Post on 11-Jan-2016
description
Protein Bioinformatics CourseMatthew Betts & Rob RussellAG Russell (Protein Evolution)
Course overviewDay 1 - ModularityDay 2 - InteractionsDay 3 - Modularity & InteractionsDay 4 - StructureDay 5 - Structure & Interactions
Daily schedule10:00-11:00 lecture11:00-12:00 work on exercises in pairs12:00-13:00 lunch13:00-15:30 work on exercises in pairs16:00-17:00 presentations by you
Protein Sequence Databases
• Homologues = proteins with a common ancestor • Homology --> similar function• Sequence similarity --> homology
• Find homologues using:• BLAST• Profile Searching
Database Searching
www.proteinmodelportal.org
Scores and E-values
How similar is my sequence to one in the database?
How much would I expect to get >= this score by
chance alone?
• Alignment• Substitution matrix• Gap penalties
• cf. random sequences• E = 1: one such match by chance• E < 0.01: significant• Depends on database:
• size: larger = better• composition (random assumed)
Homology comes in two main types:
Orthology and Paralogy
What is the difference and why does this matter?
Paralogues
Duplication -
Duplication -
Paralogues
Orthologues
Speciation - - Speciation
Different FatesOrthologues:• Both copies required (one in each species)
• conservation of function (‘same gene’)• adaptation to new environment
Easier to transferknowledge of functionbetween orthologues
Paralogues:• Both copies useful
• conservation of function• One copy freed from selection
• disabled• new function
• Different parts of each free from selection• function split between them
Assignment of orthology / paralogy can be complicated by:• duplication preceding speciation• lineage-specific deletions of paralogs• complete genome duplications• many-to-one relationship• multi-domain proteins
Homology usually found by sequence similarity, but…proteins with dissimilar sequences can still be homologous
Betts, Guigo, Agarwal, Russell, EMBO J 2001
Proteins are modular
Since the early 1970s it has been observed that protein structures are divided into discrete elements or domains that appear to fold, function and evolve independently.
• Functional domains (Pfam, SMART, COGS, CDD, etc.)
• Intrinsic features– Signal peptide, transit peptides (signalP)– Transmembrane segments (TMpred, etc)– Coiled-coils (coils server)– Low complexity regions, disorder (e.g. SEG, disembl)
• Hints about structure?
Given a sequence, what should you look for?
“Low sequence complexity”(Linker regions? Flexible? Junk?
Signal peptide(secreted or membrane attached)
Transmembrane segment(crosses the membrane)
Tyrosine kinase (phosphorylates Tyr)
Immunoglobulin domains(bind ligands?)
SMART domain ‘bubblegram’ for human fibroblast growth factor (FGF) receptor 1(type P11362 into web site: smart.embl.de)
Given a sequence, what should you look for?
Protein Modularity
• discrete structural and functional units
• found in different combinations in different proteins
Receptor-related tyrosine-kinase
Non-receptor tyrosine-kinases
consider separately in predictions
Finding Protein Domains
• through partial matches to whole sequences:
• compare to databases of domains (Pfam, SMART, Interpro)
• can be separated by:• low-complexity and disordered regions (SEG)• trans-membrane regions (TMAP)• coiled-coils (COILS)
query sequence:
matchmatch
match
Repeat searches using each domain separately
12 000 domain alignments make sequence searching easier
WPP domain alignment
Alignments provide more information about a protein family and thus allow for more sensitive sequences than a single sequence.
Domain alignments also lack low-complexity or disorder (normally) and other domains that can make single sequence searches confusing.
Finding domains in a sequence
Cryptic domains:at the border of sequence
detectability
Gallego et al, Mol Sys Biol 2010
Identified using more sensitive fold recognition methods that use structure to help find weak members of sequence families.
If Pfam or SMART or similar do not find a domain, and the region is probably not disordered, then fold recognition might help.
Domain peptide interactions
Recognition of ligands or targeting signals
Post-translational modifications
3BP1_MOUSE/528-537 APTMPPPLPPPTN8_MOUSE/612-629 IPPPLPERTPSOS1_HUMAN/1149-1157 VPPPVPPRRRNCF1_HUMAN/359-390 SKPQPAVPPRPSAPEXE_YEAST/85-94 MPPTLPHRDWSH3-interacting motif PxxP
“perpetrator”
“instance”
“motif”
“victim”
Peptides interacting with a common domain often show a common pattern or motif usually 3-8 aas.
Linear motifs
Puntervol et al, NAR, 2003; www.elm.org (Eukaryotic Linear Motif DB)
Domains: large globular segments of the proteome that fold into discrete structures and belong in sequence families.
Linear motifs: small, non-globular segments that do not adopt a regular structure, and aren’t homologous to each other in the way domains are.
Motifs lie in the disordered part of the proteome.
Linear motifs versus domains
Intrinsically unstructured or disordered proteins or protein
fragments
Disorder predictors(IUPred, RONN, DisORPred,
etc)
Neduva & Russell, Curr. Opin. Biotech, 2006
Linear motif mediated interactions
are everywhere
Include motifs for:• Targeting – e.g. KDEL• Modifications – e.g.
phosphorylation• Signaling – e.g. SH3
About 200 are currentlyknown, likely many morestill to be discovered
Finding linear motifs in a sequence
Linear motifs are much harder to find than domains.
Long (>30 AA), belong to sequence families that help detect new
family members
Short (typically < 8AA), simple patterns, e.g. PxxP will occur in
most sequences randomly.
www.russelllab.org/wiki