ssaying the myosin super-relaxed state across muscle types, cells and proteins for understanding muscle biology and use in drug discovery.

The myosin super-relaxed (SRX) state is a biochemical and structural conformation of myosin that modulates contractility and energy expenditure and is in equilibrium with the disordered relaxed (DRX) state of myosin, which can hydrolyze ATP to produce force. The proportion of myosin SRX-DRX states is perturbed in various muscle disorders, and myosin SRX-DRX states have become a promising drug target. There are many approaches that can be used to interrogate myosin conformations, including X-ray diffraction, stopped-flow kinetics and electron microscopy. These techniques are highly informative but necessitate highly skilled researchers and specialist equipment, limiting wider uptake and accessibility. For this reason, we provide a set of protocols detailing established assays to measure biochemically defined myosin SRX-DRX states in skeletal muscle, cardiac muscle, induced pluripotent stem cell-derived cardiomyocytes, myofibrils, reconstituted thick filaments and isolated molecular motors by using a simple chase assay incorporating a fluorescent ATP analogue: methylanthraniloyl (Mant)-ATP. The Mant-ATP assay provides a biochemical measure of myosin states that is distinct from assays that are used to visualize myosin structure directly. These Mant-ATP assays have various protocol lengths, ranging from 1-2 d for preparation and 30 min to run an experiment. With this set of protocols, we make the Mant-ATP assay accessible to those working in biochemistry, muscle physiology and cell biology. At the end of this protocol, users should be able to ascertain a clean fluorescent decay trace that can be fit to define the ratio of SRX/DRX myosin in their sample of choice.