Dissecting Structure Function Relationships In Molecular Motors Using Protein Engineering And Single Molecule Methods PDF Download

Cytoskeletal motors convert chemical energy into work and are involved in a wide range of cellular processes, including motility, contractility and cargo transport. Engineering the motility of these molecular machines provides insights into structural-functional relationships, components for driving transport in microscale devices, and tools for controlling cellular processes that depend on these motors. I have engineered motors with novel functions that provide new levels of control over nanoscale motion, including myosin motors that can be chemically signaled to switch their direction of motion, and kinesins with light-activated gearshifts. Myosin motors generate directed motion using a powerstroke mechanism, in which a conformational change in the catalytic head domain is amplified by a rigid geometric element called the lever arm. Our controllable motor designs are based on introducing dynamic changes in lever arm geometry. I designed myosin VI variants with chimeric lever arms composed of a three-helix bundle fused to two or more calmodulin-binding IQ repeats. In low concentrations of calcium, these engineered motors move toward the ( - ) end of actin filaments; in high concentrations of calcium, the motors are (+) end directed. I confirmed the designed behavior using in vitro assays of myosin function, including single-molecule measurements. To further adapt these controllable engineered motors to future applications, it is desirable to have 1) high processivity and 2) optical control. Processivity is a requirement for building nanoscale devices that harness the transport capabilities of molecular motors, and light is a more versatile control signal than calcium concentration because it can be precisely modulated in space and time, and is generally orthogonal to cellular signaling. I have collaborated with other members of the Bryant laboratory to develop controllable processive walkers, and to characterize myosin motors that reversibly change gears -- speed up, slow down, or switch directions -- when exposed to blue light. Finally, I expanded the toolbox of engineered motors to include microtubule-based motors. Microtubules and actin filaments are used for different sets of cellular functions, and have different properties that can be exploited for building tracks or shuttles in nanoscale device applications. Optimizing properties and controllability of both sets of motors enhances our ability to select the best motor for specific applications in vivo and in vitro. I have created microtubule-based motors that can reversibly change gears in response to blue light, by porting gearshift designs from our controllable myosins to class 14 kinesins, which have a myosin-like swinging lever arm mechanism. As part of this work, in collaboration with the Nogales laboratory at Berkeley, I have started to add cryo-EM structural data to our design cycle. We have successfully reconstructed two engineered kinesins and confirmed that the structures of engineered elements are in agreement with our structural designs based on rigid recombination of modular protein domains. I have developed a set of controllable cytoskeletal motors with novel functions. With further refinements, the engineered motors described here may be useful in diagnostic devices that harness active transport, as well as for in vivo investigations of cellular processes.