Inhibitory region of troponin I: Ca(2+)-dependent structural and environmental changes in the troponin-tropomyosin complex and in reconstituted thin filaments

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 Ca(2+) and with actin in the absence of Ca(2+). 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 Ca(2+) 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 Ca(2+), 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 Ca(2+), 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 Ca(2+). 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 Ca(2+), the mean distances were 40.2 A for Cys-96 and 35.2 A for Cys-117. In the presence of Ca(2+), Cys-96 moved away from actin Cys-374 by approximately 3.6 A, while Cys-117 moved away by approximately 8 A. 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 Ca(2+)-independent manner, while the C-terminal half of TnI binds to actin in the absence of Ca(2+) or to TnC in the presence of Ca(2+). This is the first report to demonstrate structural movement of the inhibitory region of TnI in the thin filament.

Figure 1:

Effect of Ca²⁺ on acrylamide quenching of AEDANS at TnI Cys-117: (A) Tn–Tm complex and (B) reconstituted thin filaments.

Figure 2:

Effects of Ca²⁺ on the lifetime-resolved anisotropy of AEDANS at TnI Cys-117: (A) Tn–Tm complex and (B) reconstituted thin filaments.

Figure 3:

Frequency domain phase and modulation data for AEDANS-labeled TnI Cys-117 in reconstituted thin filaments in the (A) absence of Ca²⁺ and (B) presence of Ca²⁺: (black symbols) donor only and (white symbols) donor and acceptor. The phase angle increases and the modulation decreases as the frequency increases. In the presence of the acceptor, the lifetime decreases; thus, the frequency responses shift to higher frequencies. The solid lines are the best fitted curves for distance distribution analysis.

Figure 4:

Structural changes in TnI upon binding of Ca²⁺ to the regulatory sites of TnC. Schematic drawing illustrating a proposed Ca²⁺-sensitive “hinge” near the middle of the TnI polypeptide chain. In the absence of Ca²⁺ (top), the inhibitory region (residues 96–117) and a second actin-binding site (region near residue 133) of TnI interact with actin–Tm. Binding of Ca²⁺ to the N-terminal, regulatory sites of TnC (bottom) exposes a hydrophobic pocket in TnC’s N-terminal domain. TnI residues 117–133 interact with the newly exposed hydrophobic pocket of TnC, moving the C-terminal part of TnI away from actin–Tm. The environment of the AEDANS label on TnI residue 96 does not change significantly when Ca²⁺ binds to the regulatory sites of TnC, but the side chain of the AEDANS label on TnI residue 117 becomes less flexible as it moves toward the hydrophobic pocket of TnC.