error.  
           [0011]    The term “ligand” as used herein means any chemical entity, compound, or portion thereof, that is capable of binding to a protein.  
           [0012]    The term “change in chemical shifts” as used herein means the observation of an increase or decrease in chemical shift for a resonance, an increase or decrease in intensity for a resonance, or the failure to observe a resonance when comparing a resonance of an atom from the spectrum of a mixture of ligand and protein to the resonance of the same atom from the spectrum of the protein without the ligand 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0013]    [0013]FIG. 1 is an illustration of the deviations from random coil chemical shifts of  13 C α resonances (in parts per million (ppm)) with assignments for the DNA-binding and dimerization domain of papillomavirus (strain HPV-18) E2 protein as a function of residue number. Random coil chemical shift values are from Wishart et al.,  Biochem. Cell Biol.,  76:153-63 (1998). Locations of secondary structure according to the X-ray structure of BPV-1, HPV-16 and HPV-31 are shown with α (α-helix) and β (β-sheet).  
         [0014]    [0014]FIG. 2 is an illustration of the 2-dimensional  1 H- 15 N heteronuclear single quantum correlation spectrum with assignments for the DNA-binding and dimerization domain of a 0.84 mM papillomavirus (strain HPV-18) E2 protein at 300° K.  
     
    
     DETAILED DESCRIPTION  
       [0015]    Papillomaviruses are a diverse group of small DNA viruses that infect epithelial cells and cause tumor formation. All of the papillomaviruses encode a DNA-binding protein, E2, that is involved in transcriptional regulation and viral replication. E2 protein consists of a C-terminal DNA-binding and dimerization domain (E2-DBD) and N-terminal transactivation domain, separated by a flexible region. E2-DBD from bovine papillomavirus-1 (BPV-1) has been extensively studied, and the X-ray crystallographic structure of E2-DBD bound to DNA consists of a homodimer that includes an eight-stranded β-barrel and two pairs of α-helices (Hedge et al.,  Nature,  359:505-12 (1992)). The solution and/or crystal structures of homologous E2-DBDs from human papillomavirus-31 (HPV-31) (Liang et al.,  Biochemistry,  35:2095-2103 (1996), Bussiere et al.,  Acta Cryst ., D54:1367-76 (1998)) and HPV-16 (Hedge et al.,  J. Mol. Biol.,  284:1479-89 (1998)) have been reported and are similar to BPV-1.  
         [0016]    The present invention preferably relates to the E2-DBD from the high risk strain HPV-18. The E2 protein ofHPV-18 represses the expression of the major viral transforming genes E6 and E7 and is a cofactor for the replication protein E1 binding to the origin (Kasukawa et al.,  J. Virol.,  72:8166-73 (1998)). The pivotal role of E2 in transcriptional regulation and viral replication makes it a potential target for antiviral therapy.  
         [0017]    E2-DBD of HPV-18 has 55% and 60% sequence identity to HPV-16 and HPV-31, respectively, and binds to the ACCN 6 GGT recognition sequence. Preferably, two amino acid sequences are compared using the Blastp program, version 2.0.9, of the BLAST 2 search algorithm, as described by Tatusova et al.,  FEMS Microbiol Lett  174, 247-50 (1999), and available at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. Preferably, the default values for all BLAST 2 search parameters are used, including matrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gap x_dropoff=50, expect=10, wordsize=3, and filter on. In the comparison of two amino acid sequences using the BLAST search algorithm, structural similarity is referred to as “identity.” 
         [0018]    The present invention provides a papillomavirus HPV-18 strain E2 protein DNA-binding domain having the  1 H- 15 N heteronuclear single quantum correlation spectrum shown in FIG. 2. Each correlation is labeled as to the residue in the protein from which it arises if that has been determined. The process used to make the assignments is described in the examples. The chemical shifts of all assigned  1 H,  13 C, and  15 N resonances are listed in Table 1. The resonance assignments presented here provide the basis for determining sites, preferably binding site locations of ligands previously identified by other means. Chemical shift changes induced by addition of ligand to the protein sample are manifested by changes in the appearance of  1 H- 15 N HSQC spectra. Correlations that experience the largest ligand-induced chemical shift changes are preferably located near the ligand&#39;s binding site. To determine chemical shift changes, the protein  1 H,  13 C, and  15 N resonances are preferably assigned as extensively as possible.  
         [0019]    Preferably, ligand binding sites include identified atoms that exhibit changes in chemical shifts. Preferably the identified atoms include at least one proton that, upon addition of ligand to the protein, either exhibits a change in  1 H chemical shift of at least about 0.04 ppm or is no longer observed. Preferably the identified atoms includes at least one carbon atom that, upon addition of ligand to the protein, either exhibits a change in  13 C chemical shift of at least about 0.2 ppm or is no longer observed. Preferably the identified atoms include at least one nitrogen atom that, upon addition of ligand to the protein, either exhibits a change in  15 N chemical shift of at least about 0.2 ppm or is no longer observed.  
         [0020]    In order that this invention be more fully understood, the following examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.  
