TROPONIN REGULATORY INTERACTION ON THE THIN FILAMENT

In order to understand the spatial rearrangements of thin filament proteins during the regulation of muscle contraction, we used fluorescence resonance energy transfer (FRET) to measure Ca 2+ dependent, Actin- Tm(Ac-Tm), myosin-induced distance changes between troponin I (TnI) and troponin C (TnC). We labeled the single Cys-134 of sTnI and residue 10 of sTnC with Alexa C5 488 and 594 maleimide respectively. These fluorescent probes were used as donor and acceptor respectively, for the FRET measurements. We reconstituted a troponin complex which contained the Alexa 488-labeled troponin I(TnI) ,troponin T (TnT), Alexa 594 labeled troponin C (TnC). The magnitude of distance changes at low Ca 2+ is higher than at high Ca 2+ both in presence of Ac-Tm and myosin (S1). The extent of energy transfer was determined by measuring the quenching of the donor fluorescence decay. Steady state fluorescence measurements showed that in the presence of the acceptor, donor fluorescence was quenched 50 � 85% and the quenching was sensitive to calcium, actin-Tm and rigor S1 in the presence of actin-Tm. The results indicate that the distance between these two sites is not fixed, suggesting that the protein regions involved possess considerable segmental flexibility.