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Deduction and Visualization of Protein Structures

Deduction and Visualization of Protein Structures. Presented by Soodtida Tangpraphaphorn. Protein Basics. Amino acids have different charges and polarities Primary structure is the sequence of amino acids Secondary structure is the interactions between a.a. side chains

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Deduction and Visualization of Protein Structures

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  1. Deduction and Visualization of Protein Structures Presented by Soodtida Tangpraphaphorn

  2. Protein Basics • Amino acids have different charges and polarities • Primary structure is the sequence of amino acids • Secondary structure is the interactions between a.a. side chains • Tertiary structure is the folded conformation of a polypeptide • Quaternary structure is the interaction of polypeptide subunits

  3. Applying Methods • Sequence the gene • Determine primary structure (a.a. sequence) • Search for motifs, regions of hydrophobicity/hydrophilicity, salt bridges, sulfur bridges • Find similar polypeptides • Attempt to visualize structure • Speculate function if possible

  4. Our Gene of Interest 1 atgcagttcg tgaacaagca gttcaactat aaggaccctg taaacggtgt tgacattgcc 61 tacatcaaaa ttccaaacgc cggccagatg cagccggtga aggctttcaa gattcataac 121 aaaatctggg ttattccgga acgcgataca tttacgaacc cggaagaagg agacttgaac 181 ccgccgccgg aagcaaagca ggtgccagtt tcatactacg attcaaccta tctgagcaca 241 gacaacgaga aggataacta cctgaaggga gtgaccaaat tattcgagcg tatttattcc 301 actgacctgg gccgtatgct gctgacctca atcgtccgcg gaatcccatt ttggggtggc 361 agtaccattg acacggagtt gaaggttatt gacactaact gcattaacgt gatccaacca 421 gacggtagct acagatctga agaacttaac ctcgtaatca tcgggccctc cgcggacatt 481 atccagtttg agtgcaagag ctttggccac gaagtgttga acctgacgcg taacggttac 541 ggctctactc agtacattcg tttcagccca gacttcacgt tcggtttcga ggagagcctg 601 gaggttgata ccaacccgct gttgggtgca ggcaagttcg caactgatcc agcggtgacc 661 ctggcacacg agctgatcca cgccggtcat cgtctgtatg gcattgcgat taacccgaac 721 cgcgtgttca aggttaacac caacgcctac tacgagatga gtggtttaga agtaagcttc 781 gaggaactgc gcacgttcgg tggccatgat gcgaagttta tcgacagctt gcaggagaac 841 gagttccgtc tgtactacta caacaagttt aaagatattg caagtacact gaacaaggct 901 aagtccattg tgggtaccac tgcttcatta cagtatatga aaaatgtttt taaagagaaa 961 tatctcctat ctgaagatac atctggaaaa ttttcggtag ataaattaaa atttgataag 1021 ttatacaaaa tgttaacaga gatttacaca gaggataatt ttgttaagtt ttttaaagta 1081 cttaacagaa aaacatattt gaattttgat aaagccgtat ttaagataaa tatagtacct 1141 aaggtaaatt acacaatata tgatggattt aatttaagaa atacaaattt agcagcaaac 1201 tttaatggtc aaaatacaga aattaataat atgaatttta ctaaactaaa aaattttact 1261 ggattgtttg aattttataa gttgctatgt gtaagaggga taataacttc taaaactaaa 1321 tcattagata aaggatacaa taaggcatta aatgatttat gtatcaaagt taataattgg 1381 gacttgtttt ttagtccttc agaagataat tttactaatg atctaaataa aggagaagaa 1441 attacatctg atactaatat agaagcagca gaagaaaata ttagtttaga tttaatacaa 1501 caatattatt taacctttaa ttttgataat gaacctgaaa atatttcaat agaaaatctt 1561 tcaagtgaca ttataggcca attagaactt atgcctaata tagaaagatt tcctaatgga 1621 aaaaagtatg agttagataa atatactatg ttccattatc ttcgtgctca agaatttgaa 1681 catggtaaat ctaggattgc tttaacaaat tctgttaacg aagcattatt aaatcctagt 1741 cgtgtttata catttttttc ttcagactat gtaaagaaag ttaataaagc tacggaggca 1801 gctatgtttt taggctgggt agaacaatta gtatatgatt ttaccgatga aactagcgaa 1861 gtaagtacta cggataaaat tgcggatata actataatta ttccatatat aggacctgct 1921 ttaaatatag gtaatatgtt atataaagat gattttgtag gtgctttaat attttcagga 1981 gctgttattc tgttagaatt tataccagag attgcaatac ctgtattagg tacttttgca 2041 cttgtatcat atattgcgaa taaggttcta accgttcaaa caatagataa tgctttaagt 2101 aaaagaaatg aaaaatggga tgaggtctat aaatatatag taacaaattg gttagcaaag 2161 gttaatacac agattgatct aataagaaaa aaaatgaaag aagctttaga aaatcaagca 2221 gaagcaacaa aggctataat aaactatcag tataatcaat atactgagga agagaaaaat 2281 aatattaatt ttaatattga tgatttaagt tcgaaactta atgagtctat aaataaagct 2341 atgattaata taaataaatt tttgaatcaa tgctctgttt catatttaat gaattctatg 2401 atcccttatg gtgttaaacg gttagaagat tttgatgcta gtcttaaaga tgcattatta 2461 aagtatatat atgataatag aggaacttta attggtcaag tagatagatt aaaagataaa 2521 gttaataata cacttagtac agatatacct tttcagcttt ccaaatacgt agataatcaa 2581 agattattat ctacatttac tgaatatatt aagtaa http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=18251975

