The Kinetic Characterization of the Marginally Processive Motor, Dimeric Eg5/KSP
The Kinesin-5 subfamily of the kinesin superfamily of molecular motors has been shown to play an integral role in the transfer of genetic material from mother-cell to daughter-cell. These homotetrameric kinesins function by crosslinking two microtubules in the mitotic spindle and imparting a force necessary to both assemble and maintain the spindle. The purpose of this dissertation has been to gain a better understanding of how Eg5/KSP, a member of the Kinesin-5 subfamily, coordinates the biochemical activities of its motor domains, to fulfill its cellular role.
This dissertation focuses on a truncation of the human Eg5 gene that produces a dimeric motor. Analysis of this motor has indicated that the two motor domains, which interact with the same microtubule, function cooperatively. In some respects, dimeric Eg5 resembles conventional kinesin. Both motors are capable of translocating along the microtubule by taking successive steps before dissociating. To achieve this phenomenon, both motors couple the turnover of a single molecule of ATP to each advancement while maintaining the two motor domains out of phase through alternating catalytic cycles. Also, both motors have their stepping gated by ATP binding.
The mechanistic commonalities between dimeric Eg5 and conventional kinesin, however, do not reach beyond a similar mechanism of stepping. This work has uncovered a novel biphasic, microtubule associated mechanochemical cycle. Dimeric Eg5 is the first kinesin known to begin the microtubule associated phase of its ATPase cycle with both motor domains associated with the microtubule. Furthermore, the transition to this two-motor-domain-bound state is the slow step governing steady-state ATP turnover. This slow transition only occurs once in the cycle and prior to processive movement. During processive movement, the catalytic step governs the rate of motor stepping. Dimeric Eg5 is also the first kinesin motor to have a rate-limiting catalytic step.
Advisor:Susan P. Gilbert; Jeffrey Brodsky; Roger Hendrix; William Saunders; E. Michael Ostap
School:University of Pittsburgh
School Location:USA - Pennsylvania
Source Type:Master's Thesis
Date of Publication:01/25/2008