Conformational changes of the troponin-tropomyosin complex on F-actin observed by fluorescence resonance energy transfer measurements

Contraction of vertebrate striated muscle is regulated by the strong Ca2+-dependent interaction among troponin (Tn), tropomyosin (Tm), and actin on the thin filament. Using fluorescence resonance energy transfer (FRET), the interactions between Tm and the Tn complex or between Tm and the Tn subunit, TnI or TnC, with or without other troponin subunits, were characterized in the presence or absence of F-actin and Ca2+ ions. Cys-190 of Tm was selectively labeled with the acceptor probe, 4-dimethylaminophenylazophenyl 4'-maleimide. Troponin was selectively labeled at position 9 or 133 of TnI and position 98 of TnC with a donor probe, 5-(2-iodoacetylaminoethyl)aminonaphthalene 1-sulfonic acid. FRET measurements indicate that the interaction between TnI and Tm alone is very weak, but that in the presence of F-actin, TnI binds to the proper binding site on Tm even in the absence of TnT. The distances between Cys-190 of Tm on F-actin and Cys-9 or Cys-133 of TnI or Cys-98 of TnC in the reconstituted Tn were determined to be 52.8, 53.7, Angstrom and 56.5 Angstrom, respectively, in the absence of Ca2+, indicating that the TnI-TnC complex, the globular portion of Tn, is located near Cys-190 of Tm on the reconstituted thin filaments. Upon binding of Ca2+ to TnC, these distances increased by 5.6 and 1.4 Angstrom or decreased by 5.4 Angstrom, respectively. These Ca2+-induced changes in Tn-Tm seem to occur only when F-actin is present, suggesting that the stable complex formation of TnI with the outer domain of F-actin upon removal of Ca2+ is a very important event during inhibition.