| Molecular motor proteins are essential mechanochemical enzymes that power cell division, muscle contraction, DNA and RNA unwinding and synthesis, and transport of various membranous organelles in the cell. The kinesin superfamily of molecular motors plays a pivotal role in these concerted movements. In this thesis, I will present kinetic and thermodynamic characterizations of two of these kinesin motors, Ncd and Kar3, and mutational studies on conventional kinesin that extends our understanding of the alternating site mechanism for ATP hydrolysis. The work on Ncd, a homodimeric molecular motor in Drosophila melanogaster, outlines a mechanism of ATP turnover significantly different than conventional kinesin where ADP release is rate-limiting and the two heads of the dimer bind ADP with different affinities in solution. Also, my work shows that cooperative interactions between the motor domains are necessary to promote dissociation of the Ncd motor domain from the microtubule. The next molecular motor to be examined in this thesis, Kar3, is important for meiosis and mitosis in the budding yeast Saccharomyces cerevisiae . Kar3 is unique in that it heterodimerizes with Cik1 or Vik1, both nonmotor polypeptides, thereby producing a motor protein with a single motor domain. It is not well understood how motility can be promoted by a single motor domain. Work on three constructs of Kar3, the motor domain of Kar3, a GSTKar3 construct reported to promote motility, and a Kar3-Cik1 heterodimeric construct indicate that Kar3 may not produce very robust motility within the cell, leading to the hypothesis that perhaps the role of Kar3 is to provide stability to the spindle rather than position microtubules within the spindle through motility. The last part of my thesis will examine two mutants in the conserved Switch I region of conventional kinesin. These results order the steps in the ATPase pathway for the homodimeric motor where ATP hydrolysis at the rearward head occurs after ADP release at the forward head. Also, these results implicate a structural pathway for microtubule binding that extends through several salt bridges between Switch I (α-helix 3a, loop 9, and α-helix 3) and Switch II (β-sheet 7, loop 11, and α-helix 4a) to the microtubule interface (α-helix 4, loop 12, and α-helix 5). |
