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Protein analysis and proteomics
September 27, 2006
Introduction to BioinformaticsJ. Pevsner
Many of the images in this powerpoint presentationare from Bioinformatics and Functional Genomicsby J Pevsner (ISBN 0-471-21004-8). Copyright © 2003 by Wiley.
These images and materials may not be usedwithout permission from the publisher.
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Outline for the course
Today (Sept. 27): protein analysis and proteomics, part IFriday, Sept. 29: lab 4 (proteomics)Monday, October 2: no classWednesday October 4: protein structure, part I
October 9: protein structure, part II (Ingo Ruczinski)October 11: multiple sequence alignment (Sarah Wheelan)October 16, 18: phylogeny
Wednesday, October 25: final exam; find-a-gene due
Outline for today
Protein analysis and proteomics
Individual proteinsProtein familiesPhysical propertiesLocalization Function
Large-scale protein analysis2D protein gelsYeast two-hybridRosetta Stone approachPathways
protein
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RNADNA
protein
[1] Protein families
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protein
[1] Protein families
[2] Physical properties
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protein
[1] Protein families
[2] Physical properties
[3] Protein localization
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protein
[1] Protein families
[4] Protein function
[2] Physical properties
[3] Protein localization
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protein
[1] Protein families
[4] Protein function
[2] Physical properties
[3] Protein localization
Gene ontology (GO):--cellular component--biological process--molecular function
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Perspective 1: Protein domains and motifs
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Definitions
Signature:• a protein category such as a domain or motif
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Definitions
Signature:• a protein category such as a domain or motif
Domain:• a region of a protein that can adopt a 3D structure• a fold• a family is a group of proteins that share a domain• examples: zinc finger domain
immunoglobulin domain
Motif (or fingerprint):• a short, conserved region of a protein• typically 10 to 20 contiguous amino acid residues
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15 most common domains (human)
Zn finger, C2H2 type 1093 proteinsImmunoglobulin 1032EGF-like 471Zn-finger, RING 458Homeobox 417Pleckstrin-like 405RNA-binding region RNP-1 400SH3 394Calcium-binding EF-hand 392Fibronectin, type III 300PDZ/DHR/GLGF 280Small GTP-binding protein 261BTB/POZ 236bHLH 226Cadherin 226 Page 227
15 most common domains (various species)
The European Bioinformatics Institute (EBI) offers many key proteomics resources at the Integr8 site:
http://www.ebi.ac.uk/proteome/
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Definition of a domain
According to InterPro at EBI (http://www.ebi.ac.uk/interpro/):
A domain is an independent structural unit, found aloneor in conjunction with other domains or repeats.Domains are evolutionarily related.
According to SMART (http://smart.embl-heidelberg.de):
A domain is a conserved structural entity with distinctivesecondary structure content and a hydrophobic core.Homologous domains with common functions usuallyshow sequence similarities.
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Varieties of protein domains
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Extending along the length of a protein
Occupying a subset of a protein sequence
Occurring one or more times
Example of a protein with domains: Methyl CpG binding protein 2 (MeCP2)
MBD
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TRD
The protein includes a methylated DNA binding domain(MBD) and a transcriptional repression domain (TRD).MeCP2 is a transcriptional repressor.
Mutations in the gene encoding MeCP2 cause RettSyndrome, a neurological disorder affecting girlsprimarily.
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Result of an MeCP2 blastp search:A methyl-binding domain shared by several proteins
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Are proteins that share only a domain homologous?
Example of a multidomain protein: HIV-1 pol
• 1003 amino acids long
• cleaved into three proteins with distinct activities:-- aspartyl protease-- reverse transcriptase-- integrase
We will explore HIV-1 pol and other proteins at theExpert Protein Analysis System (ExPASy) server.
Visit www.expasy.org/
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SwissProt entry for HIV-1 pol links to many databases
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ProDom entry for HIV-1 pol shows many related proteins
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Proteins can have both domains and patterns (motifs)
Domain(aspartylprotease)
Domain(reversetranscriptase)
Pattern(severalresidues)
Pattern(severalresidues)
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Definition of a motif
A motif (or fingerprint) is a short, conserved region of a protein. Its size is often 10 to 20 amino acids.
Simple motifs include transmembrane domains andphosphorylation sites. These do not imply homologywhen found in a group of proteins.
