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Transcript of Protein analysis and proteomics (Part 1 of 2). Many of the images in this powerpoint presentation...
![Page 1: Protein analysis and proteomics (Part 1 of 2). Many of the images in this powerpoint presentation are from Bioinformatics and Functional Genomics by Jonathan.](https://reader033.fdocuments.us/reader033/viewer/2022052701/56649ddf5503460f94ad806e/html5/thumbnails/1.jpg)
Protein analysis and proteomics(Part 1 of 2)
![Page 2: Protein analysis and proteomics (Part 1 of 2). Many of the images in this powerpoint presentation are from Bioinformatics and Functional Genomics by Jonathan.](https://reader033.fdocuments.us/reader033/viewer/2022052701/56649ddf5503460f94ad806e/html5/thumbnails/2.jpg)
Many of the images in this powerpoint presentationare from Bioinformatics and Functional Genomicsby Jonathan Pevsner (ISBN 0-471-21004-8). Copyright © 2003 by John Wiley & Sons, Inc.
These images and materials may not be usedwithout permission from the publisher. We welcomeinstructors to use these powerpoints for educationalpurposes, but please acknowledge the source.
The book has a homepage at http://www.bioinfbook.orgIncluding hyperlinks to the book chapters.
Copyright notice
![Page 3: Protein analysis and proteomics (Part 1 of 2). Many of the images in this powerpoint presentation are from Bioinformatics and Functional Genomics by Jonathan.](https://reader033.fdocuments.us/reader033/viewer/2022052701/56649ddf5503460f94ad806e/html5/thumbnails/3.jpg)
Outline for today
Protein analysis and proteomics
Individual proteinsProtein familiesPhysical propertiesLocalization Function
Large-scale protein analysis2D protein gelsYeast two-hybridRosetta Stone approachPathways
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protein
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RNADNA
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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
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[3] Protein localization
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protein
[1] Protein families
[4] Protein function
[2] Physical properties
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[3] Protein localization
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protein
[1] Protein families
[4] Protein function
[2] Physical properties
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[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
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15 most common domains (various species)
The European Bioinformatics Institute (EBI)offers many key proteomics resources:
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
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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?
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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
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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 LocusLink 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 a database including2,900 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)
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This lecture continues in part 2 with a discussion of two dimensional gels and the yeast two-hybrid system