Ca2+- and S1-induced movement of troponin T on reconstituted skeletal muscle thin filaments observed by fluorescence energy transfer spectroscopy

Troponin T (TnT) is an essential component of troponin (Tn) for the Ca2+-regulation of vertebrate striated muscle contraction. TnT consists of an extended NH2-terminal domain that interacts with tropomyosin (Tm) and a globular COOH-terminal domain that interacts with Tm, troponin I (TnI), and troponin C (TnC). We have generated two mutants of a rabbit skeletal beta-TnT 25-kDa fragment (59-266) that have a unique cysteine at position 60 (N-terminal region) or 250 (C-terminal region). To understand the spatial rearrangement of TnT on the thin filament in response to Ca2+ binding to TnC, we measured distances from Cys-60 and Cys-250 of TnT to Gln-41 and Cys-374 of F-actin on the reconstituted thin filament by using fluorescence resonance energy transfer (FRET). The distances from Cys-60 and Cys-250 of TnT to Gln-41 of F-actin were 39.5 and 30.0 a, respectively in the absence of Ca2+, and increased by 2.6 and 5.8 a, respectively upon binding of Ca2+ to TnC. The rigor binding of myosin subfragment 1 (S1) further increased these distances by 4 and 5 a respectively, when the thin filaments were fully decorated with S1. This indicates that not only the C-terminal but also the N-terminal region of TnT showed the Ca2+- and S1-induced movement, and the C-terminal region moved more than N-terminal region. In the absence of Ca2+, the rigor S1 binding also increased the distances to the same extent as the presence of Ca2+ when the thin filaments were fully decorated with S1. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding both in the presence and absence of Ca2+. However, plots of the extent of S1-incuced conformational change vs. molar ratio of S1 to actin showed hyperbolic curve in the presence of Ca2+ but sigmoidal curve in the absence of Ca2+. FRET measurement of the distances from Cys-60 and Cys-250 of TnT to Cys-374 of actin showed almost the same results as the case of Gln-41 of actin. The present FRET measurements demonstrated that not only TnI but also TnT change their positions on the thin filament corresponding to three states of thin filaments (relaxed, Ca2+-induced or closed, and S1-induced or open states).