Studies of Ligand-induced Conformational Perturbations in Myosin Subfragment 1

Abstract

The effect of ligand binding on the environment near the SH2 and SH1 thiols in myosin subfragment 1 has been investigated by photocross-linking after specific labeling of these thiols individually with 4-(N-maleimido)benzophenone (MBP).On photolysis, cross-linking occurred from SH2-MBP to the middle BO-kDa segment, and subsequent immunopeptide mapping revealed that the cross-link was made to a peptide stretch 31-32 kDa from the N terminus in the absence of MgADP, whereas in its presence the cross-link oc- curred at about 60-61 kDa from the N terminus.Photolysis of SH1-MBP in the absence of MgADP resulted in a major cross-link to the 27-kDa N-terminal segment and minor cross-links to the 50-kDa middle segment.In the presence of MgADP, no new cross-link occurred but the amount of cross-linking to the 50-kDa segment increased at the expense of the other.Immunopeptide mapping indicated that the regions in the 27-and BO-kDa peptides that were cross-linked to SH1-MBP are at about 14-16 and 55-56 kDa from the N terminus respectively.These results indicate that when nucleotide binds to S 1, SH2 is displaced relative to the 50-kDa segment, whereas the local environment around SH1 does not change significantly because photolysis in the presence of MgADP resulted in a change at the site of cross-linking for SH2-MBP but caused only a redistribution of the relative amounts of the cross-links formed from SH1-MBP.Recent in vitro motility and force measurement studies have established that the subfragment 1 (Sl)' region of myosin alone is capable of supporting ATP-dependent movement and to generate force (1, 2).These findings indicate that structural changes in the S1 region amounting to a displacement of about 4 nm in the axial direction must be occurring (3) when S1 binds and hydrolyzes MgATP and attaches to