The rates of switching movement of troponin T between three states of skeletal muscle thin filaments determined by fluorescence resonance energy transfer.

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.

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 ϳ450 and ϳ85 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 ϳ400 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 Striated muscle contraction is regulated by troponin (Tn) 1 and tropomyosin (Tm) on actin filaments in response to intracellular Ca 2ϩ levels (1,2). Tm, an extended ␣-helical coiled-coil dimmer, binds end to end along the actin filament and covers seven actin monomers. Tn is a complex of three proteins: TnC, TnI, and TnT. TnI inhibits actin-myosin interaction, and TnC suppresses the inhibitory effect of TnI by binding Ca 2ϩ . TnT 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 Ca 2ϩ -independent manner, whereas the C-terminal domain forms globular domain with other troponin subunits (TnI and TnC) and interacts with Tm in a Ca 2ϩ -dependent manner (3). The Ca 2ϩ -binding to TnC triggers a series of conformational changes of thin filaments, and then the active cross-bridge cycle is turned on (4,5).
Early explanation for the regulation mechanism is that the skeletal muscle thin filament has two states, "on" and "off" states depending on Ca 2ϩ concentrations. Recent kinetic studies proposed an additional state of the thin filament induced by the strong binding of myosin. Instead of a two-state model based on Ca 2ϩ -induced on-off switching, a three-state model has been proposed in which a thin filament exists in rapid equilibrium between three states, "blocked," "closed," and "open" (6). 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). However, it is still not clear whether the conformational change observed by both x-ray and three-dimensional image reconstructions of electron micrographs measurements could be entirely attributed to movement of Tm or to a change in the Tn position and/or in actin structure (9). Fluorescence resonance energy transfer (FRET) also provides structural information based on distances between probes attached to specific residues on proteins. This method can easily detect a conformational change in tertiary and quaternary structure of proteins because the transfer efficiency is a function of the inverse of the sixth power of the distance between probes. FRET between probes attached to TnI and actin showed a significant extent of Ca 2ϩ -induced and also S1-induced movement of TnI on the reconstituted thin filament (10 -14). Not only TnI but also TnT changes positions on the actin filament corresponding to three states of the thin filament (15). The movements of TnI and TnT between three states are impaired on the thin filament reconstituted with a functionally deficient mutant Tm (D234Tm) (14,16). D234Tm, in which three of seven repeats have been deleted, inhibits actomyosin-MgATPase even in the presence of Ca 2ϩ and Tn (17). The transitions between three positions of TnI and TnT on the thin filament are closely related to the regulation mechanism. Thus, FRET measurements provided a structural evidence for three states of thin filament (14 -16). The local dissociation of TnI from actin was demonstrated by use of the excimer fluorescence of pyrene-labeled Tm (18). TnI and TnI-TnC (ϪCa 2ϩ ) bind to the closed state of actin-Tm, and their binding is greatly weakened in the S1-induced open state.
For better understanding of the regulation mechanism, it is important to know the transition rates between the three states of thin filaments. Numerous stopped-flow measurements have been carried out to study the kinetics of the conformational change of the thin filament (19 -22). In these experiments, fluorescent probes were attached to Tm or actin, and the time course of the fluorescence intensity change was traced for kinetic studies of conformational changes of Tm and actin in response to three states of thin filaments. However, the fluorescence intensity change measured in these experiments indicates some environmental change around the probe, but it does not necessary mean a spatial rearrangement of thin filaments. 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. Cys-60 is located on the troponin tail domain, and Cys-250 is on the globular domain. 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.

MATERIALS AND METHODS
Reagents-Phalloidin from Amanita phalloides was purchased from Sigma. IAEDANS and DABMI were purchased from Molecular Probes. All other chemicals were of analytical grade.
Proteins-Actin, S1, and Tn were prepared from rabbit skeletal muscle as reported previously (10). ␣␣Tm was extracted from rabbit hearts as reported previously (10). Single cysteine TnT25k mutants (E60C and S250C) were prepared as reported previously (15). Actin was labeled at Cys-374 with DABMI as mentioned previously (14). The Tn complex labeled at Cys-133 of TnI with AEDANS (AEDANS-I133-Tn) was prepared as previously reported (14). TnT25k mutants were labeled with IAEDANS, and reconstitution of ternary Tn complexes with AEDANSlabeled TnT25k mutants (AEDANS-T60-Tn and AEDANS-T250-Tn) was carried out as reported previously (15). Cys-60 is located at the second residue from the N terminus, and Cys-250 is located at the seventeenth residue from the C terminus of the 25-kDa fragment of TnT. ATPase measurements indicated that Tn complexes reconstituted with the AEDANS-labeled TnT25k mutants retained the regulatory activity as well as the wild-type Tn (15). For stopped-flow measurements, equimolar phalloidin to actin was added to stabilize the actin filament.
