Proteomics

Presenting: Yaniv Loewenstein

Say What?

• Proteome - the entire complement of proteins produced in a cell or organism.

• Proteomics – an emerging scientific discipline encompassing any method for the large scale analysis of proteins.

Why proteomics?• Proteome analysis is conceptually attractive

because of its potential to determine properties of biological systems that are not apparent by DNA or mRNA sequence analysis alone.

quantity of protein expression. the subcellular location. the state of modification. the association with ligands. the rate of change with time of such properties.

Genome vs. Proteome Complexity

1 Gene various proteins

The proteome is much more complex than the genome.

Alternative Splicing. RNA editing. Alternative use of different promoters and

terminators. Post Translational Modifications.

Several genomes were solved but no eukaryotic proteomes yet.

Current efforts:

+ PPAP - the plant proteome annotation project.

(Arabidopsis thaliana)

+ D.melanogaster , M. musculus, S.cerevisiae and more.

Deinococcus radiodurans - proteome was complete recently.

Genome vs. Proteome Complexity – (continued)

Major Tools

• 2D Gels.• Pulse-Chase experiments.• Chromatography.• Mass Spectrometry.• Immunoprecipitation.• Bioinformatics and databases

YPD, CATH, SCOPE, SWISSPROT, BLAST…

2d gel

Pulse ChaseQ:What is meant by a pulse-chase

experiment? A:Pulse: A brief exposure to some labeling

condition (i.e. radioactive labeling).Chase: Removal of the label and

observation of the labeled material. => follow the fate of the labeled material. 

Today’s Topics

• Identification of in vivo substrates of the chaperonin GroEL

Walid A. Houry , Dmitirij Frishman, Christoph Eckerskorn, Friedrich Lottspeich & F. Ulrich Hartl.

• A sampling of the yeast proteome.Futcher B, Latter GI, Monardo P, McLaughlin CS, Garrels JI.

• Correlation between protein and mRNA abundance in yeast.

Gygi SP, Rochon Y, Franza BR, Aebersold R

Protein Folding

3D fold of a protein is determined by the amino-acid sequence.

• Some proteins can reach their folded states spontaneously.

• The efficiency of folding is often limited by the side reaction of aggregation.

• Misfolding and aggregation of proteins is prevented by molecular chaperones.

Molecular chaperones - what?Definition: Proteins that interact with partially folded

or improperly folded polypepetides, facilitating correct folding pathways or providing microenvironments in which folding can occur.

Hsp70

*Heat shock protection.

*escort translocation of unfolded proteins.

*Mr ~ 70 kDa

Chaperonins

* Elaborate protein complexes.

* Essential for correct folding of some newly synthesized proteins.

GroEL - I• Essential. In E. Coli.

• some proteins may require several rounds.

•Hydrophobic interactions.

• Structure:

homo-oligomer.

2 * heptamer rings.

• cofactor GroES + ATP.

GroEl cycle Mechanism.

Research Goals

• Define a Group of GroEL Subtrates.

• Search for common motifs in the GroEL substrate proteins.

Experiment synopsis• Pulse-chase labeling.• At different times of chase, cells were lysed in the

presence of EDTA.• GroEL–substrate complexes were isolated by

immunoprecipitation with anti-GroEL antibodies.• ATP + GroES added in vitro.• Total soluble cytoplasmic proteins and GroEL-

bound proteins were separated on 2D gels.• Control experiments.

Substrates• 250–300 proteins out of a total of 2,500 were

observed in complex with GroEL immediately upon labeling.

• About half of these proteins were still detectable on GroEL after 10 min of chase , though in much lower amounts relative to GroEL.

• Proteins that interact only very transiently with GroEL may have escaped detection in this analysis.

A defined set of GroEL substrates – Gel

GroEL substrates - pI and Mr

The pI distributions of total soluble cytoplasmic proteins and of GroEL substrates were found to be very similar.

E(total Mr) = 37kD

E(GroEL sub Mr) = 45kD

BUT ~20kD<Mr<~60kD

GroEL substrates - Kineticsa. 160 proteins (Mr < 60k)

released completely with time constants between 20s and 2 min.

b. 100 proteins (Mr < 60k), a fraction of the substrates remained associated.

c. Several larger proteins (Mr > 60k) were inefficiently released from the chaperonin.

A defined set of GroEL substrates – Gel

Additional experiments

• Proteins return to GroEL for additional cycles.

• Heat stress conditions.

GroEL substrates – Identification.• Analyze spots - MS.• 52 of the most abundant,

were unequivocally IDed.

* RNA poly subunits

* E.F Tu

* tRNA synthetases

in accord with previous studies.

GroEl Substrates – Common Motif.

• Run PSI-BLAST for consensus sequence or clusters of thereof

=> 0, null, gurnicht mit gurnicht!

• domains were classified independently through sequence homology to domains in SCOP & CATH.

=> 18-24 proteins.

and the plot thickens ….

A preferred structural Motif

• GroEL substrates preferentially contain several αβ domains compared with E. coli proteins.

• Of the multidomain GroEL substrates, 13 of 17 have at least two αβ domains.

• No significant preference for other 3D structures was found.

• Several model substrates of GroEL, typically used for in vitro studies, belong to this category of αβ proteins.

Graph

Substrate interaction with GroEL

• Preferred domain topology, provides insight into why and how these proteins interact with the chaperonin.

• Proteins with two or more αβ domains, may be particularly prone to aggregation.

• Hydrophobic interactions propose possible mechanism.

Possible Motivation

• Biotechnolgy

Improve the folding efficiency of foreign proteins expressed in E. coli.

• Better Understanding of protein Folding Mechanisms in eukaryotes.

Coffee