@text Green Fluorescent Protein by Mark Tidwell (Navigate more easily by clicking on text within the brackets). *{ }* The Green Fluorescent Protein (GFP) has become a frequently used tool for studying gene expression, cell lineage's, intracellular protein localization and real time interactions between proteins. GFP is found in jellyfish of the Pacific Northwest among the Aequorea, Obelia, Phialidium and Renilla, only the GFP of Aequorea victoria has been cloned and it is this GFP that is referred to throughout this text. GFP is composed of 238 amino acids. Cylindrical in shape and capped at both ends, the protein is approximately 30 by 40 Angstroms. The crystal structure of GFP has been resolved by Yang, et. al. to 1.9 Angstroms, but a more recent model of 2.13 Angstroms resolution, (PDB number, 1EMB) is used most frequently here. The cylinder is composed of 11 antiparallel beta-strands that are tightly interlocked like the staves of a barrel. *{kinemage 1}* Through the center of the barrel runs an alpha-helix which contains the fluorophore. *{kinemage 3}* The combination of a helix within a cylinder of beta-sheets describes a unique protein fold referred to as a beta-can. This structure makes for a very stable structure that is highly resistant to denaturation. Folding of the protein is believed to be auto-catalytic and is thermosensitive above 30C, but very stable once folded up to 65C. There are no clefts to allow diffusable ligands to pass through the mature protein. This is especially important in preventing quenching by oxygen. The fluorophore is comprised of Ser65, Tyr66 and Gly67. The backbone of these residues undergo a post-translational cyclization to form a 4-(p-hydroxybenzylidene)-imidazolidin-5-one. The proposed mechanism of fluorophore formation is as follows. First, a nucleophilic attack by the Gly67 amino on the carbonyl of Ser65 releases a water and forms the imidazolidone ring. Then, the C-alpha C-beta bond of Tyr66 is oxidized to create a large delocalized pi system. *{kinemage 4}* This is an important contrast to the earlier statement concerning the structure of the mature protein; atmospheric oxygen is necessary for proper formation of the fluorophore but must be kept out of the folded protein to prevent quenching. The active fluorophore is composed of the 7 atoms of the phenol of Tyr66, the 6 atoms of the imidazolidone and the bridging methylene. This is unique among fluorescent proteins in that the fluorophore is made of modified amino acids and not a separately synthesized prosthetic group. The sequence of Ser,Tyr,Gly is found in other proteins but they are neither cyclized or fluorescent, indicating that fluorescence is not an intrinsic property of this tripeptide. The fluorophore by itself will not fluoresce. The cycle of fluorescence begins when the hydroxyl form of Tyr66 absorbs a photon then releases a proton, the photon is released and a proton is taken up again. His148, Tyr66, Gln94 and Arg96 form H bonds with the benzyl oxygen of Tyr66 and/or the carbonyl Oxygen of the imidazolidone. This presumably stabilizes and delocalizes the fluorescent charge. Polar residues near the pocket have a H bond network to Tyr66 that may abstract protons in the oxidation process. *{kinemage 5}* Absorption of wild type GFP is at 395nm with a minor peak at 475nm. Emission is at 508nm. In vivo, GFP activation depends on excitation by the calcium triggered photoprotein aequorin, which generates a blue light. In A. victoria, this is thought to be a stress