| Protein motors, such as kinesin and myosin, use chemical energy in the form of ATP to physically move along filaments and perform complex mechanical tasks in the cell, such as intracellular transport, cell division and muscle contraction. Myosin and kinesin are motor proteins that move along actin and microtubules, respectively. The primary goal for this dissertation research is to determine structure-function relationships in Myo I, a class-VII myosin, and in Ncd, a member of the kinesin superfamily.;Myosin VII is a member of the myosin family that is found in human tissues and that has high homology to a D. discoideum myosin, Myo I. Work towards obtaining a crystal structure of Myo I are presented in this thesis. A Myo I structure would be the first structure of a class-VII myosin and a MyTH domain and would help discern the role myosin VIIs play in the cell. The structure of a different class of myosin may help better determine force-producing conformational changes in myosins.;Ncd is a class-14 kinesin that plays a role in spindle formation in the cell. This protein consists of a C-terminal motor domain, neck and stalk that rotate to effect movement along the microtubule. Ncd mutants, T436S and N600V, were created to disrupt nucleotide-binding and microtubule-binding, respectively, and to be able to obtain a crystal structure of Ncd in an ATP-like state. The wild-type ATPase mechanism for monomeric and dimeric Ncd is presented in order to compare with mutant Ncd kinetics. A mechanistic analysis of mutant Ncd ATPase activity may shed light on structural transitions that affect microtubule and nucleotide binding. |
