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

Tomoyoshi Kobayashi, Minae Kobayashi, Zygmunt Gryczynski, Joseph R. Lakowicz, John H. Collins

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37 Scopus citations

Abstract

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 Cys96 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'-maleimide (DABmal) on actin Cys-374 in reconstituted thin filaments. In the absence of Ca2+, the mean distances were 40.2 Å for Cys-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' 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.

Original languageEnglish
Pages (from-to)86-91
Number of pages6
JournalBiochemistry
Volume39
Issue number1
DOIs
StatePublished - 11 Jan 2000

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