Most important biological processes are mediated by """"""""protein machines,"""""""" very large multiprotein complexes whose component polypeptides often affect one anther's properties through direct interactions. Traditional biochemical methods are ill suited to study all aspects of the largest protein machines, such as RNA polymerase holoenzymes, splicosomes, and nuclear pore complexes, because classical reconstitution of the complex from purified proteins is unfeasible. Therefore, new methods are necessary to derive detailed, information about the mechanism of faction of these machines in their intact form. This proposal addresses the problem of elucidating the organization of proteins in large complexes and understanding how the array of protein-protein interactions might change during the course of the reaction in question. A novel approach noncovalent affinity modification will be developed that involves specific delivery of a small molecule with a latent reactive group to a particular protein in a large complex via noncovalent interactions. Specific delivery of the reactive agent will be achieved by fusing to the protein of interest at the genetic level a peptide (selected from a library) that binds the small molecule with high affinity and specificity. When activated, the small molecule will react with proximal proteins, resulting in their covalent modification. If a sensitive method exists to detect these products, subsequent analysis of the mixture under denaturing conditions will reveal which proteins were nearby the small molecule-docking site in the complex. Iterations of this type of experiment would allow the organization of large protein machines to be mapped out and experiments conducted at different times in the reaction cycle could reveal how the organization of the complex might change with time. While this work is directed towards the development of a new technology and is not focused on any particular disease state per se, the health-related impact of the development of this methodology would be significant. Multiprotein complexes mediate essentially all processes that are thought to lead to cancer when they malfunction, including the decoding of genetic information and regulation of the cell cycle.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058175-03
Application #
6181014
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Wehrle, Janna P
Project Start
1998-08-01
Project End
2001-07-31
Budget Start
2000-08-01
Budget End
2001-07-31
Support Year
3
Fiscal Year
2000
Total Cost
$231,606
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Udugamasooriya, D Gomika; Kodadek, Thomas (2012) On-Bead Two-Color (OBTC) Cell Screen for Direct Identification of Highly Selective Cell Surface Receptor Ligands. Curr Protoc Chem Biol 4:35-48
Liu, Bo; Archer, Chase T; Burdine, Lyle et al. (2007) Label transfer chemistry for the characterization of protein-protein interactions. J Am Chem Soc 129:12348-9
Kodadek, Thomas; Bachhawat-Sikder, Kiran (2006) Optimized protocols for the isolation of specific protein-binding peptides or peptoids from combinatorial libraries displayed on beads. Mol Biosyst 2:25-35
Liu, Bo; Burdine, Lyle; Kodadek, Thomas (2006) Chemistry of periodate-mediated cross-linking of 3,4-dihydroxylphenylalanine-containing molecules to proteins. J Am Chem Soc 128:15228-35
Lin, H-J; Kodadek, T (2005) Photo-induced oxidative cross-linking as a method to evaluate the specificity of protein-ligand interactions. J Pept Res 65:221-8
Archer, Chase T; Burdine, Lyle; Kodadek, Thomas (2005) Identification of Gal4 activation domain-binding proteins in the 26S proteasome by periodate-triggered cross-linking. Mol Biosyst 1:366-72
Burdine, Lyle; Gillette, Thomas G; Lin, Hai-Jun et al. (2004) Periodate-triggered cross-linking of DOPA-containing peptide-protein complexes. J Am Chem Soc 126:11442-3
Amini, Frank; Denison, Carilee; Lin, Hai Jui et al. (2003) Using oxidative crosslinking and proximity labeling to quantitatively characterize protein-protein and protein-Peptide complexes. Chem Biol 10:1115-27
Amini, Frank; Kodadek, Thomas; Brown, Kathlynn C (2002) Protein affinity labeling mediated by genetically encoded peptide tags. Angew Chem Int Ed Engl 41:356-9
Fancy, D A; Denison, C; Kim, K et al. (2000) Scope, limitations and mechanistic aspects of the photo-induced cross-linking of proteins by water-soluble metal complexes. Chem Biol 7:697-708

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