| Abstract
| - In muscle thin filaments, the inhibitory region (residues 96−117) of troponin I (TnI) is thoughtto interact with troponin C (TnC) in the presence of Ca2+ and with actin in the absence of Ca2+. To betterunderstand these interactions, we prepared mutant TnIs which contained a single Cys-96 or Cys-117 andlabeled 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 presenceand 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 closerinteractions with TnC under these conditions. In reconstituted thin filaments, the environment of theAEDANS on Cys-96 was not greatly affected by Ca2+, while the AEDANS on Cys-117 was more accessiblebut significantly less flexible as it moved away from actin and interacted strongly with TnC in the presenceof Ca2+. We used fluorescence resonance energy transfer (FRET) to measure distances between AEDANSon TnI Cys-96 or Cys-117 and 4-{[(dimethylamino)phenyl]azo}phenyl-4‘-maleimide (DABmal) on actinCys-374 in reconstituted thin filaments. In the absence of Ca2+, the mean distances were 40.2 Å forCys-96 and 35.2 Å for Cys-117. In the presence of Ca2+, Cys-96 moved away from actin Cys-374 by∼3.6 Å, while Cys-117 moved away by ∼8 Å. This suggests the existence of a flexible “hinge” regionnear the middle of TnI, allowing amino acid residues in the N-terminal half of TnI to interact with TnCin a Ca2+-independent manner, while the C-terminal half of TnI binds to actin in the absence of Ca2+ orto TnC in the presence of Ca2+. This is the first report to demonstrate structural movement of the inhibitoryregion of TnI in the thin filament.
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