Engineering myosins for long-range transport and directional control [electronic resource]
Keywords: Bioengineering.

Myosin motor proteins have diverse mechanical properties adapted for cellular tasks including muscle cell contraction, assembly and maintenance of cellular structures, and processive cargo transport. Protein engineering has been useful for exploring the structure-function relationships enabling these tasks and for designing myosins with novel behaviors. Engineered myosins have potential for transport and self-assembly applications in vitro and model validation and synthetic biology in vivo. During my thesis work, I have developed (1) a structural basis for controllable bidirectionality, a novel function for actomyosin; (2) modular strategies for processivity enhancement in cytoskeletal motors; and (3) new assays that increase the throughput of engineered myosin characterization. To generate unidirectional motion along the polarized actin filament, myosins rely on a directed "power stroke", in which a conformational change in the catalytic head is amplified by an extended lever arm structure. Myosin VI has a unique structural element that geometrically redirects its power stroke, reversing the directionality of the motor relative to other myosin classes. To explore how this feature might be adapted for bidirectional motion, I constructed a panel of chimeric myosin VI motors bearing artificial lever arms. I found that lever arm attachment at an intermediate angle could yield lever arm length-dependent directionality. This discovery served as the foundation for a series of controllable bidirectional motors created in collaboration with members of the Bryant laboratory. These motors are dynamically switchable, and can be assembled into dimers to processively transport cargo over short distances. Natural processive myosins are dimeric and use internal tension to coordinate detachment cycles of the two heads. I have found that processivity can be enhanced in engineered myosins using two non-natural strategies designed to optimize the effectiveness of random, uncoordinated stepping: (i) formation of three-headed and four- headed myosins, and (ii) introduction of flexible elements between heads. I quantified processivity improvements using systematic single-molecule characterization of a panel of engineered motors, and tested the modularity of these approaches by designing a robustly processive version of our controllable bidirectional myosin and the fastest measured processive cytoskeletal motor. To characterize the effects of multimerization and flexibility on stepping behavior, I developed methods for efficient labeling of myosins with gold nanoparticles, and implemented high-speed tracking to measure step sizes and dwell times. I have established protein engineering strategies for the introduction of novel behavior in myosins, and developed high-throughput methods for motor characterization, including parallelized videomicroscopy assays in multiwell plates. These engineering approaches have yielded insights into structure-function relationships in motors, including determinants of directionality and processivity. With further refinements, the engineered motors described here may be used in diagnostic devices that harness active transport, as well as for in vivo investigations of myosin-driven cellular processes.