52925 Proteiinianalyysin työt

HOW – hands-on workshop on protein analysis

Liisa Holm

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

Course web page

http://ekhidna.biocenter.helsinki.fi/how – Schedule– Talks– Exercises– Course assignments– Instructions for computer use

Topics 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

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

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

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

Introduction

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

Linear DNA

Watson & Crick (1953)

3D structure

1mbn

MyoglobinKendrew & Perutz (1957)

Function = interactions

EvolutionSequence – Structure - Function

DNA sequence

Protein sequence Protein structure

Protein functionNatural selection

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

Application: Finding Homologs

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 ClonedHuman 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 proteinHereditary Non-polyposis Colon Cancer 120436 MLH1 U07418 6.3e-196 MLH1 U07187 DNA repair proteinCystic Fibrosis 219700 CFTR M28668 1.3e-167 YCF1 L35237 Metal resistance proteinWilson Disease 277900 WND U11700 5.9e-161 CCC2 L36317 Probable copper transporterGlycerol Kinase Deficiency 307030 GK L13943 1.8e-129 GUT1 X69049 Glycerol kinaseBloom Syndrome 210900 BLM U39817 2.6e-119 SGS1 U22341 HelicaseAdrenoleukodystrophy, X-linked 300100 ALD Z21876 3.4e-107 PXA1 U17065 Peroxisomal ABC transporterAtaxia Telangiectasia 208900 ATM U26455 2.8e-90 TEL1 U31331 PI3 kinaseAmyotrophic Lateral Sclerosis 105400 SOD1 K00065 2.0e-58 SOD1 J03279 Superoxide dismutaseMyotonic Dystrophy 160900 DM L19268 5.4e-53 YPK1 M21307 Serine/threonine protein kinaseLowe Syndrome 309000 OCRL M88162 1.2e-47 YIL002C Z47047 Putative IPP-5-phosphataseNeurofibromatosis, Type 1 162200 NF1 M89914 2.0e-46 IRA2 M33779 Inhibitory regulator protein

Choroideremia 303100 CHM X78121 2.1e-42 GDI1 S69371 GDP dissociation inhibitorDiastrophic Dysplasia 222600 DTD U14528 7.2e-38 SUL1 X82013 Sulfate permeaseLissencephaly 247200 LIS1 L13385 1.7e-34 MET30 L26505 Methionine metabolismThomsen Disease 160800 CLC1 Z25884 7.9e-31 GEF1 Z23117 Voltage-gated chloride channelWilms Tumor 194070 WT1 X51630 1.1e-20 FZF1 X67787 Sulphite resistance proteinAchondroplasia 100800 FGFR3 M58051 2.0e-18 IPL1 U07163 Serine/threoinine protein kinaseMenkes Syndrome 309400 MNK X69208 2.1e-17 CCC2 L36317 Probable copper transporter

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

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 functionVerify conservation of functional motifs

NoMotif searchUse other data

Known structure in family?Yes

Comparative modellingValidate motifs against 3D model

NoSecondary structure prediction

No: use single sequence methodsNo: single sequence methods

Motif search Secondary structure predictionUse other data

Flowchart

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.

Teams

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

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