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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Arch Biochem Biophys. Author manuscript; available in PMC Jan 18, 2007.
Published in final edited form as:
PMCID: PMC1776856
NIHMSID: NIHMS15575
Structural transition of the inhibitory region of troponin I within the regulated cardiac thin filament
Wen-Ji Dong,a* Jianli An,b Jun Xing,b and Herbert C. Cheungb
a School of Chemical Engineering and Bioengineering and Department of Veterinary and Comparative Anatomy Pharmacology and Physiology, Washington State University, Pullman, WA 99164, USA
b Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA
*Corresponding author. Fax: +1 509 335 4650. E-mail address: wdong/at/vetmed.wsu.edu (W.-J. Dong).
Contraction and relaxation of cardiac muscle are regulated by the inhibitory and regulatory regions of troponin I (cTnI). Our previous FRET studies showed that the inhibitory region of cTnI in isolated troponin experiences a structural transition from a β-turn/coil motif to an extended conformation upon Ca2+ activation. During the relaxation process, the kinetics of the reversal of this conformation is coupled to the closing of the Ca2+-induced open conformation of the N-domain of troponin C (cTnC) and an interaction between cTnC and cTnI in their interface. We have since extended the structural kinetic study of the inhibitory region to fully regulated thin filament. Single-tryptophan and single-cysteine mutant cTnI(L129W/S151C) was labeled with 1,5-IAEDANS at Cys151, and the tryptophan-AEDANS pair served as a donor–acceptor pair. Labeled cTnI mutant was used to prepare regulated thin filaments. Ca2+-induced conformational changes in the segment of Trp129-Cys151 of cTnI were monitored by FRET sensitized acceptor (AEDANS) emission in Ca2+ titration and stopped-flow measurements. Control experiments suggested energy transfer from endogenous tryptophan residues of actin and myosin S1 to AEDANS attached to Cys151 of cTnI was very small and Ca2+ independent. The present results show that the rate of Ca2+-induced structural transition and Ca2+ sensitivity of the inhibitory region of cTnI were modified by (1) thin filament formation, (2) the presence of strongly bound S1, and (3) PKA phosphorylation of the N-terminus of cTnI. Ca2+ sensitivity was not significantly changed by the presence of cTm and actin. However, the cTn–cTm interaction decreased the cooperativity and kinetics of the structural transition within cTnI, while actin filaments elicited opposite effects. The strongly bound S1 significantly increased the Ca2+ sensitivity and slowed down the kinetics of structural transition. In contrast, PKA phosphorylation of cTnI decreased the Ca2+ sensitivity and accelerated the structural transition rate of the inhibitory region of cTnI on thin filaments. These results support the idea of a feedback mechanism by strong cross-bridge interaction with actin and provide insights on the molecular basis for the fine tuning of cardiac function by β-adrenergic stimulation.
Keywords: Cardiac troponin I, FRET, Inhibitory region, Ca2+ activation, Stopped-flow, Kinetics
Troponin (Tn)1 is a ternary assembly of proteins that are bound to actin and involved in activation and regulation in striated muscle. Troponin C (TnC) is the Ca2+-binding component, troponin I (TnI) binds to actin (A) and inhibits actomyosin ATPase in relaxed muscle, and troponin T (TnT) is bound to tropomyosin (Tm) and anchors the trimeric troponin complex to the actin filament [1,2]. Together this system is referred to as the regulated thin filament with the stoichiometry Tn-Tm-A7. Upon activation, regulatory Ca2+ binds to specific sites located in the N-domain of TnC and the affinity of TnC for TnI is enhanced by two orders of magnitude [3]. Concomitant with these changes, the affinity of TnI for actin is weakened, possibly disrupting contacts between actin and a specific site on TnI, which removes inhibition of the ATPase and facilitates a strong interaction between actin and the motor domain of myosin (S1) [4]. The latter interaction results in tension development.
