Probing the conformational states of the SH1-SH2 helix in myosin: A cross-linking approach

Previous biochemical studies have shown that the SH1 (Cys707) and SH2 (Cys6 97) groups on rabbit skeletal myosin subfragment 1 (S1) can be cross-linked by using reagents of different crosslinking lengths. In the presence of nu cleotide, this cross-linking is accelerated. In the crystal structure of S1 , the SH1 rind SH2 residues are located on an alpha-helix, 19 Angstrom apar t. Thus, the cross-linking results could be indicative of helix melting or increased flexibility in the presence of nucleotides. Nucleotide-induced ch anges in this region were examined in this study by monitoring the cross-li nking of SH1 and SH2 on S1 with dimaleimide reagents of spans ranging from 5 to 15 Angstrom. A method was devised to directly measure the kinetic effe cts of nucleotides on the rates of cross-linking reactions. The slow and re agent-insensitive rates of the SH1-SH2 cross-linking in the absence of nucl eotides reveal that the equipartitioning of the SH1-SH2 helix among states with different SH1-SH2 separations occurs infrequently. Tn the presence of MgADP, MgATP, and MgATP gamma S, the rates of SH1 and SH2 cross-linking wer e increased similar to 2-7-fold for the shortest reagent (5-8 Angstrom). Ra te accelerations were much greater for the longer reagents (9-15 Angstrom): 40-50-fold for MgADP, 25-40-fold for MgATP, and 80-270-fold for MgATP gamm a S. To account for any nucleotide-dependent differences in the reactivitie s of the reagents toward SH2, the rates of monofunctional SH2 modification on SH1-labeled S1 were also measured for each reagent. These experiments sh owed that the nucleotide-induced increases in the rates of SH2 modification were similar for ail of the reagents. Thus, the changes observed in the cr oss-linking rates are due not only to the type of nucleotide bound in the a ctive site but also to the span of the cross-linking reagent. These finding s are interpreted in terms of nucleotide-induced shifts in the equilibria a mong conformational states of the SH1-SH2 helix.