| Kinesin is the smallest known motor protein. It transports intracellular cargo by using its two motor domains to walk along cytoskeletal microtubules in 8-nm steps. Despite only being discovered two decades ago, this molecule has become a model system for understanding motor protein function, and the technological innovations developed to probe this system helped establish the modern field of single-molecule biophysics. This dissertation will focus on advances in high-resolution optical trapping technology with the goal of understanding the kinesin stepping mechanism.; Using an optical trapping apparatus, we made the surprise observation that some kinesin molecules alternated between slow and fast dwell times between steps, i.e., they "limp," even though they are composed of two motor domains identical in peptide sequence. The phenomenon of limping breaks 'step equivalence,' implying that kinesin molecules must strictly alternate between two different configurations as they step. Such alternation is a hallmark of an asymmetric, hand-over-hand mechanism. We used the temporal signature of limping as a means of sorting the "left" from "right" steps, and thereby resolved a long-standing controversy about the kinesin step size: do all kinesin steps measure 8 mn (the tubulin repeat distance along a microtubule protofilament), or do steps alternate between 7- and 9-nm sizes (suggested by some alternative stepping models)? Our data show that kinesin molecules always take 8-nm steps, a result that strongly constrains the allowed stepping models.; To learn more about the underlying cause of asymmetry in homodimeric kinesin, we tested competing models that seek to explain limping. We found that the degree of limping depends on the angle of load applied to the kinesin stalk, but this finding does not itself suggest which elements in the structure are asymmetric. Moreover, we found that mutations to the N-terminus of the stalk, where the motor domains are linked, was a strong determinant of limping. Taking these results together, we hypothesize that the N-terminal region of the stalk develops a structural asymmetry, which the angle of applied load accentuates or diminishes, leading to a difference in the stepping rates for the two motor domains. |