       EXAMPLES  
       [0021]    The HPV-18 E2 protein consists of 410 amino acids with the DBD residing at the C-terminus (amino acids #329-410). E2-DBD cloning procedures resulted in the addition of methionine before amino acid 329 and six histidine residues after amino acid 410. Amino acid sequencing indicated that the N-terminal des-Met form of the E2-DBD protein was the major species produced.  
         [0022]    E2-DBD was over-expressed in BL21 (DE3)  E. coli  cells using the pSRtac vector. Isotopically labeled samples were prepared in M9 glucose media containing  15 NH 4 Cl and unlabeled or U- 13 C-glucose. Cell pellets were lysed with intermittent mechanical disruption with a Tissuemizer (Tekmar Co., Cincinatti, OH). Clarified cell lysates were passed over Ni 2+ -NTA agarose (Qiagen, Inc., Valencia, Calif.), and further purified using Source 30Q anion exchange chromatography (Amersham Pharmacia Biotech, Inc.; Piscataway, N.J.). The resulting E2-DBD exists as a homodimer of molecular weight 20.6 kDa under the conditions used for the NMR experiments.  
         [0023]    The NMR samples typically consisted of 0.8 mM protein in buffer containing 20 mM phosphate, 50 mM NaCl, and 1 mM [ 2 H 10 ] dithiothreitol (DTT) at pH 6.5 in 90%  1 H 2 O/10%  2 H 2 O by volume. All NMR spectra were recorded at 27° C. on a Bruker DRX-600 spectrometer (BRUKER NMR, Rheinstetten, Germany) using a 5 mm triple-resonance probe with 3-axis gradients. HNC α , HN(CO)C α , C β C α (CO)NH, H β H α (CO)NH, HNCO and HCCH-total correlation spectroscopy (HCCH-TOCSY) (mixing times 16 and 23 milliseconds) data sets were acquired using gradient-enhanced versions of the pulse sequences. Two-dimensional  1 H- 15 N Heteronuclear Single Quantum Correlation (HSQC) and  15 N edited Nuclear Overhauser Effect Spectroscopy-HSQC (NOESY-HSQC) (mixing time 80 milliseconds) spectra were also acquired. Proton chemical shifts were referenced to the  1 H 2 O signal at 4.70 parts per million (ppm) (tetramethylsilane (TMS)=0 ppm). The  15 N and  13 C chemical shifts were referenced indirectly in a manner similar to that known in the art (e.g., Bax et al.,  J. Magn. Reson.,  67:565-69 (1986)). Carrier frequencies were 4.70 ppm for  1 H, 118 ppm for  15 N, 54 ppm for  13 C α , 40 ppm for aliphatic  13 C, and 174 ppm for  13 C′. A combination of water flip-back (e.g., Grzesiek et al.,  J. Am. Chem. Soc.,  115:12593-94 (1993)) and WATERGATE (e.g., Piotto et al.,  J. Biomol. NMR,  2:661-65 (1992)) techniques were used to eliminate the water resonance. NMR data were processed using NMRPipe and NMRDraw software from Molecular Simulations, Inc. (San Diego, Calif.).  
         [0024]    Sequence-specific backbone resonance assignments were accomplished using primarily 3-dimensional HNC α , HN(CO)C α , and C β C α (CO)NH data sets. The  13 C′ and  1 H α ,  1 H β  chemical shifts were determined using HNCO and H β H α (CO)NH data sets, respectively. The side chain  1 H and  13 C spin systems were assigned using the 3-dimensional HCCH-TOCSY experiments.  
         [0025]    The assigned  1 H- 15 N HSQC spectrum of HPV-18 E2-DBD is shown in FIG. 2. Chemical shift values for all  1 H N ,  1 H α ,  13 C α ,  13 C β ,  13 C and  15 N α  resonances except for the first four residues, the C-terminal five histidine residues, and Glu58 and Thr59 were assigned. Approximately 60% of the side chain  1 H and  13 C resonances were also assigned. Assigned  1 H,  13 C, and  15 N chemical shifts are listed in Table 1. The locations of secondary structure in the linear amino acid sequence predicted based on  13 C α  chemical shifts (see Wishart et al.,  J. Biomol. NMR,  4:171-80 (1994)) are shown in FIG. 1 and are consistent with the crystal structures of BPV-1, HPV-16 and HPV-31.  
         [0026]    The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.  