Spectroscopic Measurements-Absorption was measured with a Hitachi U-3310 spectrophotometer. Steady state fluorescence was measured with a PerkinElmer LS50B fluorometer. Protein concentrations were determined by use of absorption coefficients of A 290 nm ϭ 0.63 (mg/ml) Ϫ1 cm Ϫ1 for G-actin, A 280 nm ϭ 0.24 for Tm, 0.45 for Tn, and 0.67 (mg/ml) Ϫ1 cm Ϫ1 for TnT25k. The concentration of labeled protein was measured with the BCA protein assay reagent by using the nonlabeled protein as the standard. Relative molecular masses of 42 kDa for actin, 66 kDa for Tm, and 64 kDa for reconstituted Tn with TnT25k mutants were used. The absorption coefficients of 24,800 M Ϫ1 cm Ϫ1 at 460 nm for DABMI (25) and 6100 M Ϫ1 cm Ϫ1 at 337 nm for IAEDANS (26) were used to determine the labeling ratios. The typical labeling ratios were 1.0 for DAB-F-actin, 0.75 for AEDANS-T250-Tn, and 0.74 for AEDANS-T60-Tn. pCa values were calculated from the concentrations of added EGTA and CaCl 2 using the numerical constants of Schwarzenbach et al. (27).
Stopped-flow Measurements-Kinetic measurements were performed using an Applied Photophysics Ltd. Model SX.18 MV stoppedflow spectrofluorometer. The excitation monochromator was set at 340 nm for fluorescence or 500 nm for the light scattering measurements, and the light from 420 -540 nm was collected through a broad band cut filter (Chroma Technology Co.) placed at the emission side. The instrumental dead time was 1.63 ms under the present experimental conditions. For each experiment, the reaction curves are the average of 10 reaction traces. The data were fitted with a nonlinear least-squares procedure to a single or a double exponential expression from which the rate constants were calculated. Prior to the stopped-flow experiments, the pH of the protein sample solutions were adjusted to the experimental values, and the pH of the mixing buffer solutions (Ca 2ϩ or EGTA solutions) was adjusted by KOH to give the values of the experimental pH after rapid mixing with the protein solutions. Data sets were fit to the single and double exponential equation with a floating end point using the Applied Photophysics Ltd. SX.18MV kinetic spectrometer work station software and Microsoft Excel.

RESULTS
Ca 2ϩ -dependent Conformational Change-In the present study, IAEDANS attached to Cys-60 or Cys-250 of TnT was used as the FRET donor, and DABMI attached to Cys-374 of actin was as the energy acceptor. From steady-state fluorescence measurements, we reported previously that a large difference in the transfer efficiency between these donor and acceptor probes was observed between the presence and absence of Ca 2ϩ (15). The change in the transfer efficiency upon Ca 2ϩ binding corresponds to the increase in the distance by 3.6 Å for AEDANS-T60-Tn and 7 Å for AEDANS-T250-Tn (15). This Ca 2ϩ -dependent conformational change was measured by changing the free Ca 2ϩ concentrations. Fig. 1 shows the fluorescence titration curves of AEDANS-T250-Tn/Tm/DAB-F-actin (in the presence of the acceptor) and AEDANS-T250-Tn/Tm/ F-actin (in the absence of the acceptor) as a function of the free Ca 2ϩ concentration. The curve in the presence of the acceptor (FRET) shows a large increase in the donor fluorescence with increasing free Ca 2ϩ concentration, whereas the curve in the absence of acceptor shows a decrease in the donor fluorescence. The curve of FRET shows a very sharp transition with a midpoint at pCa 6.6. The results indicate that the rearrangement of the spatial relationship between TnT and actin in reconstituted thin filaments occurs with a highly cooperative mode. The change in FRET between probes attached to TnT and actin directly reflects the change of the physiological conditions of the reconstituted thin filament (i.e. active and inhibitory states).
