Protein analysis and proteomics (Part 1 of 2). Many of the images in this powerpoint presentation...

Protein analysis and proteomics(Part 1 of 2)

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Protein analysis and proteomics

Individual proteinsProtein familiesPhysical propertiesLocalization Function

Large-scale protein analysis2D protein gelsYeast two-hybridRosetta Stone approachPathways

protein

RNADNA

protein

[1] Protein families

protein

[2] Physical properties

protein

[3] Protein localization

protein

[4] Protein function

protein

[4] Protein function

Gene ontology (GO):--cellular component--biological process--molecular function

Perspective 1: Protein domains and motifs

Definitions

Signature: • a protein category such as a domain or motif

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

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:

http://www.ebi.ac.uk/proteome/

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.

Varieties of protein domains

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)

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.

Result of an MeCP2 blastp search:A methyl-binding domain shared by several proteins

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/

SwissProt entry for HIV-1 pol links to many databases

ProDom entry for HIV-1 pol shows many related proteins

Proteins can have both domains and patterns (motifs)

Domain(aspartylprotease)

Domain(reversetranscriptase)

Pattern(severalresidues)

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.

Perspective 2: Physical properties of proteins

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.

Syntaxin, SNAP-25 and VAMP are three proteins that interact via coiled-coil domains

Introduction to Perspectives 3 and 4: Gene Ontology (GO) Consortium

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.

GO terms are assigned to LocusLink entries

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

Perspective 3: Protein localization

protein

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.

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.

Perspective 4: Protein function

Protein function

Function refers to the role of a protein in the cell.We can consider protein function from a varietyof perspectives.

1. Biochemical function(molecular function)

RBP binds retinol,could be a carrier

2. Functional assignmentbased on homology

RBPcould bea carrier

Othercarrier

proteins

3. Functionbased on structure

RBP forms a calyx

4. Function based onligand binding specificity

RBP binds vitamin A

5. Function based oncellular process

DNA RNA

RBP is abundant,soluble, secreted

6. Function basedon biological process

RBP is essential for vision

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

Functional assignment of enzymes:the EC (Enzyme Commission) system

Oxidoreductases 1,003Transferases 1,076Hydrolases 1,125Lyases 356Isomerases 156Ligases 126

Functional assignment of proteins:Clusters of Orthologous Groups (COGs)

Information storage and processing

Cellular processes

Metabolism

Poorly characterized

Functional assignment of proteins:Clusters of Orthologous Groups (COGs)

Information storage and processing

Cellular processes

Metabolism

Poorly characterized

(Most useful for prokaryotes)

This lecture continues in part 2 with a discussion of two dimensional gels and the yeast two-hybrid system

Protein analysis and proteomics (Part 1 of 2). Many of the images in this powerpoint presentation...

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