response. Mutations in GFP can yield different emission peaks. A Tyr66His mutation causes a shift in emission to the blue at 448nm. Red shift mutants have been created as well as yellow fluorescing proteins. GFP dimers have been seen in crystals but they have not yet been observed in vivo. *{kinemage 6}* Crystals show a large number of hydrophilic contacts between the dimers. A large patch of hydrophobic residues nearby may be required for interaction with Aequorin. *{kinemage 2}* It is possible to make multiple mutations and even large deletions without interfering with folding or fluorescence, with some notable exceptions. Truncating more than 7 amino acids off of the C-terminus or removal of the Met N-terminus will result in a total loss of fluorescence. This is probably due to a defect in post-translational processing. The last 11 residues of the C-terminus are not part of a beta-strand, but do make up a cap on one end. Fusions with other proteins however, can be at either end without affecting the fold or the fluorescence. NAVIGATION: *{kinemage 1}* for ribbon overview *{kinemage 2}* for wireframe, cysteine placement and hydrophobic connections with Aequorin *{kinemage 3}* the alpha helix and the fluorophore *{kinemage 4}* fluorophore cyclization *{kinemage 5}* the fluorophore and groups associated with the fluorescent cycle and stabilization. *{kinemage 6}* the dimer of GFP and associated residues. References: Yang, T., Moss, L.G. and Phillips, G.N., "The molecular structure of green fluorescent protein" Nature Biotechnology, 14:1246 (1996) Tsien, R.Y., "The green fluorescent protein" Annual Review of Biochemistry 67:509 (1998) PDB files: 1EMB, 1YFP, 1GFL, 1BFP, 1BVA @kinemage 1 @caption This ribbon form of GFP shows the tight "barrel-stave" like fit of the Beta Strands (in green), the central helix (in gold), and the protected fluorophore (in red) located in the center. *{view 1}* GFP Seen from the edge *{view 2}* GFP Seen from the N-terminus @onewidth @viewid {ribbon side} @zoom 1.00 @zslab 200 @ztran 0 @center 27.090 29.786 37.329 @matrix 0.517801 -0.443959 0.731288 -0.581850 0.443896 0.681474 -0.627163 -0.778368 -0.028468 @2viewid {N terminal} @2zoom 1.00 @2zslab 200 @2ztran 0 @2center 27.090 29.786 37.329 @2matrix 0.463316 0.836313 0.293118 -0.415167 0.497057 -0.761952 -0.782927 0.231332 0.577504 @master {edge} @master {beta} @master {face} @master {coil} @master {alpha} 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}12.435 36.019 22.674 {R1 gly 51 }P 14.157 34.836 22.403 {R2 gly 51 }13.007 36.435 22.536 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R1 lys 52 }P 14.157 34.836 22.403 {R1 lys 52 }14.807 35.256 22.350 {"}15.456 35.663 22.465 {"}16.075 36.077 22.721 {"}16.635 36.518 23.084 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R2 lys 52 }P 13.007 36.435 22.536 {R2 lys 52 }13.592 36.783 22.563 {"}14.172 37.089 22.789 {"}14.729 37.365 23.185 {"}15.241 37.621 23.703 @ribbonlist {ribbon} color= gold nobutton master= {face} master= {alpha} {R1 lys 52 }P 14.157 34.836 22.403 {R2 lys 52 }13.007 36.435 22.536 {R1 lys 52 }P 14.807 35.256 22.350 {R2 lys 52 }13.592 36.783 22.563 {R1 lys 52 }P 15.456 35.663 22.465 {R2 lys 52 }14.172 37.089 22.789 {R1 lys 52 }P 16.075 36.077 22.721 {R2 lys 52 }14.729 37.365 23.185 {R1 lys 52 }P 16.635 36.518 23.084 {R2 lys 52 }15.241 37.621 23.703 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R1 leu 53 }P 16.635 36.518 23.084 {R1 leu 53 }17.106 37.013 23.517 {"}17.469 37.574 23.992 {"}17.748 38.185 