52925 Proteiinianalyysin työt HOW – hands-on workshop on protein analysis

1. 52925 Proteiinianalyysin työt HOW – hands-on workshop on protein analysis Liisa Holm

2. Instructors • Patrik Koskinen • Samuli Eldfors • Xuan Hung Ta • Martin Heger • Petri Törönen • Jussi Nokso-Koivisto

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

4. Week I Monday Introduction Pairwise alignments Tuesday Manual editing of sequence alignment Wednesday Secondary structure prediction Thursday Structure visualisation Friday Comparative modelling Week II Monday Phylogenomics Tuesday Sequence classifications Wednesday Protein-protein interactions Thursday Structure classifications Friday Review day Week III Work on course assignments Topics

5. Course organization • 1st and 2nd week (Structured) • Demonstrations (12 -…) • Practical exercises (…-17) • 3rd week (Self-organized) • Course assignment • Instructor available two hours daily • Discussion on Tuesday 13-15 • Written report due 8th December

6. Mode of work • Demonstrations • Practical exercises • Structured questions • You should first try yourself, then ask team mate, then ask instructor • Discuss results with team mate • Course assignments • Written reports, due 8 December • Two sequence assignments per team • Course grade based on report

7. 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

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

9. Linear DNA Watson & Crick (1953)

10. 3D structure Myoglobin Kendrew & Perutz (1957) 1mbn

11. Function = S interactions

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

13. 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

14. Application: Finding Homologs

15. 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

16. 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

17. 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

18. Course assignments • Goal: using the flowchart, what can you say, with what confidence, about the structure and function of the protein? • Max length of report ~10 pages. No need to show negative results. • More detailed guidelines given on Day 10.

19. Team n, n=1,…,12, works on both sequence_nA and sequence_nB A and B sequences have been selected to present different challenges, therefore it is strongly recommended that the team members work together on both sequences Sequences are here: http://ekhidna.biocenter.helsinki.fi/how/proteinlist.fasta Teams

20. Preparing sequence reports • Week 3 is reserved for preparing the reports. • Experience has shown that students progress at different speeds. Fast students may try the tools out on their sequence assignments during weeks 1-2. • Checkpoint on Tuesday (Day 12) • It is expected that sequence database searches and some downstream analyses have been done by then • The purpose is to summarize progress and discuss strategies forward