                                                                   TABLE 1                             1 H,  13 C, and  15 N chemical shifts of human papillomavirus E2-DBD.       HA, HB, HG, HD, HE, CA, CB, CG, CD, CE refer to       H α , H β , H γ , H δ , H ε ,       C α , C β , C γ , C δ , and C ε  respectively.                #Atom   #RES   RES   ATOMS   ppm                        1   4   THR   HA   H   5.01       2   4   THR   HB   H   3.91       3   4   THR   HG1   H   0.98       4   4   THR   HG2   H   0.98       5   4   THR   CA   C   59.95       6   4   THR   CB   C   67.75       7   4   THR   CG2   C   19.93       8   5   THR   H   H   9.18       9   5   THR   C   C   171.68       10   5   THR   CA   C   57.48       11   5   THR   N   N   124.16       12   6   PRO   HA   H   4.73       13   6   PRO   CA   C   60.10       14   6   PRO   CB   C   29.24       15   7   ILE   H   H   8.49       16   7   ILE   HA   H   5.85       17   7   ILE   HB   H   1.82       18   7   ILE   HG2   H   0.92       19   7   ILE   HD1   H   0.49       20   7   ILE   C   C   173.65       21   7   ILE   CA   C   57.29       22   7   ILE   CB   C   42.10       23   7   ILE   CG2   C   16.79       24   7   ILE   CD1   C   12.90       25   7   ILE   N   N   115.39       26   8   ILE   H   H   8.90       27   8   ILE   HA   H   5.01       28   8   ILE   HB   H   1.88       29   8   ILE   HG2   H   0.82       30   8   ILE   C   C   174.83       31   8   ILE   CA   C   58.93       32   8   ILE   CB   C   39.92       33   8   ILE   CG2   C   15.73       34   8   ILE   N   N   115.93       35   9   HIS   H   H   8.91       36   9   HIS   HA   H   5.68       37   9   HIS   HB2   H   2.81       38   9   HIS   HB3   H   2.57       39   9   HIS   C   C   173.19       40   9   HIS   CA   C   51.27       41   9   HIS   CB   C   32.38       42   9   HIS   N   N   119.91       43   10   LEU   H   H   8.98       44   10   LEU   HA   H   5.17       45   10   LEU   HB2   H   1.66       46   10   LEU   HB3   H   0.92       47   10   LEU   HG   H   1.47       48   10   LEU   HD1   H   0.82       49   10   LEU   HD2   H   0.71       50   10   LEU   C   C   172.40       51   10   LEU   CA   C   50.25       52   10   LEU   CB   C   40.76       53   10   LEU   CG   C   23.68       54   10   LEU   N   N   122.16       55   11   LYS   H   H   8.76       56   11   LYS   HA   H   5.29       57   11   LYS   HB2   H   1.65       58   11   LYS   HB3   H   1.44       59   11   LYS   HG2   H   1.40       60   11   LYS   HG3   H   1.21       61   11   LYS   HD2   H   1.62       62   11   LYS   HD3   H   1.62       63   11   LYS   HE2   H   2.70       64   11   LYS   HE3   H   2.70       65   11   LYS   C   C   172.59       66   11   LYS   CA   C   51.76       67   11   LYS   CB   C   33.58       68   11   LYS   CG   C   22.68       69   11   LYS   CD   C   27.38       70   11   LYS   CE   C   39.54       71   11   LYS   N   N   120.73       72   12   GLY   H   H   8.30       73   12   GLY   HA2   H   4.43       74   12   GLY   HA3   H   4.19       75   12   GLY   C   C   173.46       76   12   GLY   CA   C   42.96       77   12   GLY   N   N   109.97       78   13   ASP   H   H   8.50       79   13   ASP   HA   H   4.59       80   13   ASP   HB2   H   2.77       81   13   ASP   HB3   H   2.61       82   13   ASP   C   C   168.61       83   13   ASP   CA   C   52.23       84   13   ASP   CB   C   40.03       85   13   ASP   N   N   120.16       86   14   ARG   H   H   8.61       87   14   ARG   HA   H   3.58       88   14   ARG   HB2   H   1.72       89   14   ARG   HB3   H   1.68       90   14   ARG   HG2   H   1.47       91   14   ARG   HG3   H   1.47       92   14   ARG   HD2   H   3.07       93   14   ARG   HD3   H   3.02       94   14   ARG   C   C   174.68       95   14   ARG   CA   C   58.64       96   14   ARG   CB   C   27.87       97   14   ARG   CG   C   26.01       98   14   ARG   CD   C   40.85       99   14   ARG   N   N   122.34       100   15   ASN   H   H   8.64       101   15   ASN   HA   H   4.46       102   15   ASN   HB2   H   2.87       103   15   ASN   HB3   H   2.76       104   15   ASN   C   C   176.39       105   15   ASN   CA   C   54.42       106   15   ASN   CB   C   35.59       107   15   ASN   N   N   118.46       108   16   SER   H   H   8.35       109   16   SER   HA   H   3.86       110   16   SER   HB2   H   4.17       111   16   SER   HB3   H   3.63       112   16   SER   C   C   175.96       113   16   SER   CA   C   59.80       114   16   SER   CB   C   59.96       115   16   SER   N   N   118.74       116   17   LEU   H   H   8.10       117   17   LEU   HA   H   3.84       