1. Announcements: Note that there will be presentations and associated paper summaries for both Thursday and Tuesday classes. The Exam II mean is 81.6 and median is 88. Proposal resubmissions are due 4/23. It is recommended that students set up a meeting to discuss modifications for the final step of the assignment.

2. The Proteome Using high-throughput methods to identify proteins and to understand their function

3. What is proteomics? An organism’s proteome –A catalog of all proteins Expressed throughout life Expressed under all conditions The goals of proteomics –To catalog all proteins –To understand their functions –To understand how they interact with each other

4. The challenges of proteomics Splice variants create an enormous diversity of proteins –~25,000 genes in humans give rise to 200,000 to 2,000,000 different proteins –Splice variants may have very diverse functions Proteins expressed in an organism will vary according to age, health, tissue, and environmental stimuli Proteomics requires a broader range of technologies than genomics

5. Protein modifications –Phosphorylation: activation and inactivation of enzymes –Acetylation: protein stability, used in histones –Methylation: regulation of gene expression –Acylation: membrane tethering, targeting –Glycosylation: cell–cell recognition, signaling –GPI anchor: membrane tethering –Hydroxyproline: protein stability, ligand interactions –Sulfation: protein–protein and ligand interactions –Disulfide-bond formation: protein stability –Deamidation: protein–protein and ligand interactions –Pyroglutamic acid: protein stability –Ubiquitination: destruction signal –Nitration of tyrosine: inflammation

6. 2-D gel electrophoresis Polyacrylamide gel Voltage across both axes –pH gradient along first axis neutralizes charged proteins at different places –pH constant on a second axis where proteins are separated by weight x–y position of proteins on stained gel uniquely identifies the proteins BasicAcidic High MW Low MW

7. Differential in gel electrophoresis Label protein samples from control and experimental tissues –Fluorescent dye #1 for control –Fluorescent dye #2 for experimental sample Mix protein samples together Identify identical proteins from different samples by dye color with benzoic acid Cy3 without benzoic acid Cy5

8. Protein-protein interactions “The Interactome” Yeast two-hybrid analysis Other “protein complementation” methods Biochemical purification/Mass spectrometry

9. Yeast two-hybrid method Goal: Determine how proteins interact with each other Method –Use yeast transcription factors –Gene expression requires the following: A DNA-binding domain An activation domain A basic transcription apparatus –Attach protein 1 to DNA-binding domain (bait) –Attach protein 2 to activation domain (prey) –Reporter gene expressed only if protein 1 and protein 2 interact with each other

10. A schematic of the yeast two-hybrid method m n

11. Results from a yeast two-hybrid experiment Goal: To characterize protein–protein interactions among 6,144 yeast ORFs –5,345 were successfully cloned into yeast as both bait and prey –Identity of ORFs determined by DNA sequencing in hybrid yeast –692 protein–protein interaction pairs –Interactions involved 817 ORFs

12. Yeast two-hybrid results for flies & worms Worms: –Created >3000 bait constructs –Tested against two AD libraries –Mapped 4000 interactions Flies: Flies: Screened 10,000 predicted transcripts Screened 10,000 predicted transcripts Found 20,000 interactions Found 20,000 interactions Statistically assigned 4800 as “high quality” interactions Statistically assigned 4800 as “high quality” interactions

13. Caveats associated with the yeast two-hybrid method There is evidence that other methods may be more sensitive Some inaccuracy reported when compared against known protein–protein interactions –False positives –False negatives

14. Protein Complementation Enzymatic complementation –  -galactosidase reconstitution Fluorescence complementation –GFP or YFP reconstitution –FRET (fluorescence resonance energy transfer)

15. Enzymatic Complementation

16. Blue=DAPI Green=BGAL Red=Actin FKBP12-Δω and FRAP-Δα (Interaction mediated by Rapamycin)

17. Bimolecular Fluorescence Complementation (or Split GFP)

19. FRET (fluorescence resonance energy transfer)

22. Purification of interacting proteins Immunoprecipitation –Impractical on large scale (identification of unknowns) Affinity purification –Biochemically practical, but too dirty Tandem affinity purification –Sufficient yield & purity for identification of unknown proteins