Striking advances in the areas of bioinformatics, mass spectrometry and parallel yeast two-hybrid analyses have made it possible to deduce the protein components of almost any multi-protein complex. However, there remains no reliable method by which the organization of the proteins in the complex can be deduced, a central issue in eventually understanding the mechanism of action of any given complex. A related problem is to understand how proteins that regulate a complex through direct physical interactions dock with the protein machine. The major goal of this project is to develop a general protocol to address these problems. This will be done by elaborating on a novel protein cross-linking chemistry developed in my laboratory over the last two years. In this chemistry, water-soluble metal complexes are activated by brief photolysis with visible light in the presence of an electron acceptor. This results in the photo-oxidation of the complex, which in turn is thought to oxidize accessible tyrosine or tryptophan residues in proteins, creating radical intermediates. These radicals can couple to nearby side chains from partner proteins, probably by a variety of mechanisms, thus leading to covalent protein cross-linking. The reaction is fast, efficient and cross-links only proteins that are closely associated. We propose to employ this chemistry in combination with two-dimensional gel electrophoresis/mass spectrometry techniques to elucidate the architecture of the yeast 26S proteasome. In parallel, the cross-linking protocol (and related oxidative labeling reactions) will be employed to identify the specific sub-units contacted by a variety of exogenous ligands that are known to bind to the proteasome. Finally, a new aspect application of this oxidative chemistry is proposed: the development of a general method by which to identify the protein target(s) of bioactive small molecules.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058175-06
Application #
6682711
Study Section
Special Emphasis Panel (ZRG1-SSS-B (01))
Program Officer
Basavappa, Ravi
Project Start
1998-08-01
Project End
2005-11-30
Budget Start
2003-12-01
Budget End
2004-11-30
Support Year
6
Fiscal Year
2004
Total Cost
$312,000
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
800771545
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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