Abstract The goal of this R37-supported research program is to obtain a fundamental understanding of how myosin motors function and interact with proteins, lipid membranes, and other biomolecules to power structural arrangements and motile events that are crucial for eukaryotic life. Our strategy has been to define the physical properties of the myosin family to better model and test function. To this end, we made fundamental discoveries into the mechanisms of chemomechanical coupling of all myosins and provided new insights into the mechanical attachments of membrane interactions. We will continue along the lines of the original specific aims, and we will build from our discoveries to address new and exciting questions related to these Aims. Aim 1: Determine the structural origin of myosin force sensing. Our focus is to determine how myosins sense and respond to mechanical load. Recent progress has given us an unprecedented look into the myosin-I structural states that span the force-sensing transition that control exit from force-bearing states. Using our new structural "know-how," we engineered Myo1b tension-sensing properties into mechanically divergent Myo1c, and we were able to engineer a low duty ratio myo1b into a high duty ratio motor. Our newest work has resulted in specific predictions regarding the roles of key residues conserved in most members of the myosin superfamily in tuning tension sensitivity. Chemomechanical tuning via these allosteric connections will be tested, which will allow us to (1) understand the basic molecular biophysics of energy transduction (the holy grail of myosin biophysics), (2) understand the effect of disease causing mutations that are on this allosteric path that impact tension sensing, and (3) design, engineer, and express myosins of altered mechanosensitivity for cell biological experiments to probe the molecular functions of myosin. Our development of a high-speed optical trapping system (µs time resolution, Woody et al, 2018), will allow us to probe the entry into the force-bearing states, including the phosphate-release step. Mechano-diversity in this transition is controversial and virtually unexplored. We will continue our cryo-EM work to determine the structure of Myo1c, a motor with highly divergent tension sensing properties. We will also use cryo-EM to determine the structure of mechanically strained myosin, which will be facilitated by the novel cryo-EM analysis techniques developed in our recent paper (Mentes et al. (2018)), and the generation of engineered Myo1b dimers that, when actin-bound, have a positive and negative mechanical strains that substantially affect ADP release. Investigation of myosin-I function in living cells, under working conditions, has been extraordinarily difficult, as identifying the population of proteins that are actively generating force has not been possible. Thus, we are very excited about the development of FRET-force-sensors for expl...