Deduction and Deduction and Visualization of Protein Visualization of Protein

StructuresStructures

Presented by Soodtida Tangpraphaphorn

Protein BasicsProtein 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

Applying MethodsApplying Methods

1. Sequence the gene2. Determine primary structure

(a.a. sequence)3. Search for motifs, regions of

hydrophobicity/hydrophilicity, salt bridges, sulfur bridges

4. Find similar polypeptides5. Attempt to visualize structure6. Speculate function if possible

Our Gene of InterestOur Gene of Interest1 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

Amino Acid SequenceAmino Acid Sequence

MQFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKY VDNQRLLSTFTEYIK

Examine PropertiesExamine Properties

Check for regions with like charges or polarity

Try to locate sulfur-sulfur and salt bridges

Look for possible -helices, -sheets

Examine PropertiesExamine Properties

MQFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKY VDNQRLLSTFTEYIK

Check for HomologyCheck 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

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

Construct ModelConstruct 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

Construct ModelConstruct 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

ImagesImages 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.

Protein Data BankProtein 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

Generate HypothesesGenerate 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.

Generate HypothesesGenerate 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

Our “novel” gene was none other Our “novel” gene was none other than…than…

Botulinum toxin type Botulinum toxin type A A

(aka Botox!)(aka Botox!)