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 tropon in 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 inte ractions, we prepared mutant TnIs which contained a single Cys-96 or Cys-11 7 and labeled them with the thiol-specific fluorescent probe N-(iodoacetyl) -N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS). We characterized the mic roenvironments of the AEDANS labels on TnI in the presence and absence of C a2+ by measuring the extent of acrylamide quenching of fluorescence and lif etime-resolved anisotropy. In the troponin-tropomyosin (Tn-Tm) complex, the AEDANS labels on both Cys-96 and Cys-117 were less accessible to solvent a nd less flexible in the presence of Ca2+, reflecting closer interactions wi th TnC under these conditions. In reconstituted thin filaments, the environ ment of the AEDANS on Cys-96 was not greatly affected by Ca2+, while the AE DANS on Cys-117 was more accessible but significantly less flexible as it m oved away from actin and interacted strongly with TnC in the presence of Ca 2+. We used fluorescence resonance energy transfer (FRET) to measure distan ces between AEDANS on TnI Cys-96 or Cys-117 and 4-{[(dimethylamino)phenyl]a zo}phenyl-4'-malemmide (DABmal) on actin Cys-374 in reconstituted thin fila ments. 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 a way 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-term inal half of TnI to interact with TnC in a Ca2+-independent manner, while t he C-terminal half of TnI binds to actin in the absence of Ca2+ or to TnC i n the presence of Ca2+. This is the first report to demonstrate structural movement of the inhibitory region of TnI in the thin filament.