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 (Cys697) groups on rabbit skeletal myosin subfragment 1 (S1) can be cross-linked by using reagents of different cross-linking lengths. In the presence of nucleotide, this cross-linking is accelerated. In the crystal structure of S1, the SH1 and SH2 residues are located on an alpha-helix, 19 A apart. Thus, the cross-linking results could be indicative of helix melting or increased flexibility in the presence of nucleotides. Nucleotide-induced changes in this region were examined in this study by monitoring the cross-linking of SH1 and SH2 on S1 with dimaleimide reagents of spans ranging from 5 to 15 A. A method was devised to directly measure the kinetic effects of nucleotides on the rates of cross-linking reactions. The slow and reagent-insensitive rates of the SH1-SH2 cross-linking in the absence of nucleotides reveal that the equipartitioning of the SH1-SH2 helix among states with different SH1-SH2 separations occurs infrequently. In the presence of MgADP, MgATP, and MgATPgammaS, the rates of SH1 and SH2 cross-linking were increased approximately 2-7-fold for the shortest reagent (5-8 A). Rate accelerations were much greater for the longer reagents (9-15 A): 40-50-fold for MgADP, 25-40-fold for MgATP, and 80-270-fold for MgATPgammaS. To account for any nucleotide-dependent differences in the reactivities of the reagents toward SH2, the rates of monofunctional SH2 modification on SH1-labeled S1 were also measured for each reagent. These experiments showed that the nucleotide-induced increases in the rates of SH2 modification were similar for all of the reagents. Thus, the changes observed in the cross-linking rates are due not only to the type of nucleotide bound in the active site but also to the span of the cross-linking reagent. These findings are interpreted in terms of nucleotide-induced shifts in the equilibria among conformational states of the SH1-SH2 helix.
Nitao
L K
LK
Department of Chemistry and Biochemistry, Molecular Biology Institute, University of California, Los Angeles 90095, USA.
Reisler
E
E
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D016658:Psoas Muscles; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism
D003432:Cross-Linking Reagents; D008301:Maleimides; D015879:Myosin Subfragments; D013438:Sulfhydryl Compounds; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C011328:N,N'-4-phenylenedimaleimide; D003545:Cysteine