Characterization of the ethenoadenosine diphosphate binding site of myosin subfragment. 1. Energetics of the equilibrium between two states of nucleotide.cntdot.S1 and vanadate-induced global conformation changes detected by energy transfer

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Abstract
The fluorescence decay of 1,N6-ethenoadenosine diphosphate (.epsilon.ADP) bound to myosin subfragment 1 (S1) was studied as a function of temperature. The decay was biexponential, and the two lifetimes were quenched relative to the single lifetime of free .epsilon.ADP. The temperature dependence of the fractional intensities of the decay components showed two states of the S1.cntdot..epsilon.ADP complex. At pH 7.5 in 30 mM TES, 60 mM KCl, and 3 mM MgCl2, the equilibrium constant for the conversion of the low-temperature state (S1L.cntdot..epsilon.ADP) to the high-temperature state (S1H.cntdot..epsilon.ADP) was 40 at physiological temperatures, and .DELTA.H.degree. = 13 kcal.cntdot.mol-1 and .DELTA.S.degree. = 49 cal.cntdot.deg-1.cntdot.mol-1. At 10.degree. C the equilibrium constant of S1 for .epsilon.ADP was 5, indicating that S1H.cntdot..epsilon.ADP was the dominant state, and that for the vanadate complex .epsilon.ADP.cntdot.Vi was 0.7, suggesting that in S1.cntdot..epsilon.ADP.cntdot.Vi the dominant state of the S1-nucleotide complex was converted from S1H.cntdot..epsilon.ADP to S1L.cntdot..epsilon.ADP. The single rotational correlation time of bound .epsilon.ADP at 10.degree. C decreased from 107 ns in S1.cntdot..epsilon.ADP to 74 ns in S1+.cntdot..epsilon.ADP.cntdot.Vi. Conversion of the binary complex to the ternary vanadate complex resulted in a 3-.ANG. decrease in the energy transfer distance between bound .epsilon.ADP and N-[4-(dimethylamino)-3,5-dinitrophenyl]maleimide attached to SH1 and a decrease of the average distance between bound .epsilon.ADP and bound Co2+ from 12.6 to 8.3 .ANG.. On the assumption that S1+.cntdot.ADP.cntdot.Vi is a good stable analogue of S1.cntdot.ADP.cntdot.Pi, it is suggested that the transition S1L.cntdot..epsilon.ADP .fwdarw. S1H.cntdot..epsilon.ADP is involved in the power stroke of the contractile cycle. The structural changes that S1 experience during this transition may include a small increase in dimensional asymmetry and movements of two regions of the heavy chain toward the adenine-binding site.

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