Skeletal TnC has two sites for regulatory Ca2+ (sites I and II). The cardiac muscle isoform (cTnC) has only one active Ca2+-binding site (site II) because site I is inactive due to mutations [5]. Cardiac TnI (cTnI) has a unique N-terminal extension that is absent in the skeletal isoform, and this extension contains two targets (Ser23 and Ser24) for PKA phosphorylation [6,7]. In cardiac myocytes, this phosphorylation is stimulated by β-adrenergic agonists and is a regulatory device in cardiac contractility.
In the initial step in Ca2+ activation of cardiac muscle, Ca2+ binds to the single site in the N-domain of cTnC. This binding results in rearrangements of the helices leading to a relatively more open conformation of the N-domain. This open conformation exposes a hydrophobic patch within the domain and makes possible a strong interaction of this patch with the regulatory region of the cTnI (residues 150–165) [8-10]. This hydrophobic interaction is facilitated by a large conformational change of a downstream segment of cTnI (residues 130–149, the inhibitory region) from a β-turn/coil motif to an extended conformation [11,12]. This conformational change is a consequence of the release of the inhibitory region from actin triggered by Ca2+ activation. An alternate view is that the regulatory region simply slides into the open N-domain to interact with the exposed hydrophobic pocket thus stabilizing the Ca2+-saturated N-domain. This movement drags along the downstream inhibitory region which in turns breaks its contact with actin.
We recently reported the size and the rate of the reversible conformational changes of the cTnI inhibitory and regulatory regions induced by Ca2+ binding to and dissociation from reconstituted cTn, using FRET in equilibrium and kinetic measurements [11,12]. These studies have now been extended to fully regulated thin filaments containing strongly bound S1 and PKA phosphorylated cTnI. FRET sensitized acceptor emission was used to track conformational changes to avoid optical interference from endogenous tryptophans in actin and S1.
Protein preparations
A recombinant tryptophanless cTnT mutant (W239F/W289F) was generated from a full-length rat cTnT clone subcloned into a pET-17b vector, and a recombinant wild-type cTnC was generated from a cTnC cDNA clone from chicken slow skeletal muscle as previously reported [13]. A mutant containing a single tryptophan and a single cysteine, (L129W/S151C/C81I/C98S/W192F), was generated from a mouse cDNA clone subcloned into a pET-3d vector as previously described [12,14]. All plasmids were transformed into BL21(DE3) cells (Invitrogen) and expressed under isopropyl-1-thio-d-galactopyranoside induction. The expressed proteins were purified as previously described [12]. cTm [15], actin [16], and myosin subfragment 1 (S1) from chymotryptic digestion of myosin [17] were obtained from bovine cardiac tissue. Protein purification and modification of single cysteine residue with IAEDANS were performed according to previously described procedures [12,13]. Briefly, the lyophilized protein was re-suspended in a labeling buffer containing 50 mM Mops (pH 7.2), 3 M urea, 100 mM KCl, 1 mM EDTA, 1 mM DTT, then stepwise dialyzed against the labeling buffer to reduce [DTT] to ~10 μM. cTnI(129W/151C) (70–100 μM) was labeled with a 2.5 M excess of the acceptor probe IAEDANS under constant stirring overnight at 4 °C. Unreacted probes were reduced by adding DTT to a final concentration of 2 mM from a 1 M stock solution, and then removed through a combination of dialysis and G-25 gel Wltration. Reconstituted cTn were obtained using a previously described procedure [18] with modifications. cTnC, cTnI, and cTnT were separately dialyzed against a reconstitution buffer (50 mM Tris–HCl (pH 8.0), 6 M urea, 500 mM KCl, 5 mM CaCl2, 5 mM DTT), then mixed at a 1.5:1:1.2 molar ratio (15, 10, and 12 μM final concentrations). The mixture was gently shaken for two hours at room temperature, then stepwise dialyzed against a high salt buffer containing 1 M KCl, 20 mM Mops (pH 7.0), 1.25 mM MgCl2, 1.25 mM CaCl2, 1.5 mM DTT to successively reduce the urea concentration (6, 4, 2, and 0 M). The KCl concentration was subsequently reduced to 1, 0.7, 0.5, 0.3, and 0.15 M by stepwise dialysis against a working buffer containing 150 mM KCl, 50 mM Mops (pH 7.0), 5 mM MgCl2, 2 mM EGTA, and 1 mM DTT. cTn–cTm complexes were formed by mixing cTn and cTm 1:1 (2 μM each) in the same working buffer plus a sufficient volume of 3 M KCl to bring the final KCl concentration to 300 mM. Excessive salt was then reduced to 150 mM KCl in two dialysis steps. cTn–cTm–A7 was prepared by mixing cTn–cTm, and polymerized actin 1:7 (2:14 μM) in the working buffer with 300 mM KCl, followed by reducing [KCl] to 150 mM as for the cTn–cTm complex. cTn–cTm–A7S1 was prepared by adding S1 to the cTn–cTm–A7 mixture at ratio of one cTn–cTm, seven actin and seven S1 after KCl had been decreased to 150 mM. ADP was added to the preparation immediately prior to measurements to a final concentration of 4 mM from an MgADP stock solution prepared from MgCl2 (200 mM) and Na2ADP (200 mM) in the working buffer.
