Mechanism and kinetics of human kinesin-5 motor protein
- Area of Honors:
- Bachelor of Science
- Document Type:
- Thesis Supervisors:
- William O Hancock, Thesis Supervisor
- Peter J Butler, Honors Advisor
- Kinesin-5 (kin-5) is a minimally processive motor protein that walks along antiparallel microtubules to separate the two poles of the mitotic spindle and pull apart chromosomes during mitosis. With the discovery of an inhibitor molecule, kin-5 is a target for antimitotic drugs for cancer treatment; however, compared to conventional kinesin, little is known about kin-5's chemomechanical cycle and mechanism of stepping. It was found that engineered kin-5 motors with a shortened neck linker domain, which undergoes the primary conformational change in each step, exhibit substantially higher processivity, similar to that of conventional kinesin. To better characterize the effects of the length of the neck linker domain, published rates were simulated in the consensus kin-5 chemomechanical pathway and alternate pathways that included combinations of gating, strain-dependent rates, and a novel pathway involving hydrolysis following tethered-head attachment. Kinetics assays were performed to determine the microtubule dissociation rates of kin-5 in different nucleotide states to help rule out or support the proposed pathways. The nucleotide-free and ATP-bound states were confirmed to be strongly-bound to the microtubule, thus rejecting the alternate hydrolysis pathway. Simulations failed to account for the effects on processivity from shortening the neck linker domain.