Actin and Temperature Effects on the Cross-Linking of the SH1-SH2 Helix in Myosin Subfragment 1

Past biochemical work on myosin subfragment 1 (S1) has shown that the bent α-helix containing the reactive thiols SH1 (Cys 707) and SH2 (Cys 697) changes upon nucleotide and actin binding. In this study, we investigated the conformational dynamics of the SH1-SH2 helix in two actin-bound states of myosin and examined the effect of temperature on this helix, using five cross-linking reagents that are 5–15 Å in length. Actin inhibited the cross-linking of SH1 to SH2 on both S1 and S1.MgADP for all of the reagents. Because the rate of SH2 modification was not altered by actin, the inhibition of cross-linking must result from a strong stabilization of the SH1-SH2 helix in the actin-bound states of S1. The dynamics of the helix is also influenced by temperature. At 25°C, the rate constants for cross-linking in S1 alone are low, with values of ∼0.010 min −1 for all of the reagents. At 4°C, the rate constants, except for the shortest reagent, range between 0.030 and 0.070 min −1. The rate constants for SH2 modification in SH1-modified S1 show the opposite trend; they increase with the increases in temperature. The greater cross-linking at the lower temperature indicates destabilization of the SH1-SH2 helix at 4°C. These results are discussed in terms of conformational dynamics of the SH1-SH2 helix.