Biology 224 Dr. Tom Peavy Sept 28 & 30

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Biology 224Dr. Tom Peavy

Sept 28 & 30

Protein Structure &Analysis

protein

Protein families

Protein function

Physical properties

Protein localization

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

The Human Proteome Organisation (HUPO) Proteomics Standards Initiative (PSI)

Work groups

• Gel Electrophoresis• Mass Spectrometry• Molecular Interactions• Protein Modifications• Proteomics Informatics• Sample Processing

Themes

• Controlled vocabularies• MIAPE: Minimum information about a proteomics experiment

The Human Proteome Organisation (HUPO) Proteomics Standards Initiative (PSI)

http://www.psidev.info/

Protein domains, motifs& signatures

Definitions

Signature: • a protein category such as a domain or motif(a defining property of the protein or family)

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

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.

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

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)

MBD 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.

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

Are proteins that share only a domain homologous?

Proteins can have both domains and patterns (motifs)

Domain(aspartylprotease)

Domain(reversetranscriptase)

Pattern(severalresidues)

The SwissProt entry for any protein provideshighly useful information…

SwissProt entry for HIV-1 pol links to many databases

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. Profiles are found in Pfam, ProDom, SMART, and other databases.

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

ExPASy Proteomics ServerThe ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB) is dedicated to the analysis of protein sequences and structures as well as 2-D PAGE (Disclaimer / References).

http://ca.expasy.org/

InterPro is a database of protein families, domains and functional sites in which identifiable features found in known proteins can be applied to unknown protein sequences.

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

InterPro

PROSITEDatabase of protein families and domainshttp://ca.expasy.org/prosite/

Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains. http://www.sanger.ac.uk/Software/Pfam/index.shtml

PRINTS is a compendium of protein fingerprints http://umber.sbs.man.ac.uk/dbbrowser/PRINTS/

The ProDom protein domain database consists of an automatic compilation of homologous domains.http://prodes.toulouse.inra.fr/prodom/current/html/home.php

ProDom entry for HIV-1 pol shows many related proteins

SMART (a Simple Modular Architecture Research Tool) allows the identification and annotation of genetically mobile domains and the analysis of domain architectures.

http://smart.embl-heidelberg.de/

Houses the PIRSF, ProClassand ProLINK databaseshttp://pir.georgetown.edu/

www.uniprot.org

Three protein databases recently merged to form UniProt:

• SwissProt

• TrEMBL (translated European Molecular Biology Lab)

• Protein Information Resource (PIR)

You can search for information on your favorite protein there; a BLAST server is provided.

1. Go to ExPASy (http://www.expasy.ch/)2. If you know the SwissProt accession of your protein, enter it at top.3. Otherwise go into Swiss-Prot/TrEMBL, click SRS (Sequence Retrieval System), click Start, then click continue, then search for your protein of interest.

Protein family classification and databases

PIRSF TIGRFAMs

SUPERFAMILY Gene3D

PANTHER

http://pir.georgetown.edu/iproclass/http://www.tigr.org/TIGRFAMs/index.shtml

http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/ http://www.biochem.ucl.ac.uk/bsm/cath/Gene3D/

http://www.pantherdb.org/

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.

http://www.expasy.ch/

Access a variety of protein analysis programsfrom the top right of the ExPASy home page

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

Two-dimensional protein gels

First dimension: isoelectric focusing

Second dimension: SDS-PAGE

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)

Second dimension: SDS-PAGE

Electrophorese proteins through an acrylamidematrix

Proteins are charged and migrate through an electric field

Conditions are denaturing (SDS) and reducing (2-mercaptoethanol)

Can resolve hundreds to thousands of proteins

Proteins identified on 2D gels (IEF/SDS-PAGE)

Direct protein microsequencing byEdman degradations

-- done at many core facilities (e.g. UC Davis)-- 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

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

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 component

GO terms are assigned to Entrez Gene entries

Gene product cytochrome c GO entry terms:

molecular function = electron transporter activity,

the biological process = oxidative phosphorylation and induction of cell death

the cellular component = mitochondrial matrix and mitochondrial inner membrane.

Example

GO consortium (http://www.geneontology.org)No centralized GO database. Instead, curatorsof organism-specific databases assign GO termsto gene products for each organism.

AmiGO is the searchable portion of the GO--Gene Symbol, name, UniProt access numbers, and Text searches can be used to find GO 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

Biology 224 Dr. Tom Peavy Sept 28 & 30

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