PROSITE (www.expasy.org/prosite) is a dictionary of motifs (there are currently 1600 entries). In PROSITE,a pattern is a qualitative motif description (a proteineither matches a pattern, or not). In contrast, a profileis a quantitative motif description. We will encounterprofiles in Pfam, ProDom, SMART, and other databases.
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Perspective 2: Physical properties of proteins
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Physical properties of proteins
Many websites are available for the analysis ofindividual proteins. ExPASy and ISREC are twoexcellent resources.
The accuracy of these programs is variable. Predictions based on primary amino acid sequence (such as molecular weight prediction) are likely to be more trustworthy. For many other properties (such asposttranslational modification of proteins by specific sugars), experimental evidence may be required rather than prediction algorithms.
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Syntaxin, SNAP-25 and VAMP are three proteins that interact via coiled-coil domains
Introduction to Perspectives 3 and 4: Gene Ontology (GO) Consortium
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The Gene Ontology Consortium
An ontology is a description of concepts. The GOConsortium compiles a dynamic, controlled vocabularyof terms related to gene products.
There are three organizing principles: Molecular functionBiological processCellular compartment
You can visit GO at http://www.geneontology.org.There is no centralized GO database. Instead, curatorsof organism-specific databases assign GO termsto gene products for each organism.
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GO terms are assigned to Entrez Gene entries
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The Gene Ontology Consortium: Evidence Codes
IC Inferred by curatorIDA Inferred from direct assayIEA Inferred from electronic annotationIEP Inferred from expression patternIGI Inferred from genetic interactionIMP Inferred from mutant phenotypeIPI Inferred from physical interactionISS Inferred from sequence or structural similarityNAS Non-traceable author statementND No biological dataTAS Traceable author statement
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Perspective 3: Protein localization
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protein
Protein localization
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Protein localization
Proteins may be localized to intracellular compartments,cytosol, the plasma membrane, or they may be secreted. Many proteins shuttle between multiple compartments.
A variety of algorithms predict localization, but thisis essentially a cell biological question.
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Localization of 2,900 yeast proteins
Michael Snyder and colleagues incorporated epitopetags into thousands of S. cerevisiae cDNAs,and systematically localized proteins (Kumar et al., 2002).
See http://ygac.med.yale.edu for the TRIPLES database including 2,900 fluorescence micrographs.
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Perspective 4: Protein function
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Protein function
Function refers to the role of a protein in the cell.We can consider protein function from a varietyof perspectives.
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1. Biochemical function(molecular function)
RBP binds retinol,could be a carrier
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2. Functional assignmentbased on homology
RBPcould bea carrier
too
Othercarrier proteins
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3. Functionbased on structure
RBP forms a calyx
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4. Function based onligand binding specificity
RBP binds vitamin A
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5. Function based oncellular process
DNA RNA
RBP is abundant,soluble, secreted
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6. Function basedon biological process
RBP is essential for vision
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7. Function based on “proteomics”or high throughput “functional genomics”
High throughput analyses show...
