Conformational Motions of DHFR during Catalysis

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DESCRIPTION (provided by applicant): It is suspected that an enzyme's conformational motions may correlate closely to its catalytic function. Appreciation of how protein conformational fluctuations relate to catalysis will have profound implications for drug design and protein engineering efforts. The current proposal is centered on the implementation of novel single molecule fluorescence resonance energy transfer (FRET) methods to probe the dynamic motions of dihydrofolate reductase from Escherichia coli (DHFR), during catalysis. Two probes will be placed on DHFR, one in a stationary loop and the other on the Met20 loop, which undergoes conformational changes in the course of the DHFR catalytic cycle. The distance separating the tags will vary depending on the conformation of the moving Met20 loop. Single DHFR molecules will be attached to a glass slide via a biotinylated N-terminus. The conformational motions of the Met20 loop in a single DHFR molecule can thus be monitored during catalysis (equilibrium conditions) on the millisecond time-scale by changes in FRET efficiency, which is sensitive to the distance changes between the fluorescent tags. DHFR is an important target of several anticancer and antibacterial drugs and progress toward understanding its conformational fluctuations during catalysis will contribute to the design of new DHFR inhibitors that can bind to areas other than the active site to effectively target human diseases.