  5. Amino Acid Sequence MQFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKY VDNQRLLSTFTEYIK

  6. Examine Properties • Check for regions with like charges or polarity • Try to locate sulfur-sulfur and salt bridges • Look for possible a-helices, b-sheets

  7. Examine Properties MQFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKY VDNQRLLSTFTEYIK

  8. Check for Homology • Does your novel protein have and close relatives? • Similar proteins often have similar structures and functions • Input a.a. sequence into BLAST to find matches

  9. Check for Homology • BLAST http://www.ncbi.nlm.nih.gov/BLAST

  10. Construct Model • Capture images of the protein • X-ray crystallography • Pure protein crystal is exposed to x-ray, resulting diffraction patterns form spots corresponding with e- density • NMR Spectroscopy • Sample is immersed in magnetic field, causing nuclei to spin; variations in spin denote different connectivity of atoms

  11. Construct Model • Mathematical algorithms • Software packages can calculate thermodynamically stable conformations of molecules • Generates Cartesian coordinates of atoms and outputs them as file • Common molecular graphics programs include • Rasmol, Rastop and Cn3D • Takes coordinate files and generates image

  12. Images • RasMol viewer and ASN files becoming standard, Cn3D growing in popularity • 2D *.png adapted from 3D binary *.asn Chen J, Anderson JB, DeWeese-Scott C, Fedorova ND, Geer LY, He S, Hurwitz DI, Jackson JD, Jacobs AR, Lanczycki CJ, Liebert CA, Liu C, Madej T, Marchler-Bauer A, Marchler GH, Mazumder R, Nikolskaya AN, Rao BS, Panchenko AR, Shoemaker BA, Simonyan V, Song JS, Thiessen PA, Vasudevan S, Wang Y, Yamashita RA, Yin JJ, Bryant SH, "MMDB: Entrez's 3D-structure database", Nucleic Acids Res. 2003 Jan; 31(1): 474-7.

  13. Protein Data Bank • Online repository of 3D protein structures www.rcsb.org/pdb • Search for related proteins and structures • Upload structure of your novel protein with PDB information

  14. Generate Hypotheses • What do proteins similar to this do? • What purposes can large patches of like residues serve? • Charged patches can direct ion flow • Multiple sulfur and salt bridges strengthen protein, resist high temp.

  15. Generate Hypotheses • Our novel sequence is similar to: bontoxilysin; botulinum neurotoxin serotypes A, B & E, HHV7 alkaline exonuclease and Zn finger protein 577 • Some are known neurotoxins • Exonucleases cleave DNA • Zinc fingers bind DNA • So what might our novel protein do? • Most search hits suggest neurotoxin

  16. Our “novel” gene was none other than… Botulinum toxin type A (aka Botox!)

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