Abstract
Troponin (Tn) plays the key roles in the regulation of striated muscle contraction. Tn consists of three subunits (TnT, TnC, and TnI). In combination with the stopped-flow method, fluorescence resonance energy transfer between probes attached to Cys-60 or Cys-250 of TnT and Cys-374 of actin was measured to determine the rates of switching movement of the troponin tail domain (Cys-60) and of the TnT-TnI coiled-coil C terminus (Cys-250) between three states (relaxed, closed, and open) of the thin filament. When the free Ca(2+) concentration was rapidly changed, these domains moved with rates of approximately 450 and approximately 85 s(-1) at pH 7.0 on Ca(2+) up and down, respectively. When myosin subfragment 1 (S1) was dissociated from thin filaments by rapid mixing with ATP, these domains moved with a single rate constant of approximately 400 s(-1) in the presence and absence of Ca(2+). The light scattering measurements showed that ATP-induced S1 dissociation occurred with a rate constant >800 s(-1). When S1 was rapidly mixed with the thin filament, these domains moved with almost the same or slightly faster rates than those of S1 binding measured by light scattering. In most but not all aspects, the rates of movement of the troponin tail domain and of the TnT-TnI coiled-coil C terminus were very similar to those of certain TnI sites (N terminus, Cys-133, and C terminus) previously characterized (Shitaka, Y., Kimura, C., Iio, T., and Miki, M. (2004) Biochemistry 43, 10739-10747), suggesting that a series of conformational changes in the Tn complex during switching on or off process occurs synchronously.
Highlights
Binds to Tm and integrates the whole Tn complex into the thin filament
In combination with the stopped-flow method, fluorescence resonance energy transfer between probes attached to Cys-60 or Cys-250 of TnT and Cys-374 of actin was measured to determine the rates of switching movement of the troponin tail domain (Cys-60) and of the TnT-TnI coiled-coil C terminus (Cys250) between three states of the thin filament
Ca2ϩ-dependent Conformational Change—In the present study, IAEDANS attached to Cys-60 or Cys-250 of TnT was used as the Fluorescence resonance energy transfer (FRET) donor, and DABMI attached to Cys-374 of actin was as the energy acceptor
Summary
Binds to Tm and integrates the whole Tn complex into the thin filament. The extended N-terminal domain of TnT interacts with Tm in a Ca2ϩ-independent manner, whereas the C-terminal domain forms globular domain with other troponin subunits (TnI and TnC) and interacts with Tm in a Ca2ϩ-dependent manner [3]. The equilibrium between the blocked and closed states is calcium-sensitive, and strong myosin binding stabilizes the open state Structural studies, such as three-dimensional image reconstructions of electron micrographs and x-ray diffraction, have shown three positions of Tm corresponding to three states of the thin filament [7, 8]. The change in the transfer efficiency of FRET between probes attached to TnI and actin was monitored by donor fluorescence intensity to determine the rates of Ca2ϩ- and S1-induced movements of TnI on the reconstituted thin filament [23, 24]. The rates of Ca2ϩ- and S1-induced movements of TnT were determined by measuring FRET between probes attached to Cys-374 of actin and Cys-60 or Cys-250 of the point-mutated TnT25k fragment on the reconstituted thin filaments. As compared with the rate constants of S1 binding or dissociation determined by light scattering measurements, these measurements provide a better understanding for a switching mechanism by Tn in striated muscle regulation
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