24.495 {"}17.977 38.820 25.020 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R2 leu 53 }P 15.241 37.621 23.703 {R2 leu 53 }15.686 37.867 24.295 {"}16.052 38.113 24.916 {"}16.369 38.359 25.533 {"}16.676 38.605 26.113 @ribbonlist {ribbon} color= gold nobutton master= {face} master= {alpha} {R1 leu 53 }P 16.635 36.518 23.084 {R2 leu 53 }15.241 37.621 23.703 {R1 leu 53 }P 17.106 37.013 23.517 {R2 leu 53 }15.686 37.867 24.295 {R1 leu 53 }P 17.469 37.574 23.992 {R2 leu 53 }16.052 38.113 24.916 {R1 leu 53 }P 17.748 38.185 24.495 {R2 leu 53 }16.369 38.359 25.533 {R1 leu 53 }P 17.977 38.820 25.020 {R2 leu 53 }16.676 38.605 26.113 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R1 pro 54 }P 17.977 38.820 25.020 {R1 pro 54 }18.190 39.452 25.559 {"}18.421 40.052 26.104 {"}18.707 40.575 26.635 {"}19.086 40.974 27.130 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R2 pro 54 }P 16.676 38.605 26.113 {R2 pro 54 }17.012 38.851 26.625 {"}17.409 39.091 27.042 {"}17.873 39.307 27.361 {"}18.404 39.476 27.581 @ribbonlist {ribbon} color= gold nobutton master= {face} master= {alpha} {R1 pro 54 }P 17.977 38.820 25.020 {R2 pro 54 }16.676 38.605 26.113 {R1 pro 54 }P 18.190 39.452 25.559 {R2 pro 54 }17.012 38.851 26.625 {R1 pro 54 }P 18.421 40.052 26.104 {R2 pro 54 }17.409 39.091 27.042 {R1 pro 54 }P 18.707 40.575 26.635 {R2 pro 54 }17.873 39.307 27.361 {R1 pro 54 }P 19.086 40.974 27.130 {R2 pro 54 }18.404 39.476 27.581 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R1 val 55 }P 19.086 40.974 27.130 {R1 val 55 }19.594 41.201 27.566 {"}20.249 41.219 27.926 {"}20.993 41.043 28.207 {"}21.750 40.699 28.412 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R2 val 55 }P 18.404 39.476 27.581 {R2 val 55 }19.000 39.575 27.702 {"}19.657 39.584 27.728 {"}20.352 39.501 27.675 {"}21.058 39.326 27.559 @ribbonlist {ribbon} color= gold nobutton master= {face} master= {alpha} {R1 val 55 }P 19.086 40.974 27.130 {R2 val 55 }18.404 39.476 27.581 {R1 val 55 }P 19.594 41.201 27.566 {R2 val 55 }19.000 39.575 27.702 {R1 val 55 }P 20.249 41.219 27.926 {R2 val 55 }19.657 39.584 27.728 {R1 val 55 }P 20.993 41.043 28.207 {R2 val 55 }20.352 39.501 27.675 {R1 val 55 }P 21.750 40.699 28.412 {R2 val 55 }21.058 39.326 27.559 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R1 pro 56 }P 21.750 40.699 28.412 {R1 pro 56 }22.444 40.214 28.545 {"}23.018 39.613 28.609 {"}23.494 38.921 28.627 {"}23.918 38.160 28.621 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R2 pro 56 }P 21.058 39.326 27.559 {R2 pro 56 }21.747 39.060 27.399 {"}22.402 38.708 27.215 {"}23.029 38.284 27.038 {"}23.648 37.806 26.899 @ribbonlist {ribbon} color= gold nobutton master= {face} master= {alpha} {R1 pro 56 }P 21.750 40.699 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@vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R1 thr 59 }P 27.520 35.915 31.112 {R1 thr 59 }27.433 36.348 31.827 {"}26.951 36.627 32.490 {"}26.219 36.703 33.062 {"}25.437 36.547 33.508 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R2 thr 59 }P 27.094 36.930 29.732 {R2 thr 59 }26.837 37.218 30.405 {"}26.346 37.280 30.981 {"}25.743 37.123 31.451 {"}25.182 36.785 31.817 @ribbonlist {ribbon} color= gold nobutton master= {face} master= {alpha} {R1 thr 59 }P 27.520 35.915 31.112 {R2 thr 59 }27.094 36.930 29.732 {R1 thr 59 }P 27.433 36.348 31.827 {R2 thr 59 }26.837 37.218 30.405 {R1 thr 59 }P 26.951 36.627 32.490 {R2 thr 59 }26.346 37.280 30.981 {R1 thr 59 }P 26.219 36.703 33.062 {R2 thr 59 }25.743 37.123 31.451 {R1 thr 59 }P 25.437 36.547 