118   17   LEU   HB2   H   1.64       119   17   LEU   HB3   H   1.17       120   17   LEU   HD1   H   0.45       121   17   LEU   HD2   H   0.38       122   17   LEU   C   C   175.25       123   17   LEU   CA   C   55.37       124   17   LEU   CB   C   38.75       125   17   LEU   CD1   C   23.04       126   17   LEU   CD2   C   19.79       127   17   LEU   N   N   121.15       128   18   LYS   H   H   7.83       129   18   LYS   HA   H   3.91       130   18   LYS   HB2   H   1.97       131   18   LYS   HB3   H   1.97       132   18   LYS   HG2   H   1.39       133   18   LYS   HG3   H   1.27       134   18   LYS   HD2   H   1.70       135   18   LYS   HD3   H   1.60       136   18   LYS   HE2   H   2.95       137   18   LYS   HE3   H   2.95       138   18   LYS   C   C   175.74       139   18   LYS   CA   C   57.85       140   18   LYS   CB   C   29.95       141   18   LYS   CD   C   27.55       142   18   LYS   CE   C   39.77       143   18   LYS   N   N   120.70       144   19   CYS   H   H   7.59       145   19   CYS   HA   H   4.20       146   19   CYS   HB2   H   3.02       147   19   CYS   HB3   H   2.95       148   19   CYS   C   C   177.01       149   19   CYS   CA   C   60.14       150   19   CYS   CB   C   24.32       151   19   CYS   N   N   116.91       152   20   LEU   H   H   8.03       153   20   LEU   HA   H   4.09       154   20   LEU   HB2   H   1.80       155   20   LEU   HB3   H   1.54       156   20   LEU   HD1   H   0.90       157   20   LEU   HD2   H   0.82       158   20   LEU   C   C   175.16       159   20   LEU   CA   C   55.39       160   20   LEU   CB   C   39.82       161   20   LEU   CD1   C   21.58       162   20   LEU   CD2   C   25.17       163   20   LEU   N   N   121.40       164   21   ARG   H   H   8.58       165   21   ARG   HA   H   3.61       166   21   ARG   HB2   H   1.95       167   21   ARG   C   C   175.45       168   21   ARG   CA   C   58.16       169   21   ARG   CB   C   27.32       170   21   ARG   N   N   118.96       171   22   TYR   H   H   7.43       172   22   TYR   HA   H   3.91       173   22   TYR   C   C   175.54       174   22   TYR   CA   C   59.04       175   22   TYR   CB   C   35.58       176   22   TYR   N   N   116.61       177   23   ARG   H   H   7.88       178   23   ARG   HA   H   4.04       179   23   ARG   HB2   H   2.04       180   23   ARG   HB3   H   2.04       181   23   ARG   HG2   H   1.70       182   23   ARG   HG3   H   1.70       183   23   ARG   HD2   H   3.26       184   23   ARG   HD3   H   3.26       185   23   ARG   C   C   176.67       186   23   ARG   CA   C   57.11       187   23   ARG   CB   C   28.01       188   23   ARG   CG   C   25.77       189   23   ARG   CD   C   41.55       190   23   ARG   N   N   119.89       191   24   LEU   H   H   8.59       192   24   LEU   HA   H   4.18       193   24   LEU   HB2   H   1.89       194   24   LEU   HB3   H   1.46       195   24   LEU   HD1   H   0.80       196   24   LEU   HD2   H   0.60       197   24   LEU   C   C   177.05       198   24   LEU   CA   C   55.00       199   24   LEU   CB   C   38.81       200   24   LEU   CD1   C   21.32       201   24   LEU   CD2   C   22.99       202   24   LEU   N   N   117.28       203   25   ARG   H   H   7.75       204   25   ARG   HA   H   4.26       205   25   ARG   HB2   H   1.91       206   25   ARG   HB3   H   1.91       207   25   ARG   HG2   H   1.82       208   25   ARG   HG3   H   1.82       209   25   ARG   HD2   H   3.11       210   25   ARG   HD3   H   3.11       211   25   ARG   C   C   177.46       212   25   ARG   CA   C   56.71       213   25   ARG   CB   C   27.46       214   25   ARG   CG   C   25.14       215   25   ARG   CD   C   41.30       216   25   ARG   N   N   120.30       217   26   LYS   H   H   7.28       218   26   LYS   HA   H   4.17       219   26   LYS   HB2   H   1.60       220   26   LYS   HB3   H   1.60       221   26   LYS   HG2   H   1.22       222   26   LYS   HG3   H   1.22       223   26   LYS   HD2   H   1.57       224   26   LYS   HD3   H   1.57       225   26   LYS   HE2   H   2.86       226   26   LYS   HE3   H   2.88       227   26   LYS   C   C   175.55       228   26   LYS   CA   C   54.84       229   26   LYS   CB   C   29.70       230   26   LYS   CG   C   22.19       231   26   LYS   CD   C   26.73       232   26   LYS   CE   C   39.22       233   26   LYS   N   N   115.77       234   27   HIS   H   H   7.82       235   27   HIS   HA   H   5.01       236   27   HIS   HB2   H   3.40       237   27   HIS   HB3   H   2.87       238   27   HIS   C   C   174.21       239   27   HIS   CA   C   52.56       240   27   HIS   CB   C   27.78       