Hydroxyl radical mapping of protein interfaces

DESCRIPTION (provided by applicant): Identifying amino acid residues of a protein that form the binding site for a drug, or that are present at the interface in a protein-protein complex are difficult to identify from the crystal structures of the individual partner proteins. Carta Proteomics is committed to the premise that monitoring isotope exchange into proteins by mass spectrometry will permit the rapid identification of contact residues of proteins that form macromolecular complexes. Prior work and proprietary development has established amide-proton solvent exchange as one facet of Carta Proteomics. This proposal is to develop a complementary method of hydroxyl radical (-OH) induced 1I-TJ2H exchange into the aliphatic carbons of the amino acid side chains. This proposal takes advantage of the basic underlying chemistry that has been recently well characterized. The goal of this proposal is to demonstrate the capability of this chemistry to use the multiple binding sites present on thrombin to validate the ability of this methodology to correctly identify the active site in a BABIM thrombin complex and to identify the allosteric binding site for hirudin, an inhibitory oligopeptide. This demonstration will validate the application of the method to inhibitors identified from combinatorial chemistry screens. PROPOSED COMMERCIAL APPLICATION: Structure based drug design is severely hampered by the lack of a low-cost, rapid method of determining structure. Our technology of determining structures of a small molecule/protein target interaction can be easily adapted to high throughput and very fast turn-around at very low cost. Because of these unique features we expect the technology to be readily integrated into the small molecule drug discovery process. Carta Proteomics will participate by providing the proprietary expertise through research partnership agreements.