Abstract
Using electron spin resonance, we have studied dynamic structures of myosin neck domain and troponin C by site-directed spin labeling. We observed two broad but distinct orientations of a spin label attached specifically to a single cysteine (cysl56) on the regulatory light chain (RLC) of myosin in relaxed skeletal muscle fibers. The two probe orientations, separated by a 25° axial rotation, did not change upon muscle activation, but orientational distributions became narrower substantially, indicating that a fraction of myosin heads undergoes a disorder-to-order transition of the myosin light chain domain upon force generation and muscle contraction. These results provide insight into the mechanism how myosin heads move their domains to translocate an actin filament.
Site-directed spin-labeling was achieved by cy steine residues of human cardiac troponin C (TnC). Spin dipole-dipole interaction showed that free TnC undergoes a global structural change (extended-to-compact) by Ca2+ or Mg2+. The spectra from the spin labels at N-terminal half domain were broad and almost identical in parallel and perpendicular orientations of fiber, suggesting that the N-terminal of TnC molecule is flexible or disoriented with respect to the filament axis. We also succeeded, for the first time, in fixing the newly-synthesized bifunctional spin label rigidly on TnC molecule in solution (either in ±Ca2+), giving a promise that we can determine the precise coordinate of the spin principal axis on protein surface.
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References
T. Arata, Orientation of spin-labeled light chain 2 of myosin heads in muscle fibers, J. Mol. Biol. 214, 471–478 (1990).
T. Arata, The use of spin probes (Chapter 9), in: Current Methods in Muscle Physiology, edited by H. Sugi (Oxford University Press, 1998), pp. 223–239.
T. Arata, and H. Shimizu, Spin-label study of actin-myosin-nucleotide interactions in contracting glycerinated muscle fibers, J. Mol. Biol. 151, 411–437 (1981).
J. E. Baker, I. Brust-Mascher, S. Ramachandran, L. E. LaConte, and D. D. Thomas, A large and distinct rotation of the myosin light chain domain occurs upon muscle contraction, Proc. Natl. Acad. Sci. U S A. 95, 2944–2949 (1998).
C. M. Slupsky, and B. D. Sykes, NMR solution structure of calcium-saturated skeletal muscle troponin C, Biochemistry 34, 15953–15960 (1995).
S. R. Hubbard, K. O. Hodgson, and S. Doniach, Small-angle x-ray scattering investigation of the solution structure of troponin C, J. Biol. Chem. 263, 4151–4158 (1988).
T. Fujisawa, T. Ueki, and S. Iida, Structural change of the troponin C molecule upon Ca2+ binding measured in solution by the X-ray scattering technique, J. Biochem (Tokyo) 105, 377–383 (1989)
D. A. Martyn, M. Régnier, D. Xu, and A. M. Gordon, Ca2+-and cross-bridge-dependent changes in N-and C-terminal structure of troponin C in rat cardiac muscle. Biophys. J. 80, 360–370 (2001).
H. C. Li, and P. G. Fajer, Orientational changes of troponin C associated with thin filament activation, Biochemistry 33, 14324–14332 (1994).
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Arata, T. et al. (2003). Orientation and Motion of Myosin Light Chain and Troponin in Reconstituted Muscle Fibers as Detected by ESR with a New Bifunctional Spin Label. In: Sugi, H. (eds) Molecular and Cellular Aspects of Muscle Contraction. Advances in Experimental Medicine and Biology, vol 538. Springer, Boston, MA. https://doi.org/10.1007/978-1-4419-9029-7_26
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DOI: https://doi.org/10.1007/978-1-4419-9029-7_26
Publisher Name: Springer, Boston, MA
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