1. Protein domains vs. structure domains - an example.

2. Genome of the week Nanoarchaeum equitans - archaea –Hyperthermophile –Diverged early in evolution from other archaea –New kingdom of archaea? Obligate symbiont with Ignicoccus Smallest completely sequenced genome –<500kB –Genome reduction observed in symbionts (Schmidt) –Is N. equitans a “primitive” archaea or is the genome undergoing reductive evolution?

3. N. equitans lacks genes necessary for many aspects of central metabolism. –Can’t make lipids, vitamins, amino acids, etc. –Parasite, not symbiont? First archaea Genome is quite compact. –95% of genome codes for genes. 552. Not primitive. –Has complete set of information pathway and cell cycle genes found in archaea. No longer undergoing reductive evolution. –Normally would find pseudogenes - not found.

4. Protein complexes - why? Proteins often function as large, multi- subunit complexes. –ribosomes Can get clues about the function of a protein by knowing what other proteins it contacts.

5. Protein:protein interactions Genetic approach –Yeast 2-hybrid Biochemical approach –Co-immunoprecipitation –Fusion protein affinity chromatography Cell-biology –FRET - fluorescence resonance energy transfer Computational –Rosetta Stone –Co-regulation –Phylogenetic analysis

6. Yeast 2-hybrid approach Based on the fact some transcriptional activators have separable DNA binding (BD) and transcriptional activation domains (AD). –GAL4, LexA Protein you are studying = Bait –Fused to the DNA binding domain of GAL4 Protein(s) you are screening = Fish or Prey –Fused to the activation domain of GAL4 Transform Bait and Fish plasmids into yeast, measure the expression of a reporter gene. –Usually a gene can be selected for when expressed.

7. Image from: http://www.bioteach.ubc.ca/MolecularBiology/AYeastTwoHybridAssay/http://www.bioteach.ubc.ca/MolecularBiology/AYeastTwoHybridAssay/

8. Yeast 2-hybrid on a genome wide scale Clone every gene in your genome into both the “bait” and “fish” vectors. Systematically screen each gene for interactions. –Mate individual yeast strains. Many false positives.

9. Interactome Term to define all of the protein interactions that take place in the cell. Book example - predicting human interactions. Based on data that only 10% of the measured interactions are physiological

10. Yeast 2-hybrid False-positives –Some baits are “sticky” leading to non-functional interactions False negatives –Binding not tight enough to detect interaction –Fusion proteins often do not fold correctly Works best when comparing two proteins suspected of interacting Bacterial 2-hybrid systems

11. Co-immunoprecipitation Using an antibody to isolate and purify a protein from a whole cell lysate. Normally you will only purify the protein the antibody recognizes. Any additional proteins that co-purify are candidates for interacting proteins.

12. Hirano et al, 1997 Cell, Vol 89, 511-521, 16 May 1997

13. Fusion protein affinity chromatography Express the protein of interest as a fusion protein. –6-8X His residues –Glutathione S-transferase (GST) –Other “tags” Bind and purify the protein of interest –Poly His residues will bind Ni2+ –GST will bind glutathione

14. Image from: Sigma-Aldrich

15. Fusion proteins - identifying interactions. In vivo - express fusion protein in vivo –Purify complexes from the cell In vitro - overexpress protein in vitro –Bind fusion protein to a column and run whole cell lysate through the column. Identify proteins that “stick” to the fusion protein.

16. Difficulties when using biochemical approaches Stability of protein:protein interactions. –Many are not stable enough to survive purification. Is the fusion protein functional? –Many times fusions will not be functional. Quality of the antibody. –Is it good enough to precipitate enough protein for analysis?

17. Computational methods Rosetta stone analysis –Search for proteins that are separate in one organism but are fused into one protein in another organism.

18. Computational methods Co-expression –Genes that are in operons are often functionally linked. (not always true). –Determine if the structure of an operon is conserved, indicating co-expression. –Candidates for interaction. –Not a great method.

19. Phylogenetic analysis Search for the presence of a protein in all organisms. Determine the distribution. Identify other proteins that also show this distribution. Functionally interact? Physically?

20. PLEX Protein Link EXplorer. Uses phylogenetic profiles to predict possible associations.