Kinesin is a biped motor protein that walks along microtubule tracks in a cell and performs diverse tasks, including intracellular cargo transport and cell division. To date, it is the smallest known processive motor that directly converts the chemical energy of ATP into mechanical energy. A deeper insight into how kinesin functions is thus not only important for advancing fundamental knowledge of molecular motors, but also critical for developing novel therapeutics against diseases involving impaired intracellular transport. Although past biochemical, biophysical, and structural experiments revealed a significant amount of information about kinesin, the basic mechanism by which it operates as a mechanical amplifier to generate a walking stroke remains unknown. To elucidate the mechanism, it will be critical to develop a synergistic approach combining experimental manipulation of individual kinesin molecules and a computational model based on its atomistic structure. Only through such a combined approach will it be possible to find the molecular physical principle that governs the actual walking motion. Our recent molecular dynamics simulation identified the mechanical element responsible for kinesin's power stroke, which we named the cover strand. It works by assisting kinesin's leg, the neck linker, through forming or breaking a bundle with it depending on kinesin's mechanochemical cycle. Formation of the cover-neck bundle results in a forward conformational bias that generates the power stroke. To validate this experimentally, kinesin mutants missing the cover strand will be constructed and tested using single molecule optical trapping force measurements. At the same time, a computational model of the entire kinesin-microtubule complex will be constructed so that kinesin's whole walking step can be investigated in atomistic detail. Response of the mutant kinesin in the single molecule experiments will be interpreted using computational models. In this way, experiments will be used to refine models, while simulation will be used to interpret experimental data and further design new experiments. Such a tight coupling between experimentation and simulation will provide a clear molecular level mechanistic picture of kinesin motility, upon which a host of other motor proteins will be investigated as our long-term goal. ? ? Relevance: Deeper understanding of kinesin motility will enable better control of its behavior, which will lead to novel therapeutics that target kinesin-mediated transport. Our combined approach between computational modeling of macromolecular complexes and single-molecule manipulation experiment will also be a platform upon which a range of subcellular motor processes of biomedical importance will be investigated. ? ? ?
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