PKA phosphorylation of cTnI
cTnI was phosphorylated by the catalytic subunit of PKA, using a cTnC affinity column [19]. Briefly, a sample of purified cTnI mutant was loaded on a cTnC affinity column equilibrated in 50 mM KH2PO4 at pH 7.0, 500 mM KCl, 10 mM MgCl2, and 0.5 mM DTT, and 125 U PKA/mg cTnI was added directly to the column. ATP was added to the column to initiate the reaction. After 30 min at 30 °C, the column was washed with a buffer containing 50 mM Mops at pH 7.0, 500 mM KCl, 2 mM CaCl2 and 0.5 mM DTT. Phosphorylated cTnI was eluted with a buffer containing 6 M urea, 10 mM EDTA, 0.5 mM DTT and 50 mM Mops at pH 7.0. The extent of phosphorylation was quantified by mass spectrometry and by treatment of the sample with alkaline phosphatase, followed by determination of inorganic phosphate using the EnzChek Phosphate Assay kit [20]. Phosphorylation of the two PKA sites was >90%.
Fluorescence measurements
Steady-state measurements were carried out on an ISS PC1 photoncounting spectrofluorometer equipped with an auto titrator at 10 ± 0.1 °C [13]. FRET was used in titration experiments to monitor Ca2+ induced structural change of the inhibitory region of cTnI. For Ca2+ titration, 1.0-mL samples were used. The samples contained 1 μM of labeled troponin in a buffer containing 50 mM Mops, pH 7.0, 1 mM DTT, 2 mM EGTA, 5 mM MgCl2, and 150 mM KCl. FRET sensitized emission intensity of the acceptor (AEDANS) was measured at 480 nm with excitation of the tryptophan donor at 295 nm. In a typical titration experiment, up to 90 data points were collected after successively injecting aliquots of 5 μl of a Ca2+-EGTA buffer containing 50 mM Mops, pH 7.0, 1 mM DTT, 2 mM EGTA, 5 mM MgCl2, 150 mM KCl and 9 mM CaCl2. Free [Ca2+] was calculated by an in-house program, using stability constants given by Fabiato [21]. Fluorescence intensity was corrected for the volume dilution prior to data analysis, which was carried out using previous reported procedures [22]. Since both actin and S1 contain tryptophans, it was necessary to rule out potential energy transfer from these residues to the acceptor. For this purpose, a tryptophanless mutant cTnI(151C) labeled with AEDANS was used at the same concentration as cTnI(129W/151C)AEDANS in parallel measurements. The results showed no significant energy transfer from the intrinsic tryptophan residues of actin and S1 to cTnI(151C)AEDANS. The small signals detected in these parallel measurements were subtracted as background in each experiment. The concentration of AEDANS labeled cTnI was determined using a molar extinction coefficient of 5900 M−1cm−1 at 340 nm for AEDANS. Other protein concentrations were determined using the following extinction coefficients: cTnC 4140 M−1cm−1 (278 nm); cTnT (tryptophanless), 4470 M−1cm−1 (280 nm); cTm, 15,840 M−1cm−1 (276 nm); G-actin, 26,040 M−1cm−1 (290 nm); and S1, 86,250 M−1cm−1 (277 nm).
