This project will use developed computer simulation techniques to study dynamics and fluctuations associated with catalytic events in DFHR and beta-lactamase, working closely with experimental groups to interpret NMR and mutagenesis data. The analysis will extend conventional molecular dynamics simulations in novel ways to study correlated motions at long distance scales and to develop detailed models of catalytic pathways, using the following techniques. (a) Molecular dynamics simulations on key kinetic intermediate states characterized experimentally for the catalytic cycle of DHFR, providing a basis for the study of fluctuations and dynamics of these molecular systems and the coupling between these to catalytic function. Similar simulations will be performed for the evolutionally less mature enzyme system of metallo beta-lactamase. (b) Models for catalytic pathways in DHFR generated by novel trajectory techniques generation techniques that find transition states and reaction paths to connect structures of catalytic intermediates (or their analogues generated by NMR, crystallography and mutational analysis. (c) Harmonic and quasiharmonic analyses of both DHFR and beta- lactamase in free and ligand-bound forms, based on proposed structural models of catalytic intermediate states and the apo-enzymes, to study long-wavelength, low-frequency correlated motions that can affect the overall rigidity of these system. Motional models derived from these studies will be created, with adjustable parameters to fit NMR relaxation data. (d) Collaboration with Project 3 to refine a solution structure of beta-lactamase both free and in complex with inhibitors. This objective will involve first the development of quantum mechanically based models for the bimetallic active site followed by structural modeling.
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