Probing mechanisms of myosin motors using single molecule techniques [electronic resource]

Members of the myosin family of molecular motors perform a variety of essential functions in the cell, including powering muscle contraction and cell division, transporting cargo, and serving as anchors and tension generators. Myosins accomplish these tasks by transforming chemical energy from ATP hydrolysis into mechanical work, as they either move along their actin filament tracks, or power the movement of the actin filaments. Single molecule techniques have proven especially useful for understanding the biophysical mechanisms that myosins employ in order to perform their functions. In this thesis, I discuss my application of quantitative, single molecule level approaches to remaining questions about the mechanisms of myosins II, V and VI. In the first section, I describe our analysis of myosin VI processivity via structurally engineered mutant constructs, which we examined using single molecule fluorescence. Myosin VI is both structurally and functionally unusual among myosins. In order to probe our understanding of its mechanism, we replaced its lever arm with a variety of engineered artificial lever arms, and tested whether it responded as we would expect. As part of this work, I also developed a quantitative model of processivity of myosin VI, which I used to analyze the expected effects of decreasing intramolecular communication between the heads of a processive myosin. This model has implications not only for myosin VI, but for other two headed processive motors, including processive myosins, kinesins, and dyneins. In this work, I found that processivity is markedly robust to decreased inter-head communication. In order to further characterize intramolecular communication in processive myosins V and VI, I hoped to directly visualize ATP molecules binding and releasing from myosin molecules as they walked along actin filaments. This required the development of techniques to allow the resolution of single fluorescent molecules at higher concentrations of fluorophore than has previously been possible. I approached this challenge using two technological approaches: linear zero mode waveguides (ZMW) and convex lens induced confinement (CLIC). While the direct visualization of nucleotide gating remains a challenge, I made significant progress toward applying these techniques to that outstanding question. Finally, in the last section of my thesis, I discuss work on characterizing mutations in the human cardiac myosin II protein that cause hypertrophic and dilated cardiomyopathy. While many mutations that cause these diseases have been identified, their mechanism(s) of action are not understood. Characterizing these effects requires precise quantification, since the changes in myosin behavior caused by the mutations are likely to be subtle. Toward that end, I have refined our approach to the gliding filament assay to increase its precision and reproducibility, and have begun to characterize the effects of several disease-causing mutations in this motor.

Keywords: Applied Physics
Advisor: Block Steven
Spudich James A.
Bryant Zev David advisor. Thesis advisor