241   27   HIS   N   N   118.14       242   28   SER   H   H   7.50       243   28   SER   HA   H   3.46       244   28   SER   HB2   H   3.80       245   28   SER   HB3   H   3.80       246   28   SER   C   C   173.31       247   28   SER   CA   C   58.63       248   28   SER   CB   C   60.65       249   28   SER   N   N   114.42       250   29   ASP   H   H   8.46       251   29   ASP   HA   H   4.42       252   29   ASP   HB2   H   2.43       253   29   ASP   HB3   H   2.21       254   29   ASP   C   C   171.83       255   29   ASP   CA   C   52.93       256   29   ASP   CB   C   37.38       257   29   ASP   N   N   118.29       258   30   HIS   H   H   8.31       259   30   HIS   HA   H   4.90       260   30   HIS   HB2   H   3.75       261   30   HIS   HB3   H   3.33       262   30   HIS   C   C   175.04       263   30   HIS   CA   C   53.95       264   30   HIS   CB   C   29.17       265   30   HIS   N   N   116.46       266   31   TYR   H   H   7.05       267   31   TYR   HA   H   4.57       268   31   TYR   HB2   H   2.58       269   31   TYR   HB3   H   2.58       270   31   TYR   C   C   170.71       271   31   TYR   CA   C   54.00       272   31   TYR   CB   C   37.51       273   31   TYR   N   N   112.10       274   32   ARG   H   H   8.78       275   32   ARG   HA   H   4.24       276   32   ARG   HB2   H   1.90       277   32   ARG   HB3   H   1.90       278   32   ARG   HG2   H   0.50       279   32   ARG   HG3   H   0.50       280   32   ARG   HD2   H   2.44       281   32   ARG   HD3   H   2.25       282   32   ARG   C   C   170.17       283   32   ARG   CA   C   55.16       284   32   ARG   CB   C   27.64       285   32   ARG   CG   C   28.32       286   32   ARG   CD   C   41.50       287   32   ARG   N   N   119.90       288   33   ASP   H   H   7.55       289   33   ASP   HA   H   4.91       290   33   ASP   HB2   H   2.12       291   33   ASP   HB3   H   1.75       292   33   ASP   C   C   171.83       293   33   ASP   CA   C   49.82       294   33   ASP   CB   C   42.75       295   33   ASP   N   N   118.71       296   34   ILE   H   H   9.72       297   34   ILE   HA   H   5.41       298   34   ILE   HB   H   1.31       299   34   ILE   HG2   H   0.91       300   34   ILE   HD1   H   0.45       301   34   ILE   C   C   170.37       302   34   ILE   CA   C   57.10       303   34   ILE   CB   C   39.64       304   34   ILE   CG2   C   17.26       305   34   ILE   N   N   116.54       306   35   SER   H   H   9.53       307   35   SER   HA   H   5.10       308   35   SER   HB2   H   3.98       309   35   SER   HB3   H   3.98       310   35   SER   C   C   173.41       311   35   SER   CA   C   56.93       312   35   SER   CB   C   64.81       313   35   SER   N   N   127.07       314   36   SER   H   H   8.34       315   36   SER   HA   H   4.17       316   36   SER   HB2   H   2.94       317   36   SER   HB3   H   2.94       318   36   SER   C   C   171.93       319   36   SER   CA   C   56.27       320   36   SER   CB   C   61.52       321   36   SER   N   N   111.52       322   37   THR   H   H   8.87       323   37   THR   HA   H   4.42       324   37   THR   HB   H   3.98       325   37   THR   HG2   H   0.99       326   37   THR   C   C   172.22       327   37   THR   CA   C   61.50       328   37   THR   CB   C   66.25       329   37   THR   CG2   C   20.38       330   37   THR   N   N   118.94       331   38   TRP   H   H   9.25       332   38   TRP   HA   H   4.75       333   38   TRP   HB2   H   2.54       334   38   TRP   HB3   H   2.54       335   38   TRP   C   C   172.46       336   38   TRP   CA   C   52.15       337   38   TRP   CB   C   29.53       338   38   TRP   N   N   129.61       339   39   HIS   H   H   7.89       340   39   HIS   HA   H   4.44       341   39   HIS   HB2   H   2.43       342   39   HIS   HB3   H   2.43       343   39   HIS   C   C   169.88       344   39   HIS   CA   C   52.09       345   39   HIS   CB   C   30.38       346   40   TRP   H   H   8.56       347   40   TRP   HA   H   5.08       348   40   TRP   HB2   H   3.64       349   40   TRP   HB3   H   2.87       350   40   TRP   C   C   171.67       351   40   TRP   CA   C   53.85       352   40   TRP   CB   C   27.77       353   40   TRP   N   N   120.03       354   41   THR   H   H   8.67       355   41   THR   HA   H   4.42       356   41   THR   HB   H   3.92       357   41   THR   HG2   H   0.99       358   41   THR   C   C   175.17       359   41   THR   CA   C   62.27       360   41   THR   CB   C   67.99       361   41   THR   CG2   C   20.38       362   41   THR   N   N   115.31       363   42   GLY   H   H   9.77       364   42   