Identification of Macromolecular Assemblies and Protein-Protein Complexes

1. Identification of Macromolecular Assemblies and Protein-Protein Complexes Science Vol310 p. 827 (2005) In the cell, most proteins do not work independently and therefore a complete understanding of a cellular process really requires a complete understanding of how all the players interact.

2. What Are Some Important Macromolecular Complexes And Processes DNA replication and transcription- Various initiation and transcript proteins involved in timing and promotion or repression of these events. Translation of RNA and protein synthesis- The ribozyme and the ribosome complexes, proteins involved in amino acid transfer and regulation of synthesis. Immune response- Identification of epitopes and formation of degradation complexes. Cell to Cell signaling- Ligand receptor complexes and protein-kinase complexes involved in signaling cascades. Cell construction- Protein complexes involved in cell wall and or membrane construction. You may notice that each many of these subjects are central themes or targets For drug design.

3. How do you Identify and Isolate Macromolecular Complexes? Biochemists have been identifying and isolating protein-protein complexes for a long time. Eur. J. Biochem. 64 233-242 (1976) Tools; Fractionation Gel filtration Ultracentrifugation Enzyme Assay

4. Sedimentation

5. Sedimentation (cont.)

6. Calibrated Gel Filtration

7. Calibrated Gel Filtration (cont.)

8. Genetic Screening Can lead to the Isolation of Malfunctioning Protein-Protein Complexes Can you develop a way to select or screen for a specific phenotype or activity? For example, for most metabolic enzymes form a the protein-protein complex that must dissociated in order for the pathway to work. If you can experimentally Identify genetic mutations in these proteins that are inactive, then the chances are that one of those mutations may form a inactive protein-protein complex. Two examples are nitrogenase and the B12-independent glycerol dehydratase

9. Genetic Screening (Cont.) Example #1; Nitrogenase, a two-component enzyme system required for diazotrophic growth on N2. N2 + 8H+ + 8e- 16MgATP  2NH3 + H2 + 16 MgADP + 16 Pi The two components are the iron protein (FeP), a homodimeric protein that contains a single [4Fe-4S] cluster and the nucleotide binding site and the molybdenum iron protein (MoFeP) a heterotetramer (a and b subunits) that contains a unique [8Fe-7S] cluster and the site of nitrogen reduction at another unique molybdenum-iron-homocitrate (FeMoco) cluster. A central question was how MgATP binding and hydrolysis in the FeP regulates electron flow and to substrate in the MoFeP.

10. Genetic Screening (Cont.) Identification of inactive variants of the nitrogenase iron protein (FeP) 1 – Introduction of antibiotic marker and altered FeP into chromosome Inactive Variant Plate 1 Plate 2 . . . . . Replica Plate . . . . . . . . . . . . . . Media with antibiotic and nitrogen source Media with no antibiotic and no nitrogen source

11. Genetic Screening (Cont.) Is the inactive variant physiologially relevant? Several things that the wild-type Fep does; 1-Shift in midpoint potential when MgATP binds 2- This shift and associated conformational changes are required for complex formation with the MoFeP. Cluster has MgATP-Like Redox Potential Protein has MgATP-Like Conformation Cluster is intact

12. Genetic Screening (Cont.)

13. Expressing of Protein Complexes in E. coli. Tan et al. Protein Expression and Purification 40(2) 385-395 (2005) Expression of the yeast Piccolo NuA4 histone acetyltransferase complex, This polycistronic vector is necessary to insure stabilize all components and the intact complex. Sometimes answering biochemical questions requires a solid genetic approach. Tan et al. have shown that the order of expression does not matter.

14. Expressing of Protein Complexes in E. coli. (cont.) Rapid identification of deletion variants that still form complex.

15. Expressing of Protein Complexes in E. coli. (cont.)

16. Expressing of Protein Complexes in E. coli. (cont.) Multiple types of tags enhances purification and experimental flexibility.

17. Expressing of Protein Complexes in E. coli. (cont.)

18. Case Study: SAGA Binding to TBP in Yeast Transcription In yeast, the multisubunit SAGA (Spt-Ada-Gcn5 acetyltransferase) complex acts as a coactivator to recruit the TATA-binding protein (TBP) to the TATA box, a critical step in eukaryotic gene regulation. However, it is unclear which SAGA subunits are responsible for SAGA’s direct interactions with TBP. • R171E does not interact with • SAGA. Spt3 and Spt8 are also • important.

19. Case Study: SAGA Binding to TBP in Yeast Transcription Chemical cross linking is a useful tools for assessing proximity but care should be taken when interpreting specific interactions.

20. Case Study: SAGA Binding to TBP in Yeast Transcription Spt8 is sufficient for interaction with the TBP.

21. Case Study: SAGA Binding to TBP in Yeast Transcription Both Spt3 and Spt8 are required for interaction in SAGA complex.

22. Case Study: SAGA Binding to TBP in Yeast Transcription Competition for the TBP monomer is key to formation of transcription complex.

23. Case Study: SAGA Binding to TBP in Yeast Transcription

24. Identification of Macromolecular Interaction

25. Identification of Macromolecular Interaction (cont.) “Gone Fishing” – Affintiy tag isolation of peptides and peptide complexes under physiological conditions and mass spec identification of molecules that co- purify.

26. Identification of Macromolecular Interaction (cont.)

27. Identification of Macromolecular Interaction (cont.) Biacore can assist in more specific identification

28. Identification of Macromolecular Interaction (cont.)

29. Identification of Macromolecular Interaction (cont.)

30. Identification of Macromolecular Interaction (cont.) Again, chemical cross-linking is an easy, low tech, experiment that can provide information about general areas of potential interaction, but the results are not absolute.