33.508 {R2 thr 59 }25.182 36.785 31.817 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R1 leu 60 }P 25.437 36.547 33.508 {R1 leu 60 }24.804 36.130 33.791 {"}24.477 35.446 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32.563 {R2 val 61 }25.969 33.832 32.807 {"}26.574 33.305 33.179 {"}27.127 32.877 33.664 {"}27.522 32.560 34.233 @ribbonlist {ribbon} color= gold nobutton master= {face} master= {alpha} {R1 val 61 }P 24.680 33.641 33.843 {R2 val 61 }25.414 34.440 32.563 {R1 val 61 }P 25.116 32.736 33.847 {R2 val 61 }25.969 33.832 32.807 {R1 val 61 }P 25.698 31.958 33.968 {R2 val 61 }26.574 33.305 33.179 {R1 val 61 }P 26.312 31.342 34.234 {R2 val 61 }27.127 32.877 33.664 {R1 val 61 }P 26.828 30.906 34.660 {R2 val 61 }27.522 32.560 34.233 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R1 thr 62 }P 26.828 30.906 34.660 {R1 thr 62 }27.117 30.670 35.264 {"}27.080 30.631 36.033 {"}26.744 30.698 36.848 {"}26.167 30.758 37.563 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R2 thr 62 }P 27.522 32.560 34.233 {R2 thr 62 }27.652 32.368 34.857 {"}27.444 32.296 35.497 {"}26.955 32.276 36.084 {"}26.276 32.222 36.537 @ribbonlist {ribbon} color= gold nobutton master= {face} master= {alpha} {R1 thr 62 }P 26.828 30.906 34.660 {R2 thr 62 }27.522 32.560 34.233 {R1 thr 62 }P 27.117 30.670 35.264 {R2 thr 62 }27.652 32.368 34.857 {R1 thr 62 }P 27.080 30.631 36.033 {R2 thr 62 }27.444 32.296 35.497 {R1 thr 62 }P 26.744 30.698 36.848 {R2 thr 62 }26.955 32.276 36.084 {R1 thr 62 }P 26.167 30.758 37.563 {R2 thr 62 }26.276 32.222 36.537 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R1 thr 63 }P 26.167 30.758 37.563 {R1 thr 63 }25.408 30.698 38.030 {"}24.533 30.439 38.145 {"}23.647 30.032 37.970 {"}22.865 29.560 37.610 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R2 thr 63 }P 26.276 32.222 36.537 {R2 thr 63 }25.496 32.048 36.775 {"}24.703 31.695 36.747 {"}23.969 31.217 36.503 {"}23.363 30.694 36.123 @ribbonlist {ribbon} color= gold nobutton master= {face} master= {alpha} {R1 thr 63 }P 26.167 30.758 37.563 {R2 thr 63 }26.276 32.222 36.537 {R1 thr 63 }P 25.408 30.698 38.030 {R2 thr 63 }25.496 32.048 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22.403 28.007 35.514 {R2 phe 64 }23.233 29.071 34.038 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} {R1 val 68 }P 25.137 23.714 39.737 {"}25.269 23.699 39.825 {"}25.597 23.633 40.054 {"}26.024 23.487 40.376 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} {R2 val 68 }P 25.795 22.240 38.557 {"}25.929 22.227 38.649 {"}26.281 22.183 38.909 {"}26.769 22.096 39.316 @ribbonlist {ribbon} color= gold nobutton master= {face} master= {alpha} {R1 val 68 }P 25.137 23.714 39.737 {R2 val 68 }25.795 22.240 38.557 {R1 val 68 }P 25.269 23.699 39.825 {R2 val 68 }25.929 22.227 38.649 {R1 val 68 }P 25.597 23.633 40.054 {R2 val 68 }26.281 22.183 38.909 {R1 val 68 }P 26.024 23.487 40.376 {R2 val 68 }26.769 22.096 39.316 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R1 gln 69 }P 26.024 23.487 40.376 {R1 gln 69 }26.449 23.232 40.743 {"}26.792 22.849 41.104 {"}27.048 22.357 41.404 {"}27.229 21.787 41.589 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R2 gln 69 }P 26.769 22.096 39.316 {R2 gln 69 }27.314 21.955 39.847 {"}27.843 21.746 40.467 {"}28.302 21.443 41.082 {"}28.641 21.015 41.587 @ribbonlist {ribbon} color= gold nobutton master= {face} master= {alpha} {R1 gln 69 }P 26.024 23.487 40.376 {R2 gln 69 }26.769 22.096 39.316 {R1 gln 69 }P 26.449 23.232 40.743 {R2 gln 69 }27.314 21.955 39.847 {R1 gln 69 }P 26.792 