Energetics of the equilibrium between two nucleotide-free myosin subfragment 1 states using fluorine-19 NMR

Abstract

A new fluorine-containing reagent was synthesized and used to specifically label the reactive sulfhydryl (SH1) of myosin subfragment 1 (S-1). The labeled S-1 (S-1-CF3) demonstrates activated Ca and Mg ATPase activities relative to S-1 and a lower potassium EDTA ATPase activity. Maximal effect is obtained with the modification of 1 thiol/S-1. The 19F NMR spectrum of S-1-CF3 contains only 1 resonance with a line width of 110 Hz, which implies a rotational correlation time of 2.3 .times. 10-7 s. The chemical shift of this resonance is sensitive to temperature, pH, ionic strength, and nucleotides bound in the active site. The temperature dependence of the chemical shift clearly indicates 2 limiting states for the S-1-CF3 with a highly temperature-dependent equilibrium between 5 and 40.degree. C. The low-temperature state appears to be identical with the state resulting from the binding of Mg.cntdot.ADP or Mg.cntdot.AMPPNP at 25.degree. C. The energetics of the conformational change were studied under various conditions. At pH 7 in 25 mM cacodylate, 0.1 M KCl and 1 mM EDTA, .DELTA.H.degree. = 30 kcal/mol and .DELTA.S.degree. = 105 cal deg-1 mol-1. A decrease in pH to 6.5 results in an increased population of the low-temperature state with .DELTA.H.degree. = 31 kcal/mol and .DELTA.S.degree. = 107 cal deg-1 mol-1. Similarly, the low-temperature state is favored by low ionic strength. In 5.8 mM piperazine-N,N''-bis(2-ethanesulfonic acid) and 1 mM EDTA (pH 7), .DELTA.H.degree. = 8 kcal/mol and .DELTA.S.degree. = 27 cal deg-1 mol-1. 19F NMR spectra of S-1-CF3 were also obtained in D2O solution with 30% ethylene glycol at pH 7.1. Increasing concentrations of ethylene glycol progressively stabilize the high-temperature state.