GLY   HA2   H   4.03       365   42   GLY   HA3   H   4.03       366   42   GLY   C   C   173.88       367   42   GLY   CA   C   43.28       368   42   GLY   N   N   114.16       369   43   ALA   H   H   8.31       370   43   ALA   HA   H   4.32       371   43   ALA   HB   H   1.39       372   43   ALA   C   C   172.26       373   43   ALA   CA   C   50.72       374   43   ALA   CB   C   16.84       375   43   ALA   N   N   123.70       376   44   GLY   H   H   8.42       377   44   GLY   HA2   H   4.10       378   44   GLY   HA3   H   3.91       379   44   GLY   C   C   176.29       380   44   GLY   CA   C   43.25       381   44   GLY   N   N   108.16       382   45   ASN   HA   H   4.75       383   45   ASN   HB2   H   2.93       384   45   ASN   HB3   H   2.75       385   45   ASN   C   C   172.12       386   45   ASN   CA   C   50.98       387   45   ASN   CB   C   37.51       388   45   ASN   N   N   117.19       389   46   GLU   H   H   8.81       390   46   GLU   HA   H   3.98       391   46   GLU   HB2   H   1.93       392   46   GLU   HB3   H   1.87       393   46   GLU   HG2   H   2.14       394   46   GLU   HG3   H   2.14       395   46   GLU   C   C   173.36       396   46   GLU   CA   C   55.97       397   46   GLU   CB   C   27.17       398   46   GLU   CG   C   33.95       399   46   GLU   N   N   119.81       400   47   LYS   H   H   8.17       401   47   LYS   HA   H   4.19       402   47   LYS   HB2   H   1.94       403   47   LYS   HB3   H   1.76       404   47   LYS   HG2   H   1.40       405   47   LYS   HG3   H   1.33       406   47   LYS   HD2   H   1.60       407   47   LYS   HD3   H   1.60       408   47   LYS   HE2   H   2.94       409   47   LYS   HE3   H   2.94       410   47   LYS   C   C   174.43       411   47   LYS   CA   C   54.79       412   47   LYS   CB   C   30.57       413   47   LYS   CG   C   22.93       414   47   LYS   CD   C   26.73       415   47   LYS   CE   C   39.80       416   47   LYS   N   N   117.28       417   48   THR   H   H   7.49       418   48   THR   HA   H   4.37       419   48   THR   HB   H   3.99       420   48   THR   HG1   H   1.05       421   48   THR   HG2   H   1.05       422   48   THR   C   C   174.80       423   48   THR   CA   C   59.28       424   48   THR   CB   C   68.23       425   48   THR   CG2   C   19.72       426   48   THR   N   N   113.55       427   49   GLY   H   H   8.64       428   49   GLY   HA2   H   4.28       429   49   GLY   HA3   H   3.05       430   49   GLY   C   C   171.67       431   49   GLY   CA   C   42.01       432   49   GLY   N   N   111.32       433   50   ILE   H   H   8.29       434   50   ILE   HA   H   4.53       435   50   ILE   HB   H   −1.31       436   50   ILE   HG2   H   −0.31       437   50   ILE   C   C   168.12       438   50   ILE   CA   C   57.68       439   50   ILE   CB   C   37.82       440   50   ILE   N   N   119.88       441   51   LEU   H   H   8.39       442   51   LEU   HA   H   4.30       443   51   LEU   HB2   H   1.44       444   51   LEU   HB3   H   1.24       445   51   LEU   HG   H   1.44       446   51   LEU   HD1   H   0.67       447   51   LEU   C   C   171.45       448   51   LEU   CA   C   51.06       449   51   LEU   CB   C   44.03       450   51   LEU   CG   C   24.41       451   51   LEU   CD1   C   23.46       452   51   LEU   N   N   120.99       453   52   THR   H   H   8.89       454   52   THR   HA   H   5.22       455   52   THR   HB   H   3.52       456   52   THR   HG2   H   1.30       457   52   THR   C   C   173.14       458   52   THR   CA   C   59.30       459   52   THR   CB   C   72.25       460   52   THR   CG2   C   22.71       461   52   THR   N   N   120.58       462   53   VAL   H   H   8.97       463   53   VAL   HA   H   4.71       464   53   VAL   HB   H   1.65       465   53   VAL   HG1   H   0.43       466   53   VAL   HG2   H   0.16       467   53   VAL   C   C   170.60       468   53   VAL   CA   C   58.06       469   53   VAL   CB   C   31.00       470   53   VAL   CG1   C   18.20       471   53   VAL   CG2   C   20.37       472   53   VAL   N   N   127.66       473   54   THR   H   H   8.63       474   54   THR   HA   H   5.00       475   54   THR   HB   H   3.87       476   54   THR   HG2   H   1.03       477   54   THR   C   C   172.93       478   54   THR   CA   C   56.41       479   54   THR   CB   C   68.61       480   54   THR   CG2   C   19.60       481   54   THR   N   N   114.36       482   55   TYR   H   H   7.26       483   55   TYR   HA   H   4.61       484   55   TYR   HB2   H   3.55       485   55   TYR   HB3   H   3.55       486   55   TYR   C   C   171.06       487   55   TYR   