Myosin is a molecular motor that generates force on actin using energy from ATP hydrolysis. Myosin plays a key role in muscle contraction and is responsible for a variety of motility processes at the cellular level. It works cyclically, changing its conformation during the power stroke and the recovery stroke. X-ray crystallography has provided information about the structural organization of myosin in different biochemical states (as defined by bound nucleotide), inspiring several structural models that could explain the molecular mechanism of myosin's function. Spectroscopy, in combination with site-directed labeling and transient experiments, can test and refine these models and provide information about myosin's dynamic properties.
The goal of this project was to determine the structural dynamics of the myosin force-generating domain and study coupling mechanisms between this domain and the myosin active site. We have chosen Dictyostelium discoideum (Dicty) as our experimental system since it provides multiple advantages in comparison with the muscle myosin. In particular, it is possible to manipulate the Dicty DNA sequence, engineering labeling sites at desired locations and introducing functional mutations at the points of interest. As a first part of the project, we have tested Dicty myosin in comparison with myosin purified from rabbit skeletal muscle, and have shown that structural changes in the force-generating domain of Dicty and rabbit myosin are identical. We then focused on specific elements within the force-generating domain, relay helix and relay loop, as these elements appeared to be crucial for interdomain coupling and force generation. Using time-resolved EPR and FRET, we have developed a spectroscopic approach to determine the conformation of the relay helix. We have also developed a novel technique that we called transient time-resolved FRET [(TR)2FRET], which allowed us to monitor structural changes within the relay helix in real time. We then studied the relationship between the state of the myosin active site (which is determined by the bound ligand) and the structure of the relay helix. To obtain insights about regulatory mechanisms, we have investigated the effect of a mutation that is known to abolish myosin motor function, despite leaving enzymatic activity intact. These experiments revealed important coupling mechanisms between the relay loop and relay helix, providing a structural explanation for the previously observed functional effects and a model for power stroke activation in myosin.