Kinetic Modeling of mant-ATP Turnover to Interpret the Biochemically Defined Myosin SRX State

The fluorescent ATP analog mant-ATP has become a valuable tool for quantifying occupancy of the myosin super-relaxed (SRX) state, a biochemically inactive state of myosin in striated muscle. Interpretation of mant-ATP fluorescence decay kinetics is confounded by inconsistencies in state definitions and kinetic assumptions. Here, we develop a mass-action kinetic model of myosin cross-bridge cycling and mant-ATP turnover to reconcile these discrepancies and provide a mechanistic framework for interpreting SRX measurements. Our model simulates ATP label-chase experiments and demonstrates that conventional double-exponential fitting methods do not directly quantify SRX occupancy. Instead, we show that slow and fast decay phases of mant-ATP fluorescence arise from label redistribution among kinetically distinct states, not state populations in equilibrium. The model resolves several apparent paradoxes identified in recent studies by reproducing experimental observations without requiring SRX and DRX kinetic isolation or implausible equilibrium constants. Simulations further quantify the impact of experimental factors--such as ADP accumulation, photobleaching, and initial rigor state occupancy--on fluorescence kinetics and SRX estimates. These results support a revised framework for SRX quantification and suggest that label-chase experiments must be interpreted using mechanistic models to accurately assess myosin state distributions and transition kinetics. SIGNIFICANCEThe relative occupancy of myosin in the super-relaxed state (SRX) is a key determinant of basal ATP turnover in muscle. Measurement of ATP exchange using the fluorescent analog mant-ATP is used to assess the relative population of myosin in the SRX versus the disordered relaxed (DRX) state. Existing approaches to analyzing data from these experiments use double exponential fits to represent the mant-ATP decay in the chase-phase of the experiment. However, quantitative and mechanistic interpretations based on these analyses remain ambiguous. We present a mechanistic model of myosin ATP turnover that reproduces observed fluorescence decays under defined conditions. The results indicate that conventional interpretations, while qualitatively reasonable, are quantitatively inconsistent, as the observed slow and fast phases are predicted to arise from ligand redistribution rather than distinct equilibrium states. This framework enables rigorous, model-based interpretation of mant-ATP assays to help clarify how myosin kinetics are reflected in mant-ATP loading-chase experiments and experimental conditions may influence apparent SRX kinetics.