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J. Biochem, 2004, Vol. 136, No. 1 39-47
© 2004 The Japanese Biochemical Society

CELL

Fluorescence Resonance Energy Transfer between Points on Actin and the C-Terminal Region of Tropomyosin in Skeletal Muscle Thin Filaments

Masao Miki1,*, Hong Hai1, Kimiko Saeki2, Yuji Shitaka1, Ken-Ichi Sano3, Yuichiro Maéda3 and Takeyuki Wakabayashi4

1 Department of Applied Chemistry and Biotechnology, Fukui University, 3-9-1 Bunkyo, Fukui 910-8507; 2 Department of Physics, School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033; 3 Riken Harima Institute at Spring8, Mikazuki-cho, Sayo, Hyogo 679-5143; 4 Department of Biosciences, School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551

Fluorescence resonance energy transfer between points on tropomyosin (positions 87 and 190) and actin (Gln-41, Lys-61, Cys-374, and the ATP-binding site) showed no positional change of tropomyosin relative to actin on the thin filament in response to changes in Ca2+ concentration (Miki et al. (1998) J. Biochem. 123, 1104–1111). This is consistent with recent electron cryo-microscopy analysis, which showed that the C-terminal one-third of tropomyosin shifted significantly towards the outer domain of actin, while the N-terminal half of tropomyosin shifted only a little (Narita et al. (2001) J. Mol. Biol. 308, 241–261). In order to detect any significant positional change of the C-terminal region of tropomyosin relative to actin, we generated mutant tropomyosin molecules with a unique cysteine residue at position 237, 245, 247, or 252 in the C-terminal region. The energy donor probe was attached to these positions on tropomyosin and the acceptor probe was attached to Cys-374 or Gln-41 of actin. These probe-labeled mutant tropomyosin molecules retain the ability to regulate the acto-S1 ATPase activity in conjunction with troponin and Ca2+. Fluorescence resonance energy transfer between these points of tropomyosin and actin showed a high transfer efficiency, which should be very sensitive to changes in distance between probes attached to actin and tropomyosin. However, the transfer efficiency did not change appreciably upon removal of Ca2+ ions, suggesting that the C-terminal region of tropomyosin did not shift significantly relative to actin on the reconstituted thin filament in response to the change of Ca2+ concentration.

* To whom correspondence should be addressed. Tel: +81-776-27-8786, Fax: +81-776-27-8747, E-mail: masao{at}acbio2.acbio.fukui-u.ac.jp


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Troponin Is a Potential Regulator for Actomyosin Interactions
J. Biochem., February 1, 2006; 139(2): 289 - 293.
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