2. Electrophoretic separation of proteins by charge (isoelectric

focusing) and by size (SDS-PAGE)

2D-gel electrophoresis & mass spectrometry

3. Peptide fragmentation of individual protein (with proteases eg trypsin)

1. Isolate proteins from tissue (organism, condition…) of interest

Fig. 6.10 see Fig. 6.11

PROTEOMICS: LARGE-SCALE PROTEIN IDENTIFICATION & ANALYSIS

4. Determine precise peptide mass by MALDI-TOF (matrix-assisted laser desorption ionization – time of flight) mass spectrometry

5. Compare aa sequences to genomic data to correlateprotein with its gene

Fig. 6.12

STRUCTURAL PROTEOMICS

- large-scale determination of protein structures

Start with gene of interest – cloning, expression, purification of protein

- crystallize protein & X-ray diffraction analysis

NMR spectroscopy - for small proteins or domains (in solution)

Protein data bank: www.rcsb.org/pdb/

X-ray crystallography

Nov 2000 = 13,750 structures

Nov 2001 = 16,550

Nov 2006 = 40,132

Nov 2008 = 54,559

Nov 2009 = 61,418

Nov 2011 = 76,814

How similar are hemoglobin chain and -chain?

hemoglobin tetramer

Analysis of protein sequences and structures

Identification of - protein motifs, catalytic centres…

- binding to ligands, drugs

- interaction with other macromolecules

- relatedness to other proteins (homology modelling)

clues from protein sequence/structure about biological function

ExPASy (Swiss Institute of Bioinformatics) www.expasy.org/

EMBL-EBI (European Bioinformatics Institute) www.ebi.ac.uk/Tools/ppsearch/

For example:

www.ncbi.nlm.nih.gov/structure

How to find proteins that interact with protein of interest?

1. Phage display

- generate phage library producing collection of fusion proteins between phage coat protein & “ test protein” from genome of interest

- hybrid protein will be “displayed” on outer surface of phage

Fig. 6.14- then screen library to find ones having expressed protein which interacts with “test” protein of interest

mRNA

gene

RNA pol II

Transcription factors have 2 domains DNA

binding domain

Activator domain

Regulatory cis-element

2. Yeast 2-hybrid system

If TF domains physically separated, no transcription

Background info about transcription in eukaryotes

But if “bait” & “prey” interact to bring TF domains close together, then transcription occurs

mRNA

Determining protein-protein interactions using yeast 2-hybrid system

- use separate vectors to prepare [1] “bait” fused to DNA binding domain of a yeast transcription factor

- fuse (1) to gene for protein X = “bait”

Fig. 6.15

[2] shotgun library of possible “prey” fused to activation domain of yeast TF

“prey” – generate library where (2) is fused to random coding sequences from organism of interest (eg. human)

- co-transform yeast cells (which lack this transcription factor TF)

if protein X and “prey” (from library) interact, the 2 domains of yeast TF will be close together (& functional), so activate reporter gene

Fig. 6.15

eg if use lacZ reporter gene – blue colour of yeast colony

B

B

If bait protein does not interact directly with protein(s) in a complex, they may not be isolated

Fig.6.18

Fig.6.17

3. Affinity column chromatography

or use co-immunoprecipitation

- protein B (“bait”) attached on column to “fish out” the protein (or proteins) which specifically bind to it

(p.182)

If 2 genes are functionally-related, expect them to be co-inherited…

… and may be physically close in genome (as well as co-inherited)

Gene fusion/fission (2 short genes in some organisms vs. one long gene in others)

10 different organisms

Harrington FEBS Lett. 582:1251, 2008

4. Computational approaches to predict protein-protein interactions

Do genes 1 and 2 (orange & green) pass this test?

- premise that composite (naturally-fused) proteins have direct physical interaction (or functional association)

3. Bioinformatics approach to predict protein-protein interactions

- search for one large gene in organism X vs. two separate smaller genes in Y

Fig.6.19

in yeast

in E.colihis2 his10

“ HIS2 ”

Search of complete genomes of E.coli, Haemophilus, Methanococcus & yeast

- found 215 cases of “fused vs. split” state

Enright et al. Nature 402: 86, 1999

Fig. 6.20

Yeast protein-protein interaction map (from experimental data)

red dots = essential proteins (so knockout is lethal)

green = non-lethal; orange = slow growth; yellow = unknown effect

- lines connecting dots represent known protein- protein interactions

2001

2002 colour-coded forbiological function

- triplet repeat (CAG) expansion disease (p.510)

“Protein interaction network in Huntington’s disease”

Figure 2. Protein Interaction Network for Huntington’s Disease Comprehensive PPI network for htt [huntingtin protein] Y2H interactors [35 bait and 51 prey proteins & verified in pull down assays], red diamonds; previously published interactors, blue squares; interactors identified from databases HRPD, MINT, and BIND, bridging any two proteins in the extended network, green triangles Htt interactors previously reported and recapitulated in our screens.

Goehler et al. Mol Cell 15:853, 2004

Marcotte Nature 402:83, 1999

www.yeastgenome.org

RNA-seq microarray profiling

They double-checked some by RT-PCR

Papers related to questions on 2d mid-term test

Hawrylycz et al. Nature Sept. 2012

“… RNA sequencing methods, which were cost-prohibitive and technologically immature when the project was initiated, hold great promise for elucidating …[brain] transcriptional regulation in the future.”

“… a small number of high-quality, clinically unremarkable brains profiled with DNA microarrays for quantitative gene-level transcriptome coverage”

Dopamine pathway genes

Different parts of brain

Actin genes

Transcriptional profiles of multi-gene family members (in different parts of human brain)

Hawrylycz et al. Nature Sept. 2012

different parts of brain

dif

fere

nt

gen

es

Saha Haemophilia 17: e928, 2011

Factor VIII (F8) blood clotting gene

Example of highly polymorphic human gene…

Number of tandem repeats (GTGTGT…) in intron 1 varies among populations in India

3 of its introns have microsatellites which differ in copy number among individuals

Allele frequency for microsatellite in intron 1

TB: Tibeto-Burman

AA: Austro-Asiatic

“In this study, we sequenced 99% of all three [such] unfinished gaps on human chr 20..”

“The finished human genome-assemblies comprise several hundred un-sequenced euchromatic gaps, which may be rich in long polypurine/polypyrimidine stretches.

Douglas et al. G3:Genes Genomes Genet 2:1279-89, Oct. 2012

Barcode tags used in gene overexpression experiments

- similar in design to gene deletion experiments discussed in Topic 7

- to track whether having too much protein X is lethal under certain growth conditions…