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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. [ABSTRACT FROM PUBLISHER]

Rabbit skeletal muscle βa-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, 14061–14056) were used to investigate the interaction between actin and tro-pomyosin 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 fornative Tm (Miki et al. (1998) J. Biochem. 123, 1104–1111). FRET did not show any significant Sl-induced movement of Tm and D234Tm on thin filaments either. The distances between Cys-133 of Tnl, 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-10 A in the same way as on wild-type thin filaments, which corresponds to a Ca2+induced conformational change of thin filaments [Mild et al (1998) J. Biochenu 123, 324–331]. The rigor binding of myosin subfragment 1(Sl) further increased these distances by -7 A on both wild-type and mutant thin filaments when the thin filaments were fully decorated with Sl. This indicates that a further conformational change on thin filaments was induced by Sl rigor-binding (Sl-induced or open State). Plots of the extent of Sl-induced conformational change vs. molar ratio of Sl 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 Sl-induced state on mutant thin filaments is depressed with a low population of rigor Sl. 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 Sl to actinincreased 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 Sl 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 Sl-induced or open State) for the regulation mechanism of skeletal muscle contraction. [ABSTRACT FROM AUTHOR]

Two isoforms of lobster muscle tropomyosin, a fast muscle type, fTm, and a slow muscle type, sTm1, are identical except for 15 residues within the region of amino acids 39–80, which corresponds to exon 2 of the tropomyosin genes of many phyla. Although the difference in the sequence does not include the terminal regions, the two isoforms are extremely different in viscosity, which is a good measure of the head-to-tail interaction strength and should be dependent on the conformation of the terminal 7–9 residues. To determine the influence of amino-acid replacements in the internal region on the overall conformation and the functional properties of the molecule, we compared the physical properties of the two isoforms and their interactions with other proteins, such as actin and myosin subfragment 1 (S1). Limited proteolysis by trypsin and chymotrypsin showed that sTm1 is more susceptible than fTm at the sites outside the region with the replaced residues. Compared with fTm, sTm1 showed higher viscosity, had a higher actin affinity, and inhibited acto-S1 ATPase to a greater extent. Finally, the binding isotherm of S1-ADP to actin-sTm1 is less sigmoidal than that to actin-fTm. These results indicate that the amino-acid replacements in the internal region alter the conformation and the physical properties of the entire molecule as well as its interactions with actin and myosin. [ABSTRACT FROM AUTHOR]

Fluorescence resonance energy transfer (FRET) spectroscopy has been used to determine spatial relationships between residues on tropomyosin and actin in reconstituted muscle thin filament, and to detect a positional change of tropomyosin relative to actin on the thin filament in the presence and absence of Ca2+ ions. In addition to Cys-190 which is a single cysteine residue in rabbit skeletal muscle α-tropomyosin, a new site, Cys-87 which is a unique cysteine residue in a mutant α-tropomyosin, was labeled with a resonance energy donor molecule, 5-(2-iodoacetylaminoethyl)aminonaphthalene 1-sulfonic acid (IAED-ANS). On the other hand, Gln-41, Lys-61, Cys-374, and the ATP-binding site of actin were selectively labeled with acceptor probes: fluorescein cadaverine, fluorescein 5-isothiocyanate, 4-dimethyl-aminophenylazophenyl 4'-maleimide, and TNP-ATP (or TNP-ADP), respectively. The distances between probes attached to position 87 of the mutant tropomyosin and Gln-41, Lys-61, Cys-374, or the nucleotide-binding site of actin on the reconstituted thin filament in the presence of Ca2+ ion were measured to be 43.2, 49.7, 45.4, and 35.2 A, respectively, and the distance between probes attached to position 190 of tropomyosin and Gln-41 or the nucleotide-binding site of actin were 51.6 and 43.1 Å, respectively. The transfer efficiencies between these donor and acceptor molecules were large, so that the efficiency should be very sensitive to changes in distance between probes attached to tropomyosin and actin. However, the transfer efficiency did not change appreciably upon removal of Ca2+ ions, suggesting that tropomyosin does not change its position on the reconstituted thin filament in response to a change in Ca2+ ion concentration. The present results do not support the notion of tropomyosin movement on skeletal muscle thin filaments as proposed in the steric blocking theory. [ABSTRACT FROM AUTHOR]