Kinetics of the Structural Transition of Muscle Thin Filaments Observed by Fluorescence Resonance Energy Transfer

Abstract

Fluorescence resonance energy transfer showed that troponin-I changes the position on an actin filament corresponding to three states (relaxed, closed, and open) of the thin filament (Hai et al. (2002) J. Biochem. 131, 407-418). In combination with the stopped-flow method, fluorescence resonance energy transfer between probes attached to position 1, 133, or 181 of troponin-I and Cys-374 of actin on reconstituted thin filaments was measured to follow the transition between three states of the thin filament. When the free Ca(2+) concentration was increased, the transition from relaxed to closed states occurred with a rate constant of approximately 500 s(-1). For the reverse transition, the rate constant was approximately 60 s(-1). When myosin subfragment-1 was dissociated from thin filaments in the presence of Ca(2+) by rapid mixing with ATP, the transition from open to closed states occurred with a single rate constant of approximately 300 s(-1). Light-scattering measurements showed that the ATP-induced myosin subfragment-1 dissociation occurred with a rate constant of approximately 900 s(-1). In the absence of Ca(2+), the transition from open to relaxed states occurred with two rate constants of approximately 400 and approximately 80 s(-1). These transition rates are fast enough to allow the spatial rearrangement of thin filaments to be involved in the regulation mechanism of muscle contraction.

The Rates of Switching Movement of Troponin T between Three States of Skeletal Muscle Thin Filaments Determined by Fluorescence Resonance Energy Transfer

“…On the other hand, the change in the fluorescence intensity of the donor in the presence of the acceptor (TnT movement) was analyzed by a single exponential curve with a rate constant of ϳ400 s Ϫ1 both in the presence and absence of Ca 2ϩ . This rate constant also depends on the ATP concentration as in the case of TnI movement (24). It increased as the concentration of ATP increased up to 0.25 mM but became almost constant over the 0.25 mM ATP concentration.…”

“…Using time-resolved FRET between probes attached to Cys-374 of actin and positions 1, 133, or 181 of TnI, we measured the switching rates of TnI between the three states of thin filaments (24). In the present study, probes were attached to positions 60 and 250 on TnT25k fragment and Cys-374 on actin, and time-resolved FRET was measured to determine the switching rates of TnT between the three states of thin filaments.…”

“…Thus unlike TnT the Ca 2ϩ -induced movement of TnI would not be affected strongly by lowering the pH. S1-induced Movement of TnT-Recently, we determined the rates of S1-induced TnI movement by time-resolved FRET (24). Here the transition rates of S1-induced TnT movement were determined.…”

“…It increased as the concentration of ATP increased up to 0.25 mM but became almost constant over the 0.25 mM ATP concentration. In a previous study (24), the transition curve of TnI movement in the presence of Ca 2ϩ was fitted by a single exponential curve with the rate constant of ϳ300 s Ϫ1 , whereas in the absence of Ca 2ϩ the curve was fitted by a double exponential curve with the rate constants of ϳ400 and ϳ50 s Ϫ1 . These indicate that the transition of TnT from the open to relaxed states occurs directly, whereas the transition of TnI movement occurs through an intermediate state (the closed state).…”

“…On the other hand, the change in the transfer efficiency of FRET indicates a spatial rearrangement of thin filaments. 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 Ca 2ϩ -and S1-induced movements of TnI on the reconstituted thin filament (23,24). In the present study, the rates of Ca 2ϩ -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.…”