Structural Fluctuations in the Catalytic Cycle of DHFR

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DESCRIPTION (provided by applicant): Dihyrdrofolate reductase (DHFR) plays a central role in maintaining intracellular levels of tetrahydrofolate through the NADPH-dependant reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF). THF is required for normal cellular metabolism since it serves as a precursor in synthesis of several metabolites. Consequently DHFR serves as an important drug target for rapidly proliferating disease such as cancer and bacterial infections. A detailed structural picture exists for intermediate steps throughout the complex catalytic cycle providing a unique basis to correlate structure with dynamics, hence providing a deeper understanding of the catalytic cycle. NMR studies will be implemented to study relatively slow molecular motions (us-ms) for the various DHFR complexes. Highly developed Carr-PurcelI-Meiboom-Gill (CPMG) NMR experiments are well suited for studying motions at the millisecond time scale and will be implemented as such. Molecular motions occurring within this regime are thought to be of utmost importance as this is the rate at which the critical hydride transfer step occurs (~ 950/s). After establishing the backbone dynamics of the enzyme, an alternative look at motions from the substrate and cofactor will also be attempted by isotopic labeling of each for study by NMR.