Kinetics of Ca 2ϩ -induced Movement of TnT-FRET in combination with the stopped-flow method (time-resolved FRET) was used to measure the Ca 2ϩ -induced movement of TnT by rapidly changing the free Ca 2ϩ concentration. The change in the transfer efficiency after changing the solvent conditions from ϪCa 2ϩ to ϩCa 2ϩ (or vice versa) was monitored by the donor-fluorescence intensity in the presence (Fig. 2, A and C) and absence (Fig. 2, B and D) of the acceptor. The reconstituted thin filament in 30 mM KCl, 0.1 mM ATP, 2 mM MgCl 2 , 1 mM NaN 3 , and 20 mM MOPS or 20 mM MES (buffer A) with 0.5 mM EGTA (buffer A ϪCa ) or 0.1 mM CaCl 2 (buffer A ϩCa ) was mixed with the same volume of buffer A containing 4 mM CaCl 2 or 3.75 mM EGTA, respectively. Fig. 2, A and C, shows the time courses of the fluorescence intensity of AEDANS-T250-Tn/Tm/ DAB-F-actin after changing Ca 2ϩ concentrations at pH 7.0. From ϪCa 2ϩ to ϩCa 2ϩ ( Fig. 2A), the fluorescence intensity increased (the transfer efficiency decreased) very rapidly and reached a final fluorescence level within 10 ms after the flowstop point. The observed fluorescence intensity change was analyzed by a single exponential process with the rate constant of 376 Ϯ 56 s Ϫ1 . From ϩCa 2ϩ to ϪCa 2ϩ (Fig. 2C) the fluorescence intensity decreased with the rate constant of 91.0 Ϯ 14 s Ϫ1 . On the other hand, Fig. 2, B and D, shows the time courses of the fluorescence intensity of AEDANS-T250-Tn/Tm/F-actin (in the absence of the acceptor) after changing Ca 2ϩ concentrations at pH 7.0. Contrary to the case in the presence of the acceptor, the fluorescence intensity decreased with the rate constant of 345 Ϯ 38 s Ϫ1 from ϪCa 2ϩ to ϩCa 2ϩ (Fig. 2B) and increased with the rate constant of 85.9 Ϯ 10 s Ϫ1 from ϩCa 2ϩ to ϪCa 2ϩ (Fig. 2D).
The time courses of fluorescence intensity changes were followed at pH 7.5, 6.5, and 6.0. From ϪCa 2ϩ to ϩCa 2ϩ , the fluorescence intensity of AEDANS-T250-Tn/Tm/DAB-F-actin increased with rate constants of 641 Ϯ 96 s Ϫ1 at pH 7.5, 208 Ϯ 31 s Ϫ1 at pH 6.5, and 151 Ϯ 23 s Ϫ1 at pH 6.0. On the other hand, from ϩCa 2ϩ to ϪCa 2ϩ the fluorescence intensity of AE-DANS-T250-Tn/Tm/DAB-F-actin decreased with rate con-stants of 83.1.0 Ϯ 12 s Ϫ1 at pH 7.5, 109 Ϯ 16 s Ϫ1 at pH 6.5, and 150 Ϯ 23 s Ϫ1 at pH 6.0. The rate constants of Ca 2ϩ -induced TnT movement strongly depended on the pH. As the pH decreased, the transition rate from ϪCa 2ϩ to ϩCa 2ϩ became slower, but the reverse rate became faster. The same measurements using AEDANS-T60-Tn and AEDANS-I133-Tn were carried out at pH 7.5, 7.0, 6.5, and 6.0. The results were summarized in Fig. 3.