22.849 41.104 {R2 gln 69 }27.843 21.746 40.467 {R1 gln 69 }P 27.048 22.357 41.404 {R2 gln 69 }28.302 21.443 41.082 {R1 gln 69 }P 27.229 21.787 41.589 {R2 gln 69 }28.641 21.015 41.587 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R1 cys 70 }P 27.229 21.787 41.589 {R1 cys 70 }27.346 21.171 41.603 {"}27.422 20.542 41.416 {"}27.513 19.955 41.097 {"}27.685 19.468 40.739 @vectorlist {ribbon} color= gold nobutton master= {edge} master= {alpha} { R2 cys 70 }P 28.641 21.015 41.587 {R2 cys 70 }28.814 20.432 41.878 {"}28.801 19.693 41.878 {"}28.708 18.909 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{"}20.075 37.928 42.325 {"}19.788 37.206 42.601 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R2 lys 107 }P 19.377 40.205 40.508 {R2 lys 107 }19.029 39.485 40.729 {"}18.719 38.785 40.966 {"}18.442 38.086 41.207 {"}18.193 37.366 41.436 @ribbonlist {ribbon} color= sea nobutton master= {beta} master= {face} {R1 lys 107 }P 21.084 39.914 41.348 {R2 lys 107 }19.377 40.205 40.508 {R1 lys 107 }P 20.713 39.289 41.699 {R2 lys 107 }19.029 39.485 40.729 {R1 lys 107 }P 20.380 38.625 42.025 {R2 lys 107 }18.719 38.785 40.966 {R1 lys 107 }P 20.075 37.928 42.325 {R2 lys 107 }18.442 38.086 41.207 {R1 lys 107 }P 19.788 37.206 42.601 {R2 lys 107 }18.193 37.366 41.436 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R1 thr 108 }P 19.788 37.206 42.601 {R1 thr 108 }19.508 36.467 42.857 {"}19.229 35.717 43.091 {"}18.959 34.953 43.300 {"}18.712 34.168 43.479 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R2 thr 108 }P 18.193 37.366 41.436 {R2 thr 108 }17.970 36.603 41.636 {"}17.769 35.783 41.797 {"}17.585 34.925 41.923 {"}17.413 34.059 42.028 @ribbonlist {ribbon} color= sea nobutton master= {beta} master= {face} {R1 thr 108 }P 19.788 37.206 42.601 {R2 thr 108 }18.193 37.366 41.436 {R1 thr 108 }P 19.508 36.467 42.857 {R2 thr 108 }17.970 36.603 41.636 {R1 thr 108 }P 19.229 35.717 43.091 {R2 thr 108 }17.769 35.783 41.797 {R1 thr 108 }P 18.959 34.953 43.300 {R2 thr 108 }17.585 34.925 41.923 {R1 thr 108 }P 18.712 34.168 43.479 {R2 thr 108 }17.413 34.059 42.028 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R1 arg 109 }P 18.712 34.168 43.479 {R1 arg 109 }18.500 33.357 43.619 {"}18.332 32.516 43.719 {"}18.197 31.656 43.785 {"}18.084 30.791 43.829 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R2 arg 109 }P 17.413 34.059 42.028 {R2 arg 109 }17.245 33.213 42.122 {"}17.080 32.407 42.212 {"}16.929 31.622 42.291 {"}16.807 30.832 42.346 @ribbonlist {ribbon} color= sea 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34.093 38.151 {R2 leu 125 }15.618 33.976 39.802 {R1 leu 125 }P 14.575 34.919 38.160 {R2 leu 125 }15.733 34.792 39.772 {R1 leu 125 }P 14.662 35.742 38.167 {R2 leu 125 }15.876 35.628 39.740 {R1 leu 125 }P 14.771 36.564 38.166 {R2 leu 125 }16.033 36.470 39.704 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R1 lys 126 }P 14.771 36.564 38.166 {R1 lys 126 }14.899 37.388 38.147 {"}15.043 38.216 38.107 {"}15.198 39.046 38.053 {"}15.361 39.878 37.995 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R2 lys 126 }P 16.033 36.470 39.704 {R2 lys 126 }16.194 37.308 39.662 {"}16.351 38.134 39.613 {"}16.506 38.949 39.558 {"}16.669 39.759 39.499 @ribbonlist {ribbon} color= sea nobutton master= {beta} master= {face} {R1 lys 126 }P 14.771 36.564 38.166 {R2 lys 126 }16.033 36.470 39.704 {R1 lys 126 }P 14.899 37.388 38.147 {R2 lys 126 }16.194 37.308 39.662 {R1 lys 126 }P 15.043 38.216 38.107 {R2 lys 126 }16.351 38.134 39.613 {R1 lys 126 }P 15.198 39.046 