This project aims to identify and map the surface contacts between two or non-covalently bound proteins by combining three general techniques which the P.I. states that he first devised: (a) isotopic amplifications by expression and purification of proteins doubly-depleted in C-13 and N-15; (b) hydrogen/deuterium exchange of individual proteins and protein complexes in solution followed by rapid quenching, enzymatic cleavage to produce dozens of peptide fragments, and prompt ultra high-resolution mass analysts to determine extend of deuterium incorporation (and thus solvent accessibility to) the backbone of amide protons for each fragment; and (c) various Fourier transform ion cyclotron resonance (FT-ICR) techniques (micro/LC/ESI, on-line desalting, external ion cyclotron accumulation, SWIFT ion selection, digital heterodyne/digital quadrature detection, charge state deconvolution and isotopic deconvolution) needed for unequivocal interpretation of the results. The INK4 family of tumor suppressor proteins (p15,p16,p18,p19) acts in part by forming non-covalent complexes with cyclin-dependent kinases (cdk, 34-37 kDa), and cyclin (34 kDa). Only the smallest of these is accessible by high-resolution NMR. The applicants shall begin by mapping the surface contacts in binary complexes of INK4 protein, cdk, and cyclin and various recombinant variants, and then extend the same approach to epitope mapping of antigen: antibody complexes, to the troponin family of muscle proteins.
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