Inhibitory region of troponin I:: Ca2+-dependent structural and environmental changes in the troponin-tropomyosin complex and in reconstituted thin filaments

In muscle thin filaments, the inhibitory region (residues 96-117) of troponin I (TnI) is thought to interact with troponin C (TnC) in the presence of Ca2+ and with actin in the absence of Ca2+. To better understand these interactions, we prepared mutant TnIs which contained a single Cys-96 or Cys-117 and labeled them with the thiol-specific fluorescent probe N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS). We characterized the microenvironments of the AEDANS labels on TnI in the presence and absence of Ca2+ by measuring the extent of acrylamide quenching of fluorescence and lifetime-resolved anisotropy. In the troponin-tropomyosin (Tn-Tm) complex, the AEDANS labels on both Cys-96 and Cys-117 were less accessible to solvent and less flexible in the presence of Ca2+, reflecting closer interactions with TnC under these conditions. In reconstituted thin filaments, the environment of the AEDANS on Cys-96 was not greatly affected by Ca2+, while the AEDANS on Cys-117 was more accessible but significantly less flexible as it moved away from actin and interacted strongly with TnC in the presence of Ca2+. We used fluorescence resonance energy transfer (FRET) to measure distances between AEDANS on TnI Cys-96 or Cys-117 and 4-{[(dimethylamino)phenyl]azo}phenyl-4'-malemmide (DABmal) on actin Cys-374 in reconstituted thin filaments. In the absence of Ca2+ the mean distances were 40.2 Angstrom for Cys-96 and 35.2 Angstrom for Cys-117. In the presence of Ca2+, Cys-96; moved away from actin Cys-374 by similar to 3.6 Angstrom, while Cys-117 moved away by similar to 8 Angstrom. This suggests the existence of a flexible "hinge" region near the middle of TnI, allowing amino acid residues in the N-terminal half of TnI to interact with TnC in a Ca2+-independent manner, while the C-terminal half of TnI binds to actin in the absence of Ca2+ or to TnC in the presence of Ca2+. This is the first report to demonstrate structural movement of the inhibitory region of TnI in the thin filament.