Tools to study the kinesin mechanome using optical tweezers

Abstract

Molecular motors play an important role in driving some of the most complex and important tasks in biological systems, ranging from transcribing RNA from a DNA template (Polymerases) to muscle contraction (Myosin) and propelling bacteria (Flagellum). Key to the understanding of the fundamental principles and designs by which molecular motor function has been the kinesin family. Missing, however, is a clear understanding of the series of events that take place at the atomistic level when kinesin walks on a microtubule and generates force. Recent MD simulations have identified the force-generating mechanism in kinesin, the cover-neck bundle, and strongly suggest that the formation of the CNB by the N-terminal cover strand and the C-terminal neck linker of the motor head are responsible for force generation. In this thesis we present tools developed in the Lang Laboratory to further elucidate the stepping motion and force generation mechanism of kinesin using Drosophila kinesin as a model system. We demonstrate the function of a force clamp specifically designed for the laboratory and show traces of WT kinesin walking under constant load. We also purified and tested kinesin mutants running under a force load. We present two assays specifically designed to study the interaction between kinesin and the last 10-18 C-terminal residues of a-p tubulin, the E-hook. We were unable to observe kinesin - e-hook interactions, such as those suggested by the formation of tethers, when the e-hook was bound to the surface. In the case of e-hook in solution, our results indicate that 2G kinesin was still functional and its stall force approximately 3 pN just as for the case when no e-hook is present. We also propose ways that the work in this thesis can be expanded. The force clamp can be easily adapted to study novel kinesin mutants under constant load in 2D. In addition, the force clamp can be used to probe the kinesin - e-hook interactions by looking at kinesin walking over microtubules with cleaved e-hooks. The e-hook assays presented in this thesis can also be expanded to include higher concentrations of e-hook or be performed using labeled e-hook to assess single molecule interactions and concentrations.