My research is concerned with studying the dynamics of single enzyme molecules with fluorescence microscopy. The principle of the method used is to fix the active enzyme to the surface of a slide and to view the trajectory (time course) of the enzyme during the course of ligand binding and/or catalysis. The enzyme is labeled with a fluorescent tag that monitors events on the enzyme through changes in fluorescence. Methods are being developed for both the fluorescent labeling of the enzymes and their attachment to the quartz slide. Single molecule kinetics have the potential of revealing steps in the catalytic process that cannot be observed with ensemble averaged kinetics. These include direct observation of the coupling of the enzyme conformation to catalysis, monitoring the conformation of different parts of a single enzyme molecule simultaneously, and dissection of processive reactions, such as DNA synthesis, into the individual steps in the reaction. Two enzymes are being investigated, dihydrofolate reductase (DHFR) and T4 DNA polymerase. The trajectories of individual DHFR molecules reacting with substrates and methotrexate reveal conformational changes that are not seen with ensemble averaged kinetics. An isotope rate effect for the hydride transfer has been observed at the single molecule level. In the case of T4 DNA polymerase, experiments are being carried out in which the DNA template is attached to the slide. Assembly of the protein complex necessary for catalysis and the enzymatic reaction are being studied. Thse experiments are directed at understanding better how enzymes catalyze physiological reactions.
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University of Wisconsin, Madison ·