Stopped-flow measurements
Kinetic measurements were carried out at 10.0 °C in a KinTek F2004 spectrometer with a 1.5-ms dead time. To follow the FRET kinetics of Ca2+ dissociation-induced changes of the conformation of the cTnI inhibitory region, a sample saturated with Ca2+ in a buffer of 50 mM Mops, pH 7.0 containing 1 mM DTT, 5 mM MgCl2, 150 mM KCl, and 0.16 mM Ca2+ (pCa 3.8) was mixed with an equal volume of the same buffer containing 1 mM DTT, 5 mM MgCl2, 150 mM KCl, and 2 mM EGTA. After mixing, [protein] = 2 μM and [EGTA] = 1000 μM. As in equilibrium experiments, the time-dependent change of FRET sensitized acceptor emission was Wrst determined with a sample containing cTnI(129W/151C)AEDANS, followed by determination of the time-dependent acceptor AEDANS emission with a sample containing tryptophanless cTnI(151C)AEDANS, using the same excitation and emission wavelengths as for equilibrium measurements. The signals from the second set of measurement were taken as background and subtracted from the first set of measurements. Eight to 10 kinetic tracings were collected for each set of samples, and the averages from each set of samples were used to calculate the rate constants associated with structural changes of the cTnI inhibitory region triggered by Ca2+ dissociation.
Characterization of FRET sensitized acceptor emission
Our previous study showed that the addition of Ca2+ resulted in a large increase of the donor (Trp129) fluorescence and a decrease of the acceptor (AEDANS) fluorescence in cTnI(129W/151C)AEDANS which was reconstituted into cTn. These spectral changes indicated a decrease of energy transfer from Trp129 to the acceptor attached to Cys151 and suggested a change of the cTnI inhibitory region from a β-turn/coil to an extended conformation [11,12]. Similar Ca2+-induced spectral changes were observed with the cTn complexed with cTm (Fig. 1). In the absence of bound Ca2+, the Trp129 fluorescence intensity at 340 nm (solid curve, Fig. 1) was quenched by more than 50% when the FRET acceptor (AEDANS) was attached to Cys151 (solid circle). The second emission band in the 480 nm region is the acceptor emission sensitized by FRET from Trp129 to the acceptor. These large reciprocal spectral changes indicate a large energy transfer and a small separation between the donor and acceptor sites. Upon addition of Ca2+, much of the donor emission that was quenched by the acceptor was recovered and the acceptor emission band was reduced (open circle). These results indicate that Ca2+ induced a large decrease in FRET and an increase in the donor–acceptor distance. Lifetime data from Trp129 corroborated this Ca2+-induced decrease in FRET and yielded an increase of 9.3 Å in the donor–acceptor distance (data not shown). As a control experiment, the cTn–cTm complex was prepared with a cTn reconstituted with a tryptophanless cTnI labeled at Cys151 with AEDANS, cTnI(151C)AEDANS. Upon excitation at 295 nm, there was negligible emission in the region of 335 nm and only small emission in the region of the 480 nm band (solid square). This emission spectrum was insensitive to Ca2+ (open square). The small 480-nm band was due to direct excitation (295 nm) of the acceptor probe AEDANS, not FRET because no tryptophan donor was present in the control system.
Fig. 1
Fig. 1
Steady-state fluorescence emission spectra of cTnI(129W/151C) mutant labeled with AEDANS at Cys151 in the cTn–cTm complex reconstituted with cTnC, tryptophanless cTnT, and cTm. Solid line: donor only sample containing cTnI(129W/151C); solid circle: (more ...)
