This 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 quartz 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. Initially the enzyme of interest will be labeled with biotin, usually as a tag on the amino terminus of the enzyme, and attached to a slide to which avidin is tightly bound at a dilute concentration. Because biotin binds to avidin very tightly, the enzyme will become attached to the slide. The changes in fluorescence are followed either with an ICCD camera or an avalanche photodiode. 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 methotrexate reveal a conformational change that is not seen with ensemble averaged kinetics. The binding of other ligands to DHFR is being carried out and the hydride transfer reaction is being studied with single DHFR molecules. In the case of T4 DNA polymerase, experiments are being carried out in which either the DNA template or the enzyme is attached to the quartz slide. The addition of individual bases to the growing DNA chain will be monitored and the kinetics characterized. These experiments are directed at understanding better how enzymes catalyze physiological reactions. A better understanding of enzyme mechanisms is essential for understanding the physiology of disease and the development of new drugs.
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