Differential Behavior of Two Cysteine Residues on the Myosin Head in Muscle Fibers

We have previously shown that the orientation of (iodoacetamido)tetramethylrhodamine labels on SHj thiol of S-l moieties changes when MgADP is added to the fibers in rigor [Borejdo, J., Assulin, O., Ando, T., & Putnam, S. (1982) J, Mol Biol. 158, 391-414. Burghardt, T. P., Ando, T., & Borejdo, J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7515-7519]. Here we report the results of experiments in which the SH₂ of S-l was labeled with maleimidorhodamine. The specificity of modification of thiols was checked by measuring the stoichiometry of attached dye, by determining the extent of the decrease in EDTA(K⁺)- and Ca²⁺-ATPase activities, and by the localization of the dyes on peptides containing SH₁ and/or SH₂. Labeled S-1 was diffused into single glycerinated fibers of rabbit psoas muscle, and the orientation of chromophores was measured by fluorescence detected dichroism. The dye attached to SH1 was oriented at 650 with respect to the fiber axis in rigor and at 51 ° in the presence of MgADP, regardless of whether SH₂ was modified or not. The dye on SH2 was oriented near 42° both in the presence and in the absence of ADP, regardless of whether SH₁ was modified or not. Our results show that rhodamine oriented differently when attached to SH₂ compared with when attached to SH₁ and that in the former placement it was not sensitive to MgADP. We think this indicates that the SH₂-containing region has a mobility different from that of the SH₁-containing region, i.e., that this is evidence for internal flexibility of S-1.