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master= {edge} master= {beta} { R1 ile 128 }P 15.974 43.105 37.921 {R1 ile 128 }16.058 43.794 37.969 {"}16.092 44.393 38.048 {"}P 16.834 44.291 38.708 {"}15.349 44.495 37.387 {"}16.747 45.776 39.064 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R2 ile 128 }P 17.399 42.914 39.301 {R2 ile 128 }17.520 43.595 39.313 {"}17.577 44.188 39.369 {"}P 16.834 44.291 38.708 {"}18.319 44.086 40.029 {"}16.747 45.776 39.064 @ribbonlist {ribbon} color= sea nobutton master= {beta} master= {face} {R1 ile 128 }P 15.974 43.105 37.921 {R2 ile 128 }17.399 42.914 39.301 {R1 ile 128 }P 16.058 43.794 37.969 {R2 ile 128 }17.520 43.595 39.313 {R1 ile 128 }P 16.092 44.393 38.048 {R2 ile 128 }17.577 44.188 39.369 {R1 ile 128 }P 15.349 44.495 37.387 {R2 ile 128 }18.319 44.086 40.029 {R1 ile 128 }P 16.747 45.776 39.064 {R2 ile 128 }16.747 45.776 39.064 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} {R1 ser 147 }P 38.235 30.467 34.342 {"}38.206 30.499 34.499 {"}38.140 30.571 34.919 {"}38.061 30.651 35.525 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} {R2 ser 147 }P 39.325 32.137 34.190 {"}39.295 32.169 34.349 {"}39.217 32.244 34.784 {"}39.109 32.333 35.427 @ribbonlist {ribbon} color= sea nobutton master= {beta} master= {face} {R1 ser 147 }P 38.235 30.467 34.342 {R2 ser 147 }39.325 32.137 34.190 {R1 ser 147 }P 38.206 30.499 34.499 {R2 ser 147 }39.295 32.169 34.349 {R1 ser 147 }P 38.140 30.571 34.919 {R2 ser 147 }39.217 32.244 34.784 {R1 ser 147 }P 38.061 30.651 35.525 {R2 ser 147 }39.109 32.333 35.427 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R1 his 148 }P 38.061 30.651 35.525 {R1 his 148 }37.999 30.706 36.237 {"}37.971 30.709 36.991 {"}37.957 30.658 37.764 {"}37.929 30.558 38.548 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R2 his 148 }P 39.109 32.333 35.427 {R2 his 148 }38.987 32.404 36.214 {"}38.864 32.433 37.083 {"}38.726 32.416 37.989 {"}38.551 32.353 38.891 @ribbonlist {ribbon} color= sea nobutton master= {beta} master= {face} {R1 his 148 }P 38.061 30.651 35.525 {R2 his 148 }39.109 32.333 35.427 {R1 his 148 }P 37.999 30.706 36.237 {R2 his 148 }38.987 32.404 36.214 {R1 his 148 }P 37.971 30.709 36.991 {R2 his 148 }38.864 32.433 37.083 {R1 his 148 }P 37.957 30.658 37.764 {R2 his 148 }38.726 32.416 37.989 {R1 his 148 }P 37.929 30.558 38.548 {R2 his 148 }38.551 32.353 38.891 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R1 asn 149 }P 37.929 30.558 38.548 {R1 asn 149 }37.859 30.413 39.332 {"}37.726 30.231 40.110 {"}37.539 30.031 40.880 {"}37.313 29.838 41.644 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R2 asn 149 }P 38.551 32.353 38.891 {R2 asn 149 }38.320 32.248 39.748 {"}38.020 32.103 40.529 {"}37.668 31.937 41.251 {"}37.288 31.770 41.937 @ribbonlist {ribbon} color= sea nobutton master= {beta} master= {face} {R1 asn 149 }P 37.929 30.558 38.548 {R2 asn 149 }38.551 32.353 38.891 {R1 asn 149 }P 37.859 30.413 39.332 {R2 asn 149 }38.320 32.248 39.748 {R1 asn 149 }P 37.726 30.231 40.110 {R2 asn 149 }38.020 32.103 40.529 {R1 asn 149 }P 37.539 30.031 40.880 {R2 asn 149 }37.668 31.937 41.251 {R1 asn 149 }P 37.313 29.838 41.644 {R2 asn 149 }37.288 31.770 41.937 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R1 val 150 }P 37.313 29.838 41.644 {R1 val 150 }37.065 29.673 42.403 {"}36.812 29.553 43.158 {"}36.570 29.462 43.911 {"}36.357 29.381 44.663 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R2 val 150 }P 37.288 31.770 41.937 {R2 val 150 }36.904 31.622 42.613 {"}36.538 31.506 43.300 {"}36.190 