Temperature-modulated binding of ADP and adenyl-5'-yl imidodiphosphate to myosin subfragment 1 studied by calorimetric titration.

A calorimetric titration method was used to study the binding of ADP and adenyl-5"yl imidodiphosphate (AMP-PNP) to myosin subfragment 1 in 0.1 M KC1 containing 0.01 M MgClz and 0.02 M Tris/HCl (pH 8.0) at 4 K intervals between 4 and 24 "C.Binding constant (K), stoichiometry of binding (n), and heat of reaction (AH), as well as their temperature dependence were determined.Both ADP and AMP-PNP strongly bind to subfragment 1 (K = lo6 M-') with a 1:l ratio (n = 0.87) and AH c 0. Variation of AH with temperature is very large relative to that of the binding constant.For ADP binding, the (-AH)/temperature curve has a rather sharp deflection point at 13 "C, above which the slope (AC,) decreases from -2.0 to -0.4 kJ K" mol".AMP-PNP binding is less exothermic and hence more entropic in nature than ADP binding as indicated by much less negative values of AH for the former compared to the latter despite similar values of K.At temperatures below 12 "C, the entropy change for AMP-PNP binding is strongly positive in contrast with that forADP binding which is consistently negative over the temperature range studied.The deflection point of AC, is shifted to a higher temperature of 17 "C, and AC, values below and above this point are more negative than the corresponding values for ADP binding.These results suggest that subfragment 1 exists in two conformational states, the equilibrium between which is sharply temperature-dependent, and that the transition between these states modifies the thermodynamics of nucleotide binding.