Ca2+- and S1-induced conformational changes of reconstituted skeletal muscle thin filaments observed by fluorescence energy transfer spectroscopy: structural evidence for three States of thin filament.
Rabbit skeletal muscle alpha-tropomyosin (Tm) and the deletion mutant (D234Tm) in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing [Landis et al. (1997) J. Biol. Chem. 272, 14051-14056] were used to investigate the interaction between actin and tropomyosin or actin and troponin (Tn) by means of fluorescence resonance energy transfer (FRET). FRET between Cys-190 of D234Tm and Gln-41 or Cys-374 of actin did not cause any significant Ca2+-induced movement of D234Tm, as reported previously for native Tm [Miki et al. (1998) J. Biochem. 123, 1104-1111]. FRET did not show any significant S1-induced movement of Tm and D234Tm on thin filaments either. The distances between Cys-133 of TnI, and Gln-41 and Cys-374 of actin on thin filaments reconstituted with D234Tm (mutant thin filaments) were almost the same as those on thin filaments with native Tm (wild-type thin filaments) in the absence of Ca2+. Upon binding of Ca2+ to TnC, these distances on mutant thin filaments increased by approximately 10 A in the same way as on wild-type thin filaments, which corresponds to a Ca2+-induced conformational change of thin filaments [Miki et al. (1998) J. Biochem. 123, 324-331]. The rigor binding of myosin subfragment 1 (S1) further increased these distances by approximately 7 A on both wild-type and mutant thin filaments when the thin filaments were fully decorated with S1. This indicates that a further conformational change on thin filaments was induced by S1 rigor-binding (S1-induced or open state). Plots of the extent of S1-induced conformational change vs. molar ratio of S1 to actin showed that the curve for wild-type thin filaments is hyperbolic, whereas that for mutant thin filaments is sigmoidal. This suggests that the transition to the S1-induced state on mutant thin filaments is depressed with a low population of rigor S1. In the absence of Ca2+, the distance also increased on both wild-type and mutant thin filaments close to the level in the presence of Ca2+ as the molar ratio of S1 to actin increased up to 1. The curves are sigmoidal for both wild-type and mutant thin filaments. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding. For mutant thin filaments, the transition from the closed state to the open state in the presence of ATP is strongly depressed, which results in the inhibition of acto-myosin ATPase even in the presence of Ca2+. The present FRET measurements provide structural evidence for three states of thin filaments (relaxed, Ca2+-induced or closed, and S1-induced or open states) for the regulation mechanism of skeletal muscle contraction.
Hai
Hong
H
Department of Applied Chemistry and Biotechnology, Fukui University, Bunkyo, Fukui 910-8507, Japan.
Sano
Ken-Ichi
K
Maeda
Kayo
K
Maéda
Yuichiro
Y
Miki
Masao
M
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004735:Energy Transfer; D017353:Gene Deletion; D015335:Molecular Probes / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry; D009154:Mutation; D015879:Myosin Subfragments / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin / Q000378:metabolism*; D019210:Troponin I / Q000378:metabolism*
D000199:Actins; D015335:Molecular Probes; D015879:Myosin Subfragments; D014335:Tropomyosin; D019210:Troponin I; D002118:Calcium