31.401 44.001 {"}35.859 31.278 44.712 @ribbonlist {ribbon} color= sea nobutton master= {beta} master= {face} {R1 val 150 }P 37.313 29.838 41.644 {R2 val 150 }37.288 31.770 41.937 {R1 val 150 }P 37.065 29.673 42.403 {R2 val 150 }36.904 31.622 42.613 {R1 val 150 }P 36.812 29.553 43.158 {R2 val 150 }36.538 31.506 43.300 {R1 val 150 }P 36.570 29.462 43.911 {R2 val 150 }36.190 31.401 44.001 {R1 val 150 }P 36.357 29.381 44.663 {R2 val 150 }35.859 31.278 44.712 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R1 tyr 151 }P 36.357 29.381 44.663 {R1 tyr 151 }36.189 29.288 45.415 {"}36.077 29.166 46.169 {"}35.996 29.020 46.926 {"}35.914 28.860 47.686 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R2 tyr 151 }P 35.859 31.278 44.712 {R2 tyr 151 }35.544 31.107 45.431 {"}35.240 30.869 46.156 {"}34.942 30.575 46.883 {"}34.643 30.246 47.610 @ribbonlist {ribbon} color= sea nobutton master= {beta} master= {face} {R1 tyr 151 }P 36.357 29.381 44.663 {R2 tyr 151 }35.859 31.278 44.712 {R1 tyr 151 }P 36.189 29.288 45.415 {R2 tyr 151 }35.544 31.107 45.431 {R1 tyr 151 }P 36.077 29.166 46.169 {R2 tyr 151 }35.240 30.869 46.156 {R1 tyr 151 }P 35.996 29.020 46.926 {R2 tyr 151 }34.942 30.575 46.883 {R1 tyr 151 }P 35.914 28.860 47.686 {R2 tyr 151 }34.643 30.246 47.610 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R1 ile 152 }P 35.914 28.860 47.686 {R1 ile 152 }35.799 28.694 48.451 {"}35.618 28.530 49.217 {"}35.343 28.360 49.956 {"}34.945 28.175 50.636 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R2 ile 152 }P 34.643 30.246 47.610 {R2 ile 152 }34.334 29.903 48.333 {"}34.007 29.565 49.050 {"}33.651 29.241 49.769 {"}33.251 28.942 50.496 @ribbonlist {ribbon} color= sea nobutton master= {beta} master= {face} {R1 ile 152 }P 35.914 28.860 47.686 {R2 ile 152 }34.643 30.246 47.610 {R1 ile 152 }P 35.799 28.694 48.451 {R2 ile 152 }34.334 29.903 48.333 {R1 ile 152 }P 35.618 28.530 49.217 {R2 ile 152 }34.007 29.565 49.050 {R1 ile 152 }P 35.343 28.360 49.956 {R2 ile 152 }33.651 29.241 49.769 {R1 ile 152 }P 34.945 28.175 50.636 {R2 ile 152 }33.251 28.942 50.496 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R1 met 153 }P 34.945 28.175 50.636 {R1 met 153 }34.395 27.966 51.227 {"}33.675 27.734 51.700 {"}P 32.975 28.090 51.846 {"}34.376 27.377 51.554 {"}31.519 27.779 52.763 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R2 met 153 }P 33.251 28.942 50.496 {R2 met 153 }32.794 28.674 51.239 {"}32.275 28.447 51.991 {"}P 32.975 28.090 51.846 {"}31.575 28.804 52.137 {"}31.519 27.779 52.763 @ribbonlist {ribbon} color= sea nobutton master= {beta} master= {face} {R1 met 153 }P 34.945 28.175 50.636 {R2 met 153 }33.251 28.942 50.496 {R1 met 153 }P 34.395 27.966 51.227 {R2 met 153 }32.794 28.674 51.239 {R1 met 153 }P 33.675 27.734 51.700 {R2 met 153 }32.275 28.447 51.991 {R1 met 153 }P 34.376 27.377 51.554 {R2 met 153 }31.575 28.804 52.137 {R1 met 153 }P 31.519 27.779 52.763 {R2 met 153 }31.519 27.779 52.763 @vectorlist {ribbon} color= invisible nobutton master= {edge} master= {coil} off { R1 ile 161 }P 31.519 27.779 52.763 {R1 ile 161 }width4 30.761 27.765 52.988 {"}width4 30.041 27.884 53.015 {"}width4 29.418 28.124 52.867 {"}width4 28.958 28.466 52.577 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} {R1 ile 161 }P 28.958 28.466 52.577 {"}28.399 28.415 52.080 @vectorlist {ribbon} color= white nobutton master= {edge} master= {coil} off { R2 ile 161 }P 31.519 27.779 52.763 {R2 ile 161 }width4 30.761 27.765 52.988 {"}width4 30.041 27.884 53.015 {"}width4 29.418 28.124 52.867 {"}width4 