Kinetics of S1-induced Movement of TnT-According to the three-state model of thin filaments, rigor S1 binding to the regulated thin filament induces the open state (6). Steady-state FRET measurements showed that the transfer efficiency between probes attached to Cys-60 or Cys-250 of the TnT25k mutant and Cys-374 or Gln-41 of actin in the reconstituted thin filament decreases significantly by rigor S1 binding (15). In the absence of the acceptor, the donor fluorescence did not change appreciably by rigor S1 binding. The change in the transfer efficiency was monitored by the donor-fluorescence intensity in the presence of the acceptor. Using AEDANS-T60-Tn and DAB-F-actin, time courses of the light scattering and fluorescence intensity changes of the reconstituted thin filament were followed after S1 binding or dissociation in the presence of Ca 2ϩ (transition from the closed to open states or vice versa) (Fig. 4). In the case of S1 dissociation, the reconstituted thin filaments and S1 complex in 30  ϳ20% of the total change under the present experimental conditions. The whole process of the light scattering change was regarded as a single exponential process and fitted with the rate constant of 975 Ϯ 107 s Ϫ1 (Fig. 4, A (1)), although such fast rate is near the limitation of the present instrumental conditions (dead time of 1.63 ms). On the other hand, the fluorescence intensity decreased much slower than the light scattering intensity (Fig. 4, A (2)), and the trace was analyzed by a single exponential curve with the rate constant of 372 Ϯ 48 s Ϫ1 .
In the case of S1 binding, the reconstituted thin filament in 140 mM KCl, 2 mM MgCl 2 , 20 mM MOPS, pH 7.5, and 1 mM NaN 3 (buffer FЈ) with 0.1 mM CaCl 2 (buffer FЈ ϩCa ) was mixed with the same volume of S1 in buffer FЈ ϩCa at 20°C. When S1 was mixed with thin filaments, the fluorescence intensity increased slightly faster than the light scattering intensity (Fig.  4B). The curves were analyzed by a single exponential process with the rate constants of 10.3 Ϯ 1.3 s Ϫ1 for light scattering (curve 1) and 12.2 Ϯ 1.6 s Ϫ1 for fluorescence (curve 2).
Time courses of the light scattering and fluorescence intensity changes of the reconstituted thin filament after S1 binding or dissociation were followed also in the absence of Ca 2ϩ (transition from the relaxed to open states or vice versa). In the case of S1 dissociation (Fig. 5A), the reconstituted thin filament and S1 complex in buffer F with 1.0 mM EGTA (buffer F ϪCa ) was mixed with the same volume of buffer F ϪCa including 1.0 mM ATP at 20°C. The light scattering intensity decreased as rapidly as the case in the presence of Ca 2ϩ , and the trace was fitted by a single exponential process with the rate constant of 888 Ϯ 98 s Ϫ1 (Fig. 5A (1)). On the other hand, the fluorescence intensity ( Fig. 5A (2)) decreased much slower than the light scattering intensity, and the curve was analyzed by a single exponential process with the rate constant of 407 Ϯ 53 s Ϫ1 .
In the case of S1 binding, the reconstituted thin filament in buffer FЈ with 1.0 mM EGTA (buffer FЈ ϪCa ) was mixed with the same volume of S1 in buffer FЈ ϪCa at 20°C. When S1 was mixed with thin filaments, the fluorescence intensity increased slightly faster than the light scattering intensity (Fig. 5B). The curves were fitted by a single exponential process with the rate constants of 4.5 Ϯ 0.6 s Ϫ1 for light scattering (curve 1) and 5.9 Ϯ 0.7 s Ϫ1 for fluorescence (curve 2). The values were approximately half of the rate constants in the presence of Ca 2ϩ .
Using AEDANS-T250-Tn and DAB-F-actin, time courses of the light scattering and fluorescence intensity change of the reconstituted thin filaments after S1 binding and dissociation were also followed under the same experimental conditions as AEDANS-T60-Tn. Almost the same results were obtained. Results are summarized in Table I.
Effects of ATP Concentrations on Rate Constants-Using AE-DANS-T60-Tn, rate constants of the light scattering and fluorescence intensity changes of the reconstituted thin filament after S1 dissociation were determined at various ATP concentrations. Stopped-flow measurements were carried out under the same experimental conditions as shown in Figs. 4A and 5A except for the concentration of ATP. Time courses of the light scattering and fluorescence intensity changes were analyzed with a single exponential process both in the presence and absence of Ca 2ϩ . Fig. 6 shows the rate constant versus the ATP concentration in the presence and absence of Ca 2ϩ . The rate constant of the light scattering intensity change (dissociation of S1 from the thin filament) increased markedly as the ATP concentration increased up to 0.5 mM, irrespective of the presence or absence of Ca 2ϩ . On the other hand, the rate constant of the fluorescence intensity change increased as the ATP concentration increased up to 0.25 mM but did not change at more than 0.25 mM ATP.