RBP levels elevated in renal failureRBP levels decreased in liver disease
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Functional assignment of enzymes:the EC (Enzyme Commission) system
Oxidoreductases 1,003Transferases 1,076Hydrolases 1,125Lyases 356Isomerases 156Ligases 126
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Functional assignment of proteins:Clusters of Orthologous Groups (COGs)
Information storage and processing
Cellular processes
Metabolism
Poorly characterized
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Functional assignment of proteins:Clusters of Orthologous Groups (COGs)
Information storage and processing
Cellular processes
Metabolism
Poorly characterized
(Most useful for prokaryotes; we will describe COGs in the Genomics course)
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Proteomics: High throughput protein analysis
Proteomics is the study of the entire collection of proteins encoded by a genome
“Proteomics” refers to all the proteins in a celland/or all the proteins in an organism
Large-scale protein analysis2D protein gelsYeast two-hybridRosetta Stone approachPathways
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Classical biochemical approach
Identify an activityDevelop a bioassayPerform a biochemical purification
Strategies: size, charge, hydrophobicityPurify protein to homogeneityClone cDNA, express recombinant protein
Grow crystals, solve structure (next Wednesday)Page 247
Two-dimensional protein gels
First dimension: isoelectric focusing
Second dimension: SDS-PAGE
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Two-dimensional protein gels
First dimension: isoelectric focusing
Electrophorese ampholytes to establisha pH gradient
Can use a pre-made strip
Proteins migrate to their isoelectric point(pI) then stop (net charge is zero)
Range of pI typically 4-9 (5-8 most common)
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Two-dimensional protein gels
Second dimension: SDS-PAGE
Electrophorese proteins through an acrylamidematrix
Proteins are charged and migrate through an electric field v = Eq / d6πrη
Conditions are denaturing
Can resolve hundreds to thousands of proteins
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Proteins identified on 2D gels (IEF/SDS-PAGE)
Direct protein microsequencing byEdman degradations
-- done at Hopkins, other cores-- typically need 5 picomoles-- often get 10 to 20 amino acids sequenced
Protein mass analysis by MALDI-TOF
-- done at core facilities-- often detect posttranslational
modifications-- matrix assisted laser desorption/ionization
time-of-flight spectroscopy
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Evaluation of 2D gels (IEF/SDS-PAGE)
Advantages:Visualize hundreds to thousands of proteinsImproved identification of protein spots
Disadvantages:Limited number of samples can be processedMostly abundant proteins visualizedTechnically difficult
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Affinity chromatography/mass spec
Bait proteinGST
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Affinity chromatography/mass spec
Bait proteinGST
Add yeast extractProtein complexes bindMost proteins do not bind
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Affinity chromatography/mass spec
Bait proteinGST
EluteRun gelMALDI-TOFIdentify complexes
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Affinity chromatography/mass spec
Data on complexes deposited in databases
http://yeast.cellzome.comhttp://www.bind.ca
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Evaluation of affinity chromatography/mass spec
Advantages:Thousands of protein complexes identifiedFunctions can be assigned to proteins
Disadvantages:False negative resultsFalse positive results
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Affinity chromatography/mass spec
False negatives:• Bait must be properly localized and
in its native condition• Affinity tag may interfere with function• Transient protein interactions may be missed• Highly specific physiological conditionsmay be required
• Bias against hydrophobic, and small proteins
Bait proteinGST
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Affinity chromatography/mass spec
False positives:• sticky proteins
Bait proteinGST
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The yeast two-hybrid system
Reporter gene
Bait proteinDNA Binding
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The yeast two-hybrid system
Reporter gene
Prey proteinDNA activation
Prey proteinDNA activation
Prey proteinDNA activation Prey protein
DNA activation
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The yeast two-hybrid system
Reporter gene
Bait proteinDNA Binding
Prey proteinDNA activation
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The yeast two-hybrid system
Reporter gene
Bait proteinDNA Binding
Prey proteinDNA activation
Isolate and sequence the cDNAof the binding partner you have found
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red = cellular role & subcellular localization of interacting proteins are identical; blue = localiations are identical; green = cellular roles are identical
Evaluation of the yeast two-hybrid system
Advantages:Thousands of protein complexes identifiedFunctions can be assigned to proteins
Disadvantages:Detects only pairwise protein interactionsFalse-negative results (as for affinity chromatography)
-- bait may be mislocalized-- transient interactions may be missed-- some complexes require special conditions-- bias against hydrophobic proteins
False-positive results-- some proteins may be sticky-- bait protein may auto-activate a reporter
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The Rosetta Stone approach
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Marcotte et al. (1999) and other groups hypothesized that some pairs of interacting proteins are encoded by two genes in many genomes, but occasionally theyare fused into a single gene.
By scanning many genomes for examples of “fusedgenes,” several thousand protein-protein predictionshave been made.
Yeast topoisomerase II
E. coligyrase B
E. coligyrase A
The Rosetta Stone approach
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6,217 yeast proteins
Experimental data (500 links)Related metabolic function (2,000 links)Related phylogenetic profiles (20,000 links)Rosetta Stone method (45,000 links)Correlated mRNA expression (26,000 links)
Marcotte et al. (1999) Nature 402:83
Pathway maps
A pathway is a linked set of biochemical reactions
ExPASyProNetEcoCyc: E. coli pathwaysMetaCyc: 450 pathways, 158 organismsKEGG: Kyoto Encyclopedia of Genes & Genomes
Issues:Is the extrapolation between species valid?Have orthologs been identified accurately?False positive, false negative findings
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