The long range goal of this research program is to understand mechanisms of regulation in multisubunit proteins and other macromolecular assemblies. Our immediate goal is to elucidate the allosteric mechanism of E. coli aspartate transcarbamylase via analysis of the functional energetics of protein-protein and protein-ligand interactions and correlation of energetic and structural information. An analysis of the macroscopic thermodynamic properties of this system has been completed and atomic coordinates for three major conformational states are now available, making it possible to begin mapping pathways of energy transduction within the structure. Major goals of the next grant period will be to: (a) Continue the development of low and high pressure analytical gel chromatographic methods and apply them to the analysis of previously inaccessible protein- protein interactions in the catalytic subunit (c3), regulatory subunit (r2) and native enzyme (c6r6) over a range of conditions. (b) Extend the experimental analysis of electrostatic effects by (i) carrying out a comprehensive analysis of the effects of varying pH, ionic strength and specific ions on the structure, structural dynamics, energetics and function of wild type ATCase and its subunits, (ii)testing hypotheses developed by computer modelling in the previous grant period by examining the structure, energetics and functional properties of single site mutants in which groups calculated to undergo large pK changes upon assembly or ligand binding are modified, (iii) investigating the mechanism of synergistic inhibition by CTP and UTP, particularly the role of ionizable groups, by site-directed mutagenesis and enzymatic assay. (c) Develop cryoisoelectric focussing as a method for analyzing populations of ligated species and analyze the populations of PALA-ligated species over a range of pH and temperature and in the presence and absence of CTP and ATP. (d) Develop statistical thermodynamic models of the allosteric mechanism.
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