Switch action of troponin on muscle thin filament as revealed by spin labeling and pulsed EPR.
We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to measure the distance between spin labels at Cys(133) of the regulatory region of TnI (TnI133) and a native or genetically substituted cysteine of TnC (TnC44, TnC61, or TnC98). In the +Ca(2+) state, the TnC44-TnI133-T distance was 42 A, with a narrow distribution (half-width of 9 A), suggesting that the regulatory region binds the N-lobe of TnC. Distances for TnC61-TnI133 and TnC98-TnI133 were also determined to be 38 A (width of 12 A) and 22 A (width of 3.4 A), respectively. These values were all consistent with recently published crystal structure (Vinogradova, M. V., Stone, D. B., Malanina, G. G., Karatzaferi, C., Cooke, R., Mendelson, R. A., and Fletterick, R. J. (2005) Proc. Natl Acad. Sci. U.S.A. 102, 5038-5043). Similar distances were obtained with the same spin pairs on a reconstituted thin filament in the +Ca(2+) state. In the -Ca(2+) state, the distances displayed broad distributions, suggesting that the regulatory region of TnI was physically released from the N-lobe of TnC and consequently fluctuated over a variety of distances on a large scale (20-80 A). The interspin distance appeared longer on the filament than on troponin alone, consistent with the ability of the region to bind actin. These results support a concept that the regulatory region of TnI, as a molecular switch, binds to the exposed hydrophobic patch of TnC and traps the inhibitory region of TnI away from actin in Ca(2+) activation of muscle.
Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka 560-0043, Japan.
Tomoki
T
Aihara
Motoyoshi
M
Nakamura
Shoji
S
Ueki
Hideyuki
H
Hara
Masao
M
Miki
Toshiaki
T
Arata
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D018485:Muscle Fibers, Skeletal / Q000737:chemistry* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011817:Rabbits; D013113:Spin Labels; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D013113:Spin Labels; D019209:Troponin C; D019210:Troponin I; D003545:Cysteine; D002118:Calcium