To extend the measurements to fully regulate thin filaments, we need to establish the extent to which endogenous tryptophan residues in actin and S1 may interfere with FRET from Trp129 to acceptor AEDANS attached to Cys151 in cTnI. Fig. 2 shows two sets of AEDANS emission spectra determined with excitation at 295 nm. The spectra in Fig. 2A were collected from a preparation in which cTn–cTm was reconstituted from cTnI(129W/ 151C)AEDANS (donor–acceptor sample), and the spectra in Fig. 2B were collected from a preparation in which cTn was prepared from a tryptophanless cTnI(151C)AEDANS (acceptor alone). Since the samples were excited at 295 nm (donor excitation), the spectra in Fig. 2A are FRET sensitized acceptor emission. Ca2+ reduced the sensitized acceptor emission (solid curve vs. dashed curve), indicating a decrease in FRET and an increase in donor–acceptor separation. In the presence of actin (cTnC-cTm-A7) or actin plus S1 (cTn–cTm–A7–S1), and in the absence of Ca2+, the two spectra are essentially the same and are about 9% enhanced relative to cTn–cTm (solid circle and solid square). These two spectra were reduced to about the same extent upon addition of Ca2+ (open circle and open square). The observed enhancement of sensitized AEDANS emission in the presence of actin and S1 could arise from one or both possible sources. One source was an alteration of the environment of residue 151 of cTnI in fully regulated thin filaments and in the presence of strongly bound S1. The other source was energy transfer from endogenous tryptophan residues in actin and S1 to AEDANS attached to Cys151 of cTnI. The emission spectra of AEDANS excited at 295 nm (Fig. 2B) were obtained with a cTn–cTm reconstituted with a tryptophanless cTnI labeled at Cys151, cTnI(151C)AEDANS (acceptor alone). Consistent with the spectra shown in Fig. 2A, AEDANS emission is enhanced in the presence of actin and actin plus S1 relative to the cTn–cTm. We attribute the enhanced ADEANS emission observed with cTn-cTm-A7 and cTn–cTm–A7–S1 to energy transfer from tryptophan residues in actin and S1. However, these actin- and S1-sensitized AEDANS spectra are not sensitive to Ca2+. Thus, the contribution of the tryptophan residues in actin and S1 to energy transfer between Trp129 and (Cys151)AEDANS can be treated as background signal and can be adequately removed during data analysis. These results have provided a basis to use FRET sensitized acceptor (AEDANS) Xuorescence to monitor Ca2+ titration and the kinetics of conformational transitions.
Fig. 2
Fig. 2
(A) FRET sensitized acceptor emission spectra of cTnI(129W/151C)AEDANS reconstituted into cTn–cTm complex (solid and dashed curves), cTn–cTm–A7, thin filaments (circle) and cTn–cTm–A7 plus strongly bound S1 (square). (more ...)
Equilibrium conformation of the cTnI inhibitory region
Equilibrium conformations at different levels of Ca2+ saturation were studied using FRET sensed Ca2+ titration data. The titration curves were constructed from the FRET sensitized acceptor emission from cTnI(129W/151C)AEDANS at different levels of reconstitution (Fig. 3). The decrease in acceptor fluorescence with increasing [Ca2+] reflects a large Ca2+-induced decrease in FRET between donor and acceptor. These spectral changes indicate an increase in inter-site distance between residue 129 and residue 151. These curves were fitted to the Hill equation to recover pCa50 and the Hill coefficient for each reconstituted state (Table 1). The value of pCa50 indicates the sensitivity of the structural change of the cTnI inhibitory region to free Ca2+ concentration and the Hill coefficient reflects a cooperativity of the system. The presence of cTm had little effect on pCa50, but significantly decreased the Hill coefficient. On the other hand, the presence of actin decreased pCa50 from 5.69 to 5.60 and increased the Hill coefficient from 1.37 to 1.70. Though the marginal change in pCa50 was not statistically signiWcant (P > 0.05), the change in cooperativity (the Hill coefficient) was significant (P < 0.05). These changes suggest a potential role of actin filaments in cooperativity of thin filament activation. When strongly bound S1 was present, the titration curve shifted to a higher pCa (ΔpCa50 = +0.36 units) with a Hill coefficient of 1.11. Both changes in Ca2+ sensitivity and cooperativity were statistically significant (P <0.05).
