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Liisa Holm

(52925 Proteiinianalyysin työt) Bioinformatiikan syventävä harjoitustyökurssi HOW – hands-on workshop on protein analysis. Liisa Holm. Instructors. Patrik Koskinen Petri Törönen. Course web page. http://ekhidna.biocenter.helsinki.fi/how Schedule Talks Exercises Course assignments

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Liisa Holm

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  1. (52925 Proteiinianalyysin työt)Bioinformatiikan syventävä harjoitustyökurssiHOW – hands-on workshop on proteinanalysis Liisa Holm

  2. Instructors • Patrik Koskinen • Petri Törönen

  3. Course web page http://ekhidna.biocenter.helsinki.fi/how • Schedule • Talks • Exercises • Course assignments • Instructions for computer use

  4. Mode of work • Course assignments • Two sequences assigned to each team • Sessions (12-16) • Demonstrations (~ 1 hour) • Practical exercises • Structured questions • You should first try yourself, then ask team mate, then ask instructor • Discuss results with team mate • Try out tools on your assigned sequences during the course • Second-last session reserved solely for working on course assignments • Presentations • Course grade based on presentation (2 March) • Two sequence assignments per team

  5. Objectives • Infer function and/or structure starting from the amino acid sequence of a query protein • Identify related sequences, place in family • Identify conserved positions in sequence and structure • Learn to use representative web-based tools • No programming, no Unix/Linux

  6. Introduction • Most cellular functions are performed or facilitated by proteins. • Primary biocatalyst • Cofactor transport/storage • Mechanical motion/support • Immune protection • Control of growth/differentiation

  7. Linear DNA Watson & Crick (1953)

  8. 3D structure Myoglobin Kendrew & Perutz (1957) 1mbn

  9. Function = S interactions

  10. EvolutionSequence – Structure - Function Natural selection DNA sequence Protein function Protein sequence Protein structure

  11. What can sequence analysis do? • Homology • Inference of inherited complex features: what is conserved is important • Most powerful approach • Good tertiary structure prediction • Diagnostic patterns • E.g. subcellular localization signals • Physical preferences • Good secondary structure prediction • Prediction of transmembrane segments • Poor ab initio tertiary structure prediction

  12. Application: Finding Homologs

  13. Application:Finding Homologues • Find Similar Ones in Different Organisms • Human vs. Mouse vs. Yeast • Easier to do Expts. on latter! (Section from NCBI Disease Genes Database Reproduced Below.) Best Sequence Similarity Matches to Date Between Positionally Cloned Human Genes and S. cerevisiae Proteins Human Disease MIM # Human GenBank BLASTX Yeast GenBank Yeast Gene Gene Acc# for P-value Gene Acc# for Description Human cDNA Yeast cDNA Hereditary Non-polyposis Colon Cancer 120436 MSH2 U03911 9.2e-261 MSH2 M84170 DNA repair protein Hereditary Non-polyposis Colon Cancer 120436 MLH1 U07418 6.3e-196 MLH1 U07187 DNA repair protein Cystic Fibrosis 219700 CFTR M28668 1.3e-167 YCF1 L35237 Metal resistance protein Wilson Disease 277900 WND U11700 5.9e-161 CCC2 L36317 Probable copper transporter Glycerol Kinase Deficiency 307030 GK L13943 1.8e-129 GUT1 X69049 Glycerol kinase Bloom Syndrome 210900 BLM U39817 2.6e-119 SGS1 U22341 Helicase Adrenoleukodystrophy, X-linked 300100 ALD Z21876 3.4e-107 PXA1 U17065 Peroxisomal ABC transporter Ataxia Telangiectasia 208900 ATM U26455 2.8e-90 TEL1 U31331 PI3 kinase Amyotrophic Lateral Sclerosis 105400 SOD1 K00065 2.0e-58 SOD1 J03279 Superoxide dismutase Myotonic Dystrophy 160900 DM L19268 5.4e-53 YPK1 M21307 Serine/threonine protein kinase Lowe Syndrome 309000 OCRL M88162 1.2e-47 YIL002C Z47047 Putative IPP-5-phosphatase Neurofibromatosis, Type 1 162200 NF1 M89914 2.0e-46 IRA2 M33779 Inhibitory regulator protein Choroideremia 303100 CHM X78121 2.1e-42 GDI1 S69371 GDP dissociation inhibitor Diastrophic Dysplasia 222600 DTD U14528 7.2e-38 SUL1 X82013 Sulfate permease Lissencephaly 247200 LIS1 L13385 1.7e-34 MET30 L26505 Methionine metabolism Thomsen Disease 160800 CLC1 Z25884 7.9e-31 GEF1 Z23117 Voltage-gated chloride channel Wilms Tumor 194070 WT1 X51630 1.1e-20 FZF1 X67787 Sulphite resistance protein Achondroplasia 100800 FGFR3 M58051 2.0e-18 IPL1 U07163 Serine/threoinine protein kinase Menkes Syndrome 309400 MNK X69208 2.1e-17 CCC2 L36317 Probable copper transporter

  14. What you will learn • Multiple alignment • Used as input to many prediction tools • Improves sequence-structure alignment • Identify functional sites • Protein structure • Visualisation • Comparative modelling • Using phylogeny in function assignment • Family classifications

  15. Query = Protein sequence Sequence similarity to other proteins? Yes: does similarity imply homology? Yes: place query in family tree Known function(s) in family? Yes Transfer function Verify conservation of functional motifs No Motif search Use other data Known structure in family? Yes Comparative modelling Validate motifs against 3D model No Secondary structure prediction No: use single sequence methods No: single sequence methods Motif search Secondary structure prediction Use other data Flowchart

  16. Course assignments • Goal: using the flowchart, what can you say, with what confidence, about the structure and function of the protein? • Max length of presentation is 25 minutes. No need to dwell on negative results. • More detailed guidelines given in Session 7.

  17. Team n, n=1,…,12, works on both sequence_nA and sequence_nB A and B sequences have been selected to present different challenges. The team members should work together on both sequences, discussing the findings between them and making notes for the final report (presentation). Sequences are here: http://ekhidna.biocenter.helsinki.fi/how/proteinlist.fasta Teams

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