We are studying the mechanism of kinesin, a mechanoenzyme that drives microtubule-based intracellular organelle transport processes. Kinesin couples a free-energy-liberating chemical reaction (the hydrolysis of ATP) to a cycle of mechanical steps that moves the enzyme molecules and attached organelles along microtubules. The long-term goals of our research are to: 1) characterize the molecular events that are the fundamental steps in kinesin movement, and 2) determine how these steps are coupled to the reactions of ATP hydrolysis. We have developed a light microscope digital image processing technique for measuring with nanometer-scale precision the movements generated by kinesin molecules. This technique represents an opportunity to directly observe the fundamental processes of mechanoenzyme movement. Microtubule-based intracellular motility 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. Kinesin (or kinesin homologs) are thought to function in all of these categories of cellular processes. The proposed research is an exploration at the molecular level of these functions.
The specific aim of the proposed research is to directly observe and characterize the movement caused by a single catalytic turnover of an individual kinesin molecule. In the proposed experiments, we will: * express and purify a specifically biotinated kinesin fusion protein. Previous studies suggest that this species should be fully mechanochemically functional. We will characterize the functional properties of this recombinant mechanoenzyme to be sure that this is the case. * synthesize a novel enzyme-labelling reagent that consists of 100 nm diameter polystyrene beads each of which contains a single site for the attachment of a biotinated enzyme. * prepare and characterize the 1:1 mechanoenzyme:bead conjugates. The conjugates will then be used to confirm that we can reliably observe movements driven by single mechanoenzyme molecules. We will establish a quantitative motility assay which will measure the specific activity of movement by measuring the fraction of the enzyme-bead conjugates capable of movement. * search for single-turnover movements in the assay by examining the motion of the mechanoenzyme:bead complexes with nanometer-scale precision. These measurements will be conducted under conditions (e.g., limiting ATP) in which discrete """"""""jumps"""""""" corresponding to single-turnover movements should be detected. * characterize the spatial and temporal properties of the observed jumps and determine if they have the characteristics of single-turnover movements.
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