CA   C   55.21       488   55   TYR   CB   C   40.88       489   55   TYR   N   N   113.74       490   56   HIS   H   H   9.34       491   56   HIS   HA   H   4.42       492   56   HIS   HB2   H   3.08       493   56   HIS   HB3   H   2.81       494   56   HIS   C   C   173.18       495   56   HIS   CA   C   56.49       496   56   HIS   CB   C   29.81       497   56   HIS   N   N   118.21       498   57   SER   H   H   7.34       499   57   SER   C   C   173.49       500   57   SER   CA   C   54.41       501   57   SER   N   N   105.78       502   59   THR   HA   H   3.91       503   59   THR   HB   H   4.07       504   59   THR   HG2   H   1.20       505   59   THR   CA   C   64.19       506   59   THR   CB   C   66.34       507   59   THR   CG2   C   18.99       508   60   GLN   H   H   8.02       509   60   GLN   HA   H   4.06       510   60   GLN   HB2   H   2.09       511   60   GLN   HB3   H   2.09       512   60   GLN   HG2   H   3.26       513   60   GLN   HG3   H   3.26       514   60   GLN   C   C   174.20       515   60   GLN   CA   C   56.90       516   60   GLN   CB   C   27.27       517   60   GLN   CG   C   41.55       518   60   GLN   N   N   123.81       519   61   ARG   H   H   7.31       520   61   ARG   HA   H   2.99       521   61   ARG   HB2   H   1.70       522   61   ARG   HB3   H   1.70       523   61   ARG   C   C   175.22       524   61   ARG   CA   C   57.25       525   61   ARG   CB   C   27.77       526   61   ARG   N   N   119.25       527   62   THR   H   H   8.47       528   62   THR   HA   H   3.71       529   62   THR   HB   H   4.21       530   62   THR   HG2   H   1.16       531   62   THR   C   C   174.94       532   62   THR   CA   C   64.67       533   62   THR   CB   C   66.46       534   62   THR   CG2   C   19.65       535   62   THR   N   N   117.57       536   63   LYS   H   H   7.88       537   63   LYS   HA   H   4.05       538   63   LYS   HB2   H   1.90       539   63   LYS   HB3   H   1.90       540   63   LYS   HG2   H   1.29       541   63   LYS   HG3   H   1.29       542   63   LYS   HD2   H   1.59       543   63   LYS   HD3   H   1.59       544   63   LYS   HE2   H   2.84       545   63   LYS   HE3   H   2.79       546   63   LYS   C   C   173.47       547   63   LYS   CA   C   57.28       548   63   LYS   CB   C   29.34       549   63   LYS   CG   C   22.63       550   63   LYS   CD   C   26.76       551   63   LYS   CE   C   39.80       552   63   LYS   N   N   121.56       553   64   PHE   HA   H   3.94       554   64   PHE   HB2   H   3.75       555   64   PHE   HB3   H   3.75       556   64   PHE   C   C   177.53       557   64   PHE   CA   C   59.77       558   64   PHE   CB   C   35.86       559   64   PHE   N   N   122.19       560   65   LEU   H   H   8.46       561   65   LEU   HA   H   4.03       562   65   LEU   HB2   H   1.92       563   65   LEU   HB3   H   1.33       564   65   LEU   HD1   H   0.67       565   65   LEU   HD2   H   0.48       566   65   LEU   C   C   174.91       567   65   LEU   CA   C   54.86       568   65   LEU   CB   C   39.32       569   65   LEU   CD1   C   19.30       570   65   LEU   CD2   C   22.91       571   65   LEU   N   N   118.84       572   66   ASN   H   H   7.89       573   66   ASN   HA   H   4.72       574   66   ASN   HB2   H   2.84       575   66   ASN   HB3   H   2.76       576   66   ASN   C   C   176.34       577   66   ASN   CA   C   51.67       578   66   ASN   CB   C   37.26       579   66   ASN   N   N   114.93       580   67   THR   H   H   7.52       581   67   THR   HA   H   4.25       582   67   THR   HB   H   3.74       583   67   THR   HG2   H   0.96       584   67   THR   C   C   173.66       585   67   THR   CA   C   61.85       586   67   THR   CB   C   68.91       587   67   THR   CG2   C   18.92       588   67   THR   N   N   112.40       589   68   VAL   H   H   7.73       590   68   VAL   HA   H   3.39       591   68   VAL   HB   H   1.05       592   68   VAL   HG1   H   0.16       593   68   VAL   HG2   H   −0.12       594   68   VAL   C   C   171.61       595   68   VAL   CA   C   60.07       596   68   VAL   CB   C   29.25       597   68   VAL   CG1   C   18.45       598   68   VAL   CG2   C   17.60       599   68   VAL   N   N   122.00       600   69   ALA   H   H   8.12       601   69   ALA   HA   H   4.23       602   69   ALA   HB   H   1.19       603   69   ALA   C   C   172.02       604   69   ALA   CA   C   49.53       605   69   ALA   CB   C   15.99       606   69   ALA   N   N   129.17       607   70   ILE   H   H   8.40       608   70   ILE   C   C   174.04       609   70   ILE   CA   C   54.26       610   70   ILE   N   N   