DISCUSSION
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.
Ca 2ϩ -induced Movement of TnT-In contrast to the case of AEDANS attached to positions 1 and 181 on TnI (24), the fluorescence intensity of the donor, AEDANS, attached to positions 60 or 250 on TnT was sensitive to changes in Ca 2ϩ concentration. Upon Ca 2ϩ binding, the donor fluorescence intensity decreased by 9% for T60-Tn and 14% for T250-Tn in the absence of the acceptor, whereas the fluorescence intensity in the presence of the acceptor increased by 29% for T60-Tn and 40% for T250-Tn under the present experimental conditions. The environmental sensitivity of the donor fluorescence would affect the transition rates measured by time-resolved FRET. Dong et al. (28) developed a method for such correction by converting the fluorescence intensity traces in the presence and absence of acceptor to a FRET efficiency trace. In the present study, the extents of the change of the donor fluorescence in the absence of the acceptor were only 9 and 14%. Furthermore, both rate constants in the presence and absence of acceptor were almost the same to each other (376 and 345 s Ϫ1 , Fig. 2, A  and B; 91 and 86 s Ϫ1 , Fig. 2, C and D) so that the environmental sensitivity of the donor fluorescence did not seriously affect on the calculation of the rate constants of the FRET trace. Indeed, such correction changed the transition rate by Ͻ10%.
The transition rate of TnT upon Ca 2ϩ binding or detaching was almost the same as that of TnI at pH 7.0, which suggests that the troponin complex moves as a whole on the actin filament with this rate constant. Ca 2ϩ binding triggers a series of conformational changes in the Tn complex on the thin filament: (1) the opening of the TnC N domain and the exposure of the hydrophobic patch of the N lobe of TnC, (2) the release of the amphiphilic helix (residues 128 -148 in skeletal TnI) from the actin/Tm, (3) the binding of the regulatory region of TnI to TnC, (4) the binding of the C-terminal region of TnT to Tm, and (5) movement of the N-terminal region of TnT. In the Ca 2ϩ -induced conformational change of the thin filament, the series of spatial rearrangements in the Tn complex would occur without any delay in each step, i.e. synchronously. Therefore, the rate constants of TnI and TnT movements are similar to each other.  Fig. 2 were carried out at pH 6.0, 6.5, 7.0, and 7.5. Closed and open symbols represent the rate constants of transition from ϪCa 2ϩ to ϩCa 2ϩ and from ϩCa 2ϩ to ϪCa 2ϩ , respectively. MOPS for pH 7.5 and pH 7.0 and MES for pH 6.5 and pH 6.0 were used.
Using a mutant TnC (F29W) and dansylaziridine-labeled TnC, Johnson et al. (29) determined the rates of Ca 2ϩ binding to and dissociation from the Ca 2ϩ -specific site of TnC. Ca 2ϩ dissociates from the Ca 2ϩ -specific site with a rate constant of 483 s Ϫ1 . The rate of Ca 2ϩ association with TnC (F29W) increased linearly as the free Ca 2ϩ concentration increased (up to ϳ750 s Ϫ1 at 5.1 M Ca 2ϩ ), indicating that Ca 2ϩ binds as rapidly as it can diffuse to the protein (29). The rapid fluorescence intensity change of the mutant TnC (F29W) reflects the environmental change of the donor, which may come from the Ca 2ϩ binding itself or from the first event (1), opening of the TnC N domain and the exposure of the hydrophobic patch of the N lobe of TnC. After some time lag, the subsequent spatial rearrangements of the Tn subunits (from (2) to (5)) follow synchronously. Thus, the information of Ca 2ϩ binding triggers a synchronous conformational rearrangement of the Tn subunits, and consequently the Tn complex moves as a whole from the position of the relaxed state to the position of the closed state with a rate constant of ϳ450 s Ϫ1 . On the other hand, upon deactivation, Ca 2ϩ detaching triggers a series of conformational changes in the Tn complex on the thin filament: (1) TnC N domain closing, (2) separation of TnI from TnC in the central helix region of the TnC, (3) the binding of the amphiphilic helix of TnI to the actin/Tm, and (4) movement of the TnT to the position of the closed state. Dong et al. (28) measured time-resolved FRET between TnI and TnC to determine the rate of the separation of the regulatory region of TnI from TnC. The rate was determined to be 236 s Ϫ1 in the TnI-TnC binary complex and 133 s Ϫ1 in the Tn complex. This rate, if measured on the reconstituted thin filament, may be close to the rate of the TnT movement, ϳ85 s Ϫ1 . The information of Ca 2ϩ detaching triggers a synchronous conformational rearrangement of the Tn subunits, and consequently the Tn complex moves as a whole to the position of the relaxed state on the thin filament with a rate constant of ϳ85 s Ϫ1 . These rates of the Tn movement on the thin filament by the Ca 2ϩ association and dissociation are rapid enough to account for the speed of skeletal muscle contraction and relaxation.
