Kinesin is a mechanoenzyme that drives microtubule-based intracellular organelles transport processes. Kinesin couples a free-energy-liberating chemical reaction (the hydrolysis of ATP) to a cycle of mechanical processes that move the enzyme molecules and attached organelles along microtubules. We want to characterize the cycle of mechanical processes by which kinesin moves and to determine how these processes are coupled to the reactions of ATP hydrolysis. We have developed a novel experimental system that allows us to directly monitor mechanical processes and chemical steps in single kinesin molecules specifically conjugated to microscopic polystyrene beads. The system makes it possible to quantitatively compare mechanical and chemical reaction rates under identical conditions, thereby allowing direct studies of mechanochemical coupling. Intracellular organelle transport by kinesin and kinesin homologs plays an essential role in the physiology of eukaryotic cells. Its functions include transport of materials, chromosome and nuclear movements in mitosis/meiosis, and morphogenesis of membranous organelles. To explore these functions at the molecular level, we will: 1) measure the distance moved by kinesin per ATP hydrolyzed. We will measure the ATPase Vmax for bead-conjugated single kinesin molecules and compare this to the movement velocity of the conjugates under the same conditions. This study will test the validity of models in which one ATP is hydrolyzed per mechanical step. 2) measure the rate of ADP-induced release of microtubule-bound kinesin heads. This study will test the hypothesis that head release is an essential step in the kinesin movement cycle. Knowing the kinetics of head release will help us understand how single two-headed kinesin molecules remain associated with the microtubule while moving along it. 3) derive the structure of a two-headed kinesin derivative from two- dimensional molecular crystals. Structural data will help reveal the molecular conformational changes that drive kinesin movement and the nature of interactions between kinesin heads. 4) prepare one-headed kinesin derivatives and characterize their functional properties. By characterizing the steady-state ATPase, microtubule release kinetics, and single-molecule motility, this study will help reveal the role of head-head interactions in two-headed kinesin function.
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