Orientational changes of troponin C associated with thin filament activation.
We have used electron paramagnetic resonance to describe the orientational changes of troponin C (TnC) accompanying muscle activation by Ca2+. Rabbit skeletal TnC was labeled with maleimide spin label (MSL) at Cys-98 and reconstituted into an oriented skinned muscle fiber. About 70% of endogenous troponin C was replaced with labeled TnC, with a concomitant recovery of 80-90% of muscle tension. The nanosecond domain mobility present in solution, as determined from the EPR spectra of randomized samples, is fully inhibited in the reconstituted fibers. The orientational analysis revealed a bimodal orientational distribution of TnC in the absence Ca2+ and attached myosin heads. One of the components is well-ordered with its probe axis inclined at 22 degrees to the fiber axis, while the other is more disordered and inclined at 58 degrees. Ca2+ and/or cross-bridge binding significantly disordered the labeled domain and increased the average probe axis angle by 20-30 degrees away from the fiber axis. The order for the magnitude of angular tilt was Ca2+ < myosin cross-bridges < Ca2+ and cross-bridges. Thus, TnC exists in many different orientational conformations depending on which ligand is bound. We believe that these conformations reflect different activation mechanisms by Ca2+ and cross-bridge binding.
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Institute of Molecular Biophysics, Florida State University, Tallahassee 32306.
Fajer
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D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D003497:Cyclic N-Oxides; D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000187:drug effects; D009210:Myofibrils / Q000378:metabolism; D009218:Myosins / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D013113:Spin Labels; D014336:Troponin / Q000737:chemistry* / Q000378:metabolism; D019209:Troponin C
D003497:Cyclic N-Oxides; D013113:Spin Labels; D014336:Troponin; D019209:Troponin C; C017480:4-maleimido-2,2,6,6-tetramethylpiperidinooxyl; D009218:Myosins; D003545:Cysteine; D002118:Calcium