| Kinesin family motor proteins drive many essential cellular processes, including cargo transport and mitotic spindle assembly and regulation. They accomplish these tasks by converting the chemical energy released from the hydrolysis of adenosine triphosphate (ATP) directly into mechanical motion along microtubules in cells. Optical traps allow us to track and apply force to individual motor proteins, and have already revealed many details of the movement of conventional kinesin, although the precise mechanism by which chemical energy is converted into mechanical motion is unclear. Other kinesin family members remain largely uncharacterized.; This dissertation details the use of a novel optical-trapping assay to study Eg5, a Kinesin-5 family member involved in both spindle assembly and pole separation during mitosis. We demonstrate that individual Eg5 dimers are relatively slow and force-insensitive motors that take about 8 steps, on average, before detaching from the microtubule. Key differences in processivity and force-response between Eg5 and conventional kinesin suggest ways in which the two motors might have evolved to perform very different tasks in cells.; This dissertation also details efforts to unravel how chemical energy is converted into mechanical motion by simultaneously measuring mechanical transitions (with an optical trap) and nucleotide binding and release (with single-molecule fluorescence) for individual conventional kinesin motors. We constructed a combined instrument, demonstrated its capabilities by unzipping fluorescently-labeled DNA duplexes, and used this instrument to record the motion of individual conventional kinesin motors powered by the hydrolysis of fluorescent nucleotides. Preliminary data reveal the challenges inherent in such measurements and guide proposals for future experimental approaches.; Finally, this dissertation includes several chapters intended to serve as practical guides to understanding, constructing, and maintaining optical traps and single-molecule fluorescence microscopes, as well as implementing novel surface chemistry treatments for the development of new single-molecule assay geometries. |
