Conformational flexibility of the Cys 697-Cys 707 segment of myosin subfragment-1. Distance distributions by frequency-domain fluorometry

The separation between Cys 697 (SH₁) and Cys 707 (SH₂) of the heavy chain of myosin subfragment-1 was previously measured by fluorescence resonance energy transfer with a donor linked to SH₁ and an acceptor to SH₂. In the present study the distribution of the distances between the two thiols was recovered from frequency-domain fluorometry. In the native state and in the presence of ligands such as MgADP, pyrophosphate, orthovanadate (V_i) and actin, we found wide distributions of the separations between SH₁ and SH₂ (11-16 Å) comparable to that found in the random-coil state (20 Å). These results suggest that the SH₁-SH₂ segment has a high degree of conformational flexibility even in native S1. The flexibility is not much affected by the physiological state of S1. However, the ligands MgADP, V_i and MgADP + V_i decrease significantly the mean SH₁±SH₂ distance from 27 to 17 Å with the effect of MgADP + V_i being the most pronounced. The anisotropy decay of donor-labeled S1 is biphasic with two rotational correlation times. The long component is decreased by these ligands from 289 to 93 ns, suggesting a more compact symmetric structure of S1 in the presence of the ligands. The complex S1 (MgADP)V_i has been shown to be a stable analogue of S1(MgADP)P_i, an unstable intermediate that is generated in the actomyosin ATPase cycle during muscle contraction. Since the power stroke of muscle is accompanied by release of P_i from S1(MgADP)P_i, the present results are consistent with a model in which force generation can be accompanied by transition of S1 from a highly symmetric or compact structure to a more extended structure.