Differential behavior of two cysteine residues on the myosin head in muscle fibers

Abstract

We have previously shown that the orientation of (iodoacetamido)tetramethylrhodamine labels on SH1 thiol of S-1 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-1 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 65 degrees with respect to the fiber axis in rigor and at 51 degrees in the presence of MgADP, regardless of whether SH2 was modified or not. The dye on SH2 was oriented near 42 degrees 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.

On the origin and transmission of force in actomyosin subfragment 1.

“…Monofunctional attachment to Cys-707 has a variable effect, depending on substrate conformation; some crosslinkers affect, but do not extinguish, ATPase (39). The emission anisotropy of fluorophores attached to Cys-697 is much lower than that of fluorophores attached to Cys-707 (suggesting that Cys-697 is more mobile) (12,67). When ADP binds, the distance from the 50-kDa-20-kDa connector to Cys-707 changes little, but that to Cys-697 significantly increases (12).…”

Specific cross-linking of the SH1 thiol of skeletal myosin subfragment 1 to F-actin and G-actin

“…However, no production of the 140-kDa derivative could be observed in the reaction mixture containing either MBS-G-actin or MBS-Factin (Figure 5A,B, lanes c and e). In contrast, the coupling of the MBS-actins to S-l specifically blocked at the SH2 thiol with the fluorescent tetramethylrhodamine group (Kasprzak et al, 1989; Miyanishi & Borejdo, 1989) generated a fluorescent 140-kDa band, the intensity of which increased when the MBS-G-actin-S-1 cross-linking was performed in the presence of Mg2+ ions (Figure 6). An identical result was obtained by reacting MBS-G-actin with S-l intramolecularly cross-linked with pPDM between Cys-540 and SH2 the sulfhydryl of either Cys-697 (SH2) or Cys-540 did not contribute to the cross-linking process and the new 140-kDa species was resulting mainly from a specific covalent coupling of the SHI site of the S-l heavy chain (Cys-707) to the maleimidobenzoyl group of MBS-actin.…”

Nucleotide-induced changes in the interaction of myosin subfragment 1 with actin: detection by antibodies against the N-terminal segment of actin

“…41, 1991 evidence for nucleotide-induced changes in these proteins (Cooke, 1986). This evidence includes among others the perturbation of S-1 tryptophan fluorescence (Bagshaw & Trentham, 1974), changes in the environment of extrinsic probes attached to the reactive cysteine residues on S-1, SHI (Seidel & Gergely, 1971;Aguirre et al, 1986;Miyanishi & Borejdo, 1989;Fajer et al, 1990;Ajtai at al., 1990), and SH2 (Ajtai & Burghardt, 1989), changes in the chemical reactivities of these residues [e.g., see Doung and ] and their distance from each other (Rajasekharan et al, 1989;Dalbey et al, 1983;Garland et al, 1988), alterations in the crosslinking of S-1 to actin (Yamomoto, 1989) and in the conformation of the cross-linked complex (Duong & Reisler, 1989), transitions in the state of the actin-bound nucleotide (Yanagida, 1981), etc. In most cases, these observations report on local changes in actin and S-1, and it is yet to be clarified which contact sites at the actomyosin interface are impacted by nucleotides.…”

“…In agreement with this observation, the weakly bound S-1 was shown also to be mobile on a microsecond time scale (Svensson & Thomas, 1986;Bernett & Thomas, 1989) and accessible to chemical and proteolytic probes (Duong & Reisler, 1989). Clearly, the environment of the S H I cysteine on S-1 is changed upon binding of ATP or even ADP to actomyosin (Miyanishi & Borejdo, 1989;Fajer et al, 1990;Ajtai et al, 1990). Yet, it remains to be determined whether the SHI site is an actual component of the actomyosin interface (Suzuki et al, 1987;Keane et al, 1990;Chase et al, 1991) or is just located along the communication pathway between the nucleotide and actin binding sites on myosin (Botts et al, 1984).…”