Fig. 3
Fig. 3
Equilibrium Ca2+ titration of cTnI(129W/151C)AEDANS reconstituted into cTn complex (black), cTn–cTm (red), cTn–cTm–A7 (green) and cTn–cTm–A7 plus strongly bound S1 (blue). Solid lines are the best fitted curves (more ...)
Table 1
Table 1
Ca2+ Titration of reconstituted complexes containing cTnI(129W/151C)AEDANS
To investigate how PKA phosphorylation at the N-terminus of cTnI modulates the structural transitions of the inhibitory region of cTnI within the thin filament, Ca2+ titrations were carried out with reconstituted samples containing PKA phosphorylated cTnI mutants (data not shown). Recovered pCa50 and the Hill coefficients from these titration curves are also given in Table 1. At each level of reconstitution, phosphorylation of cTnI at Ser23/Ser24 desensitized the structural transitions of the inhibitory region of cTnI to Ca2+ binding by about 0.2 pCa unit. These changes were statistically significant with >99% certainty. These results are consistent with previous observations that PKA phosphorylation of cTnI decreased the Ca2+ sensitivity to the N-domain of cTnC.
Structural kinetics of cTnI inhibitory region
An initial kinetic experiment was performed in which a preparation of cTn containing cTnI(129W/151C)AEDANS but no Ca2+ was rapidly mixed with an equal volume of the same buffer which contained no proteins but an excess of Ca2+. A large initial signal change was observed within the mixing time of the instrument regardless of whether the signal was donor emission or sensitized acceptor emission. This rapid change could not be resolved and suggested a very fast conformational change of the inhibitory region induced by Ca2+ binding to cTnC. For this reason, the FRET kinetic experiments reported here are limited to those triggered by dissociation of Ca2+ from cTnC within the cTn complex. Fig. 4A shows two biphasic FRET kinetic tracings obtained by mixing a preparation of cTn containing cTnI(129W/151C) and saturated with bound Ca2+AEDANS with an equal volume of the same buffer containing an excess of EGTA and no protein. The upper tracing was obtained from sensitized acceptor emission and the lower tracing from donor emission. The fast phase was over in less than 0.1 s and the slow phase persisted to 2–3 s. Under the experimental conditions, cTnC in the preparations was initially saturated with Ca2+ in both the single regulatory site in the N-domain and the two Ca2+/Mg2+ sites in the C-domain. We previously showed that the rate of Ca2+ dissociation from the single regulatory site in the N-domain is two orders of magnitude faster than that from the two sites in the C-domain [11,12]. The previous results suggest that the fast phase of the FRET tracing in Fig. 4A is associated with conformational change of the inhibitory region triggered by Ca2+ dissociation from the regulatory site. The slow phase is associated with Ca2+ dissociation from the non-regulatory sites in the C-domain. The kinetic tracings shown in Fig. 4B support this conclusion. In this experiment, the cTn preparations were reconstituted with a cTnC mutant in which two of the six acidic residues (Asp) within the 12-residue Ca2+-binding loop in site II were substituted by hydrophobic residues (Val and Ala). These substitutions are known to inactivate the 12-residue loop for chelating Ca2+. This cTnC mutant had no bound Ca2+ in the N-domain. With this preparation, the fast kinetic phase is eliminated, indicating that the fast phase observed in Fig. 4A is associated with Ca2+ dissociation from the regulatory site.
Fig. 4
Fig. 4
FRET-based biphasic stopped-flow kinetic tracings of Ca2+ dissociation induced distance changes between residues 129 and 151 of mutant cTnI(129W/151C)AEDANS reconstituted with cTnC and tryptophanless cTnT. The two blue tracings were obtained from donor (more ...)