pH Dependence-It is well known that the Ca 2ϩ sensitivity of contraction of skinned fibers prepared from mammalian stri- Tn in buffer FЈ ϩCa was mixed with 9.6 M S1 in buffer FЈ ϩCa . The best fit exponential curve to each trace was superimposed.
FIG. 5. Stopped-flow traces for the S1-induced TnT (E60C) movement in the absence of Ca 2؉ on the reconstituted thin filament. S1 dissociation (A) or S1 binding (B) was traced with a light scattering (curve 1) or fluorescence (curve 2) signal. Sample and experimental conditions were the same as described in Fig. 4, except for 1 mM EGTA instead of 0.1 mM CaCl 2 . The best fit exponential curve to each trace was superimposed. ated muscle is reduced by acidic pH and that troponin is responsible for this acidosis. Lowering pH reduces the affinity of skeletal TnC for Ca 2ϩ binding. The pH effect on the Ca 2ϩ affinity of TnC was further modified through other troponin subunits (30). The roles of subunits in the pH dependence on the Ca 2ϩ sensitivity have been extensively studied by exchanging troponin subunits with the isoforms in permeabilized muscle fibers (31,32). To obtain further insights into the molecular mechanism determining the pH sensitivity of Ca 2ϩ regulation, the pH effects on the transition rates of TnI and TnT upon Ca 2ϩ binding to TnC were examined. The transition rate of TnT movement upon Ca 2ϩ binding decreased significantly as the pH decreased, whereas the rate of TnI movement did not change much (Fig. 3). On the other hand, after Ca 2ϩ detaching from TnC, the both transition rates of TnT and TnI movements increased appreciably as the pH decreased. The switching rate of TnT upon Ca 2ϩ binding to TnC was affected more than that of TnI by lowering pH, suggesting that the synchronous conformational change in the Tn subunits triggered by Ca 2ϩ binding is impaired. On the other hand, a deletion mutant Tm (D234Tm) in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing inhibits the thin filament-activated myosin-ATPase activity whether Ca 2ϩ is present or not (17). The Ca 2ϩinduced movement of TnT was impaired on this functionary deficient mutant Tm, although the Ca 2ϩ -induced movement of TnI was not affected (14,16). These indicate that a synchronous movement of the Tn subunits would be critical for the regulation mechanism.
The C-terminal region of TnT forms a globular portion of the Tn complex with TnC and TnI, which is located near residues 150 -180 on Tm (33). The C-terminal 17 residues of TnT contain the Tm-binding site that is critical for the Ca 2ϩ -sensitizing activity of TnT (3). This region in which there are no acidic but six basic residues is highly positive. Therefore, the ionic interaction between the Ca 2ϩ -sensitizing region of TnT and Tm would strongly depend on pH. On the other hand, the Ca 2ϩ binding induces the exposure of the hydrophobic region in TnC, which binds the regulatory region of TnI. 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. When strongly bound S1 is abruptly dissociated from the thin filament, the transition from the open to closed and relaxed states would occur in the presence and absence of Ca 2ϩ , respectively. The change in the light scattering intensity indicates the dissociation of S1 from the regulated thin filament. The rate of dissociation increased as the concentration of ATP increased (Fig. 6). At 0.5 mM ATP, it was Ͼ800 s Ϫ1 . 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. 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). That is, TnT moves differentially with TnI. The differential movement suggests a flexible joint between TnI and TnT subunits in the Tn complex.