125.89       611   71   PRO   HA   H   4.43       612   71   PRO   HB3   H   1.92       613   71   PRO   HG2   H   3.83       614   71   PRO   HG3   H   3.35       615   71   PRO   CA   C   60.85       616   71   PRO   CB   C   30.38       617   71   PRO   CG   C   25.23       618   72   ASP   H   H   8.56       619   72   ASP   HA   H   4.19       620   72   ASP   HB2   H   2.65       621   72   ASP   HB3   H   2.65       622   72   ASP   C   C   174.61       623   72   ASP   CA   C   53.85       624   72   ASP   CB   C   38.07       625   72   ASP   N   N   120.03       626   73   SER   H   H   7.48       627   73   SER   HA   H   4.26       628   73   SER   HB2   H   4.07       629   73   SER   HB3   H   3.83       630   73   SER   C   C   173.98       631   73   SER   CA   C   55.90       632   73   SER   CB   C   60.58       633   73   SER   N   N   109.69       634   74   VAL   H   H   7.83       635   74   VAL   HA   H   4.45       636   74   VAL   HB   H   1.99       637   74   VAL   HG1   H   0.66       638   74   VAL   HG2   H   0.62       639   74   VAL   C   C   171.92       640   74   VAL   CA   C   59.08       641   74   VAL   CB   C   30.98       642   74   VAL   CG1   C   20.02       643   74   VAL   CG2   C   20.02       644   74   VAL   N   N   125.42       645   75   GLN   H   H   8.94       646   75   GLN   HA   H   4.45       647   75   GLN   HB2   H   2.03       648   75   GLN   HB3   H   1.90       649   75   GLN   HG2   H   2.43       650   75   GLN   HG3   H   2.23       651   75   GLN   C   C   172.04       652   75   GLN   CA   C   53.00       653   75   GLN   CB   C   28.74       654   75   GLN   CG   C   32.19       655   75   GLN   N   N   125.65       656   76   ILE   H   H   8.83       657   76   ILE   HA   H   4.63       658   76   ILE   HB   H   1.88       659   76   ILE   HG2   H   0.67       660   76   ILE   C   C   172.76       661   76   ILE   CA   C   58.71       662   76   ILE   CB   C   37.76       663   76   ILE   CG2   C   15.81       664   76   ILE   N   N   122.43       665   77   LEU   H   H   9.07       666   77   LEU   HA   H   5.04       667   77   LEU   HB2   H   1.65       668   77   LEU   HB3   H   1.30       669   77   LEU   HG   H   1.43       670   77   LEU   HD1   H   0.74       671   77   LEU   HD2   H   0.60       672   77   LEU   C   C   172.98       673   77   LEU   CA   C   51.54       674   77   LEU   CB   C   41.98       675   77   LEU   CG   C   25.94       676   77   LEU   CD1   C   22.69       677   77   LEU   CD2   C   22.12       678   77   LEU   N   N   128.16       679   78   VAL   H   H   8.87       680   78   VAL   HA   H   4.38       681   78   VAL   HB   H   1.55       682   78   VAL   HG1   H   0.71       683   78   VAL   HG2   H   0.71       684   78   VAL   C   C   173.14       685   78   VAL   CA   C   58.45       686   78   VAL   CB   C   32.33       687   78   VAL   CG1   C   19.09       688   78   VAL   CG2   C   19.09       689   78   VAL   N   N   121.05       690   79   GLY   H   H   7.86       691   79   GLY   HA2   H   5.08       692   79   GLY   HA3   H   4.08       693   79   GLY   C   C   172.86       694   79   GLY   CA   C   44.62       695   79   GLY   N   N   111.73       696   80   TYR   H   H   8.54       697   80   TYR   HA   H   5.37       698   80   TYR   HB2   H   2.99       699   80   TYR   HB3   H   2.61       700   80   TYR   C   C   169.75       701   80   TYR   CA   C   54.23       702   80   TYR   CB   C   40.30       703   80   TYR   N   N   119.24       704   81   MET   H   H   8.60       705   81   MET   HA   H   5.35       706   81   MET   HB2   H   1.94       707   81   MET   HB3   H   1.94       708   81   MET   HG2   H   2.55       709   81   MET   HG3   H   2.50       710   81   MET   C   C   171.31       711   81   MET   CA   C   51.86       712   81   MET   CB   C   34.66       713   81   MET   CG   C   29.09       714   81   MET   N   N   117.15       715   82   THR   H   H   8.53       716   82   THR   HA   H   4.98       717   82   THR   HB   H   3.51       718   82   THR   HG2   H   1.06       719   82   THR   C   C   172.03       720   82   THR   CA   C   59.38       721   82   THR   CB   C   68.52       722   82   THR   CG2   C   19.60       723   82   THR   N   N   122.12       724   83   MET   H   H   8.25       725   83   MET   HA   H   5.19       726   83   MET   C   C   170.95       727   83   MET   CA   C   51.06       728   83   MET   CB   C   33.27       729   83   MET   N   N   122.01       730   84   HIS   H   H   8.90       731   84   HIS   C   C   173.02       732   84   HIS   CA   C   53.04       733   84   HIS   N   N   118.65