Fig. 5 shows the fast kinetic phase of the tracings shown in Fig. 4A in an expanded time scale. The two base lines suggest that 10–15% of the signals (either donor emission or sensitized acceptor emission) were lost during rapid mixing. The best fits were obtained with a biexponential function for both tracings. The kinetic parameters obtained from sensitized acceptor emission and donor emission are very similar (legends to Fig. 5), and the results suggest that both signals report the same conformational transition which was triggered by dissociation of bound Ca2+ from the regulatory site in the N-domain of cTnC. Subsequent kinetic studies with fully regulated thin filament and in the presence of strongly bound S1 were made using sensitized acceptor emission. The presence of actin and S1 had little effects on the slow rate, but greatly modified the fast rate. The two recovered kinetic rates suggest a two-step transition of the inhibitory region of cTnI within the thin filament in response Ca2+ dissociation. The fast step accounting for an average of 85% of functional structural change may be the key step for regulation, and the slow step accounting for an average of 15% of structural change may reflect protein structure relaxation after the major structural change. The rate constants of the fast phase are compared in Fig. 6 for all four preparations. Also shown in Fig. 6 are the rate constants of the fast phase for the preparations which were reconstituted with PKA phosphorylated cTnI(129W/151C)AEDANS. The changes in the rates of structural transitions induced by different levels of reconstitution and by PKA phosphorylation of cTnI were statistically significant (P <0.05).
Fig. 5
Fig. 5
The tracings in Fig. 4A are displayed in (A) in an expanded time window. The black tracing is from FRET sensitized acceptor emission, and the blue tracing is from donor emission. The black and blue flat tracings are base line for the sensitized acceptor (more ...)
Fig. 6
Fig. 6
Comparison of the rates of the fast phase of FRET kinetics triggered by dissociation of bound Ca2+ from the regulatory site in cTnC determined from sensitized acceptor fluorescence at different levels of reconstitution, with non-phosphorylated and phosphorylated (more ...)
The key events in regulation of cardiac muscle involve reversible Ca2+ binding to cTnC, structural changes within the trimeric troponin complex associated with the binding, and changes in the interaction between cTnC and cTnI in their interface. These Ca2+-dependent events trigger the contractile cycle by removing the inhibition of actomyosin ATPase and initiating strong interaction between myosin cross-bridges and the actin filament. The latter interaction results in tension development. Ca2+ binding is considered the trigger of activation and the change in the cTnC-cTnI linkage is the switch between activation and deactivation. Full understanding of the switching mechanism requires detailed knowledge of functionally important structural transitions and the dynamics and thermodynamics associated with the transitions. Toward this goal, we have developed several FRET-based conformational markers for conformational transitions. One such marker is the mutant cTnI(129W/151C) AEDANS, which reports a Ca2+-induced change in the secondary structure of cTnI in the inhibitory region. Energy transfer between tryptophan donor and AEDANS acceptor was determined from quenching of donor fluorescence. Equilibrium FRET studies show a Ca2+-induced increase of about 9 Å in the donor–acceptor separation between residues 129 and 151 in cTnI reconstituted into the cTn complex. This distance was fully recovered upon dissociation of bound Ca2+. The kinetics of this distance decrease as monitored by FRET indicates that two thirds (~6 Å) of the total change is associated with Ca2+ dissociation from the cTnC N-domain and the remainder of the change (~3 Å) is associated with dissociation of Ca2+ from the C-domain [12]. We have extended these previous studies to fully regulated thin filament both in the absence and presence of strongly bound S1, and with preparations in which cTnI is phosphorylated by PKA. Unlike in previous studies, we report here use of sensitized acceptor fluorescence to track energy transfer. This strategy minimizes potential optical interference from tryptophan residues in actin and S1, and the sensitized acceptor fluorescence proves to be a good and useful FRET signal to study structural changes in the inhibitor region of cTnI in fully regulated systems.
Ca2+ binding induces changes in tryptophan-sensitized acceptor fluorescence within the inhibitory region of cTnI in cTn. These spectral changes are not significantly affected upon reconstitution with cTm, actin and strongly bound S1. Results from Ca