When S1 binds to the thin filament, the transition to the open state from the closed and relaxed states would occur in the presence and absence of Ca 2ϩ , respectively. The light scattering measurements showed that the S1 binding occurred with the rate constant of ϳ10 and ϳ5 s Ϫ1 in the presence and absence of Ca 2ϩ , respectively, in accordance with the previous report (19). Time-resolved FRET showed that the movement of AEDANS-T60-Tn on thin filaments occurred with a slightly higher rate than that observed from the light scattering change in the presence or absence of Ca 2ϩ . On the other hand, the movement of TnI occurred with almost the same and slightly slower rates than those observed from the light scattering change in the presence and absence of Ca 2ϩ , respectively (24). The results indicate that the S1-induced movement of TnT occurs in more cooperative manner than that of TnI because TnT is an elongated molecule along the actin filament.
Kinetics with the Fluorescence Signal from Pyrene Probe Attached to Tm-Geeves and Lehrer (19) measured the rates of transition between the three states of the thin filament by monitoring the fluorescence intensity from the pyrene label attached to Tm. Although the FRET efficiencies in TnI and TnT movements correspond well to the three states of the thin filament, the excimer fluorescence of pyrene attached to Tm is sensitive to S1-induced but not to Ca 2ϩ -induced conformational changes of the thin filament. In the case of ATP-induced dissociation of S1 from the thin filament, the light scattering signals showed the single exponential transients with the rates depending on the ATP concentration linearly. The rates were almost the same as our previous (24) and present results. However, the fluorescence signal from pyrene-labeled Tm showed significant lags irrespective of Ca 2ϩ concentrations. In the case of S1 binding, the light scattering signals showed  6. Effect of ATP concentration on rate constants of S1induced conformational change. The rate constants in the presence (closed symbols) and absence (open symbols) of Ca 2ϩ are plotted against the ATP concentration after rapid mixing. Triangle and circle symbols represent the rate constants observed by the light scattering and fluorescence signal, respectively. Experimental conditions were the same as in Fig. 4A and Fig. 5A except for the ATP concentration.
almost the same results as our previous and present results. However, the fluorescence signal showed the transitions one order faster than those of the light scattering change. They explained the results by a highly cooperative conformational change of thin filaments in which a single S1 binding is sufficient to switch on the cooperative unit of 10 -12 actin subunits. Consequently, in the case of ATP-induced dissociation of S1, the lag of the fluorescence signal was observed, and in the case of S1 binding the rapid change in the pyrene fluorescence of Tm was seen. TnT is an elongated molecule of which the N-terminal region extends along the C-terminal region of Tm to the beginning of the next Tm on the actin filament. However, the FRET signal of TnT gave different aspects from the fluorescence signal of the pyrene label attached to Tm. The movement of TnT did not show the similar lag in the case of S1 dissociation nor the rapid transition rate one order faster than the light scattering signal in the case of S1 binding. Here it should be noted that the change in the fluorescence signal from pyrenelabeled Tm shows a conformational change around the environment of the probe but does not necessary mean the spatial rearrangement of the thin filament. The binding of S1 induces a cooperative conformational change of actin subunits through Tm, and the fluorescence intensity of pyrene on Tm increased faster than the scattering intensity. This conformational change would induce the movement of the N-terminal region of TnT following a further movement of the globular region of the Tn complex. On the other hand, in the case of the ATP-induced dissociation of S1, ATP binds to S1 as rapidly as it can diffuse to the protein, and the number of strongly bound S1 decreases instantaneously. Consequently, S1 dissociation, TnI, and TnT movements occur without the lag. Then, the environmental change around the pyrene probe attached to Tm follows with a lag compared with S1 dissociation and TnT movement.
Steady-state FRET measurements showed the structural evidence for the three states of thin filaments in which Tn changes the positions on the actin filament in response to the three states. Time-resolved FRET measurements determined the rates of movement of TnI and TnT on the transition between the three states of the thin filament. In most but not all aspects, the troponin tail and globular domains move with very similar rates on the transition. That is, a series of conformational changes in the troponin complex during the switching on or off process occurs synchronously. The rates are fast enough to allow the Tn movement to be directly involved in muscle regulation. The crystal structure of the core domain of Tn has been revealed (34). Based on the atomic structures of Tn and actin (35,36), more studies are necessary to clarify all of the structural changes that occur on the thin filament in combination with the switching movement of the Tn complex. It is especially important to know how the conformational change of actin subunits is related to the movement of the Tn complex.