The kinetics of a two-state transition of myosin subfragment 1 A temperature-jump relaxation study

AbstractTemperature-jump measurements were carried out on myosin subragment 1 (S1) labeled at Cys-707 with 5-(iodoacetamido)fluorescein (S1-AF). The relaxation was monitored by following the increase in the fluorescence intensity of the attached probe after a jump of 5.8°C. A single relaxation process was observed over a range of final temperatures, and the relaxation time decreased from 16.69 ms at 15°C to 3.91 ms at 27°C. The relaxation results are interpreted in terms of a two-state transition: (S1-AF)L (S1-AF)11, and the observed single relaxation time (τ) equals 1/(k+ + k−). The individual first-order rate constants, k+ and k−, were calculated from τ and the equilibrium constant previously determined. The activation energy was 21.9 kcal/mol for the forward reaction and 9.3 kcal/mol for the reverse reaction, corresponding to an enthalpy value of 12.6 kcal/mol for the two-state transition. The results provide, for the first time, direct kinetic evidence of a two-state transition of S1 in the absence of bound nucleotide, and support a two-state model of unliganded myosin subfragment 1