Structural Kinetics of Troponin during Activation of Skeletal Muscle Fibers by Photolysis of Nitrophenyl-EGTA (NP-EGTA)

Abstract

Mutants of troponin C (TnC) with bifunctional rhodamine (BR) cross-linking pairs of cysteine residues 56 and 63 or 96 and 103, along the C- and E-helices respectively, were exchanged into demembranated fibers from rabbit psoas muscle. Changes in the orientation of the two helices following a sudden increase in [Ca2+] elicited by photolysis of NP-EGTA were monitored by polarized fluorescence. The second- and fourth-rank order parameters, and respectively, that describe the orientation distribution of the BR dipoles with respect to the muscle fiber axis were calculated from the polarized fluorescence intensities. 5 ms after photolysis, when force was only 3% of maximum, the changes in and were 90% complete for the C-helix and 65% for the E-helix. The results reveal the kinetics of the first steps in the signaling pathway in the troponin complex: the [Ca2+] jump induces a fast structural change in the N-terminal lobe of TnC, containing the regulatory Ca2+ sites near the C helix, followed by a structural change in its C-lobe (containing the E helix) that is still faster than force generation. When active force development was inhibited by N-benzyl-p-toluene-sulphonamide (BTS), the kinetics and amplitude of the orientation change in the C-helix were not affected, whereas the orientation change in the E-helix was reduced. The effect of BTS on the kinetics also revealed a component of the E-helix orientation change that is synchronous with force development and accounts for the amplitude decrease, suggesting that myosin head binding to actin completes the structural change of the C-lobe initially triggered by calcium binding to the N-lobe.Supported by Medical Research Council and Wellcome Trust, UK.