Orientation and motion of myosin light chain and troponin in reconstituted muscle fibers as detected by ESR with a new bifunctional spin label.
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 (cys156) on the regulatoy light chain (RLC) of myosin in relaxed skeletal muscle fibers. The two probe orientations, separated by a 25 degrees 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 cysteine 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.
Arata
Toshiaki
T
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Nakamura
Motoyoshi
M
Akahane
Hidenobu
H
Aihara
Tomoki
T
Ueki
Shoji
S
Sugata
Kazunori
K
Kusuhara
Hiroko
H
Morimoto
Masashi
M
Yamamoto
Yukio
Y
D000199:Actins / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000737:chemistry; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D018994:Myosin Light Chains / Q000737:chemistry* / Q000378:metabolism; D009218:Myosins / Q000737:chemistry; D016011:Normal Distribution; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013113:Spin Labels; D014336:Troponin / Q000737:chemistry*; D019209:Troponin C / Q000737:chemistry
D000199:Actins; D018994:Myosin Light Chains; D013113:Spin Labels; D014336:Troponin; D019209:Troponin C; D003553:Cystine; D009218:Myosins; D008274:Magnesium; D003545:Cysteine; D002118:Calcium