1. Biology 224 Dr. Tom Peavy Sept 28 & 30
Protein Structure &
Analysis
Biology 224
Dr. Tom Peavy
Sept 28 & 30
<Images from Bioinformatics and Functional
Genomics by Jonathan Pevsner>

2. Protein families
Protein localization
protein
Protein function
Gene ontology (GO):
--cellular component
--biological process
--molecular function
Physical properties

3. 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

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

5. Protein domains, motifs
& signatures

6. 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

7. Definition of a domain
According to InterPro at EBI (
A domain is an independent structural unit, found alone
or in conjunction with other domains or repeats.
Domains are evolutionarily related.
According to SMART (
A domain is a conserved structural entity with distinctive
secondary structure content and a hydrophobic core.
Homologous domains with common functions usually
show sequence similarities.

8. 15 most common domains (human)
Zn finger, C2H2 type proteins
Immunoglobulin
EGF-like
Zn-finger, RING 458
Homeobox
Pleckstrin-like 405
RNA-binding region RNP-1 400 SH
Calcium-binding EF-hand 392
Fibronectin, type III 300
PDZ/DHR/GLGF 280
Small GTP-binding protein 261
BTB/POZ
bHLH
Cadherin

9. Varieties of protein domains
Extending along the length of a protein
Occupying a subset of a protein sequence
Occurring one or more times

10. 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 Rett
Syndrome, a neurological disorder affecting girls
primarily.

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

12. Are proteins that share only a domain homologous?

13. Proteins can have both domains and patterns (motifs)
(several
residues)
Pattern
(several
residues)
Domain
(aspartyl
protease)
Domain
(reverse
transcriptase)

14. The SwissProt entry for
any protein provides
highly useful information…

15. SwissProt entry for HIV-1 pol links to many databases

16. 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 and
phosphorylation sites. These do not imply homology
when found in a group of proteins.
PROSITE ( is a dictionary of
motifs (there are currently 1600 entries). In PROSITE,
a pattern is a qualitative motif description (a protein
either matches a pattern, or not). In contrast, a profile
is a quantitative motif description. Profiles are found
in Pfam, ProDom, SMART, and other databases.
Page

18. http://www.ebi.ac.uk/Databases/ http://www.ebi.ac.uk/interpro/
InterPro
      
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.
                              
ExPASy Proteomics Server
The 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).

19. PROSITE Database of protein families and domains
                                                        
PROSITE Database of protein families and domains
         
Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains.
         
PRINTS is a compendium of protein fingerprints
       
The ProDom protein domain database consists of an automatic compilation of homologous domains.

21. ProDom entry for HIV-1 pol shows many related proteins
Page 231

22. Houses the PIRSF, ProClass and ProLINK databases
         
SMART (a Simple Modular Architecture Research Tool) allows the identification and annotation of genetically mobile domains and the analysis of domain architectures.
                                     
Houses the PIRSF, ProClass
and ProLINK databases

23.
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.

24. 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.
Page 230

25. Protein family classification and databases PIRSF
         
PIRSF
TIGRFAMs
Gene3D
SUPERFAMILY
PANTHER

27. Physical properties of proteins
Many websites are available for the analysis of
individual proteins. ExPASy and ISREC are two
excellent 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 as
posttranslational modification of proteins by
specific sugars), experimental evidence may be
required rather than prediction algorithms.
Page 236

28.
Page 230

29. Access a variety of protein analysis programs
from the top right of the ExPASy home page
Page 235

30. Page 244

31. Page 244

32. 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 cell
and/or all the proteins in an organism
Large-scale protein analysis
2D protein gels
Yeast two-hybrid
Rosetta Stone approach
Pathways
Page 247

33. Two-dimensional protein gels
First dimension: isoelectric focusing
Second dimension: SDS-PAGE
Page 248

35. Two-dimensional protein gels
First dimension: isoelectric focusing
Electrophorese ampholytes to establish
a 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)
Page 248

36. Two-dimensional protein gels
Second dimension: SDS-PAGE
Electrophorese proteins through an acrylamide
matrix
Proteins are charged and migrate through an
electric field
Conditions are denaturing (SDS)
and reducing (2-mercaptoethanol)
Can resolve hundreds to thousands of proteins
Page 248

37. Proteins identified on 2D gels (IEF/SDS-PAGE)
Direct protein microsequencing by
Edman 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
Page 250-1

38. Page 252

39. Evaluation of 2D gels (IEF/SDS-PAGE)
Advantages:
Visualize hundreds to thousands of proteins
Improved identification of protein spots
Disadvantages:
Limited number of samples can be processed
Mostly abundant proteins visualized
Technically difficult
Page 251

40. Gene Ontology (GO) Consortium

41. The Gene Ontology Consortium
An ontology is a description of concepts. The GO
Consortium compiles a dynamic, controlled vocabulary
of terms related to gene products.
There are three organizing principles:
Molecular function
Biological process
Cellular component

42. GO terms are assigned to Entrez Gene entries
Page 241

43. Page 241

44. Example
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.

45. GO consortium (http://www.geneontology.org)
No centralized GO database. Instead, curators
of organism-specific databases assign GO terms
to 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

47. The Gene Ontology Consortium: Evidence Codes
IC Inferred by curator
IDA Inferred from direct assay
IEA Inferred from electronic annotation
IEP Inferred from expression pattern
IGI Inferred from genetic interaction
IMP Inferred from mutant phenotype
IPI Inferred from physical interaction
ISS Inferred from sequence or structural similarity
NAS Non-traceable author statement
ND No biological data
TAS Traceable author statement