28.958 28.466 52.577 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} {R2 ile 161 }P 28.958 28.466 52.577 {"}29.516 28.517 53.075 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R1 lys 162 }P 28.399 28.415 52.080 {R1 lys 162 }28.002 28.969 51.755 {"}27.942 29.572 51.332 {"}28.151 30.199 50.838 {"}28.528 30.827 50.305 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R2 lys 162 }P 29.516 28.517 53.075 {R2 lys 162 }29.444 28.815 52.605 {"}29.570 29.192 52.083 {"}29.853 29.633 51.526 {"}30.239 30.118 50.941 @ribbonlist {ribbon} color= sea nobutton master= {beta} master= {face} {R1 lys 162 }P 28.399 28.415 52.080 {R2 lys 162 }29.516 28.517 53.075 {R1 lys 162 }P 28.002 28.969 51.755 {R2 lys 162 }29.444 28.815 52.605 {R1 lys 162 }P 27.942 29.572 51.332 {R2 lys 162 }29.570 29.192 52.083 {R1 lys 162 }P 28.151 30.199 50.838 {R2 lys 162 }29.853 29.633 51.526 {R1 lys 162 }P 28.528 30.827 50.305 {R2 lys 162 }30.239 30.118 50.941 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R1 val 163 }P 28.528 30.827 50.305 {R1 val 163 }28.973 31.432 49.768 {"}29.404 31.998 49.249 {"}29.814 32.535 48.744 {"}30.212 33.059 48.236 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R2 val 163 }P 30.239 30.118 50.941 {R2 val 163 }30.675 30.629 50.338 {"}31.115 31.150 49.725 {"}31.546 31.674 49.111 {"}31.962 32.193 48.508 @ribbonlist {ribbon} color= sea nobutton master= {beta} master= {face} {R1 val 163 }P 28.528 30.827 50.305 {R2 val 163 }30.239 30.118 50.941 {R1 val 163 }P 28.973 31.432 49.768 {R2 val 163 }30.675 30.629 50.338 {R1 val 163 }P 29.404 31.998 49.249 {R2 val 163 }31.115 31.150 49.725 {R1 val 163 }P 29.814 32.535 48.744 {R2 val 163 }31.546 31.674 49.111 {R1 val 163 }P 30.212 33.059 48.236 {R2 val 163 }31.962 32.193 48.508 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R1 asn 164 }P 30.212 33.059 48.236 {R1 asn 164 }30.608 33.587 47.711 {"}31.010 34.129 47.158 {"}31.404 34.672 46.578 {"}31.776 35.197 45.974 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R2 asn 164 }P 31.962 32.193 48.508 {R2 asn 164 }32.361 32.703 47.926 {"}32.738 33.198 47.367 {"}33.095 33.684 46.812 {"}33.433 34.165 46.230 @ribbonlist {ribbon} color= sea nobutton master= {beta} master= {face} {R1 asn 164 }P 30.212 33.059 48.236 {R2 asn 164 }31.962 32.193 48.508 {R1 asn 164 }P 30.608 33.587 47.711 {R2 asn 164 }32.361 32.703 47.926 {R1 asn 164 }P 31.010 34.129 47.158 {R2 asn 164 }32.738 33.198 47.367 {R1 asn 164 }P 31.404 34.672 46.578 {R2 asn 164 }33.095 33.684 46.812 {R1 asn 164 }P 31.776 35.197 45.974 {R2 asn 164 }33.433 34.165 46.230 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R1 phe 165 }P 31.776 35.197 45.974 {R1 phe 165 }32.110 35.683 45.351 {"}32.394 36.117 44.710 {"}32.625 36.507 44.048 {"}32.806 36.864 43.357 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R2 phe 165 }P 33.433 34.165 46.230 {R2 phe 165 }33.756 34.647 45.594 {"}34.063 35.131 44.883 {"}34.337 35.599 44.114 {"}34.562 36.030 43.312 @ribbonlist {ribbon} color= sea nobutton master= {beta} master= {face} {R1 phe 165 }P 31.776 35.197 45.974 {R2 phe 165 }33.433 34.165 46.230 {R1 phe 165 }P 32.110 35.683 45.351 {R2 phe 165 }33.756 34.647 45.594 {R1 phe 165 }P 32.394 36.117 44.710 {R2 phe 165 }34.063 35.131 44.883 {R1 phe 165 }P 32.625 36.507 44.048 {R2 phe 165 }34.337 35.599 44.114 {R1 phe 165 }P 32.806 36.864 43.357 {R2 phe 165 }34.562 36.030 43.312 @vectorlist {ribbon} color= sea nobutton master= {edge} master= {beta} { R1 lys 166 }P 32.806 36.864 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