Actin and temperature effects on the cross-linking of the SH1-SH2 helix inmyosin subfragment 1

Past biochemical work on myosin subfragment 1 (S1) has shown that the bent alpha-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 Angstrom in length. Actin inhibited t he cross-linking of SH1 to SH2 on both S1 and S1.MgADP for all of the reage nts. Because the rate of SH2 modification was not altered by actin, the inh ibition 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 al so influenced by temperature. At 25 degrees C, the rate constants for cross -linking in S1 alone are low, with values of similar to 0.010 min(-1) for a ll of the reagents. At 4 degrees C, the rate constants, except for the shor test 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 degrees C. These results are discussed in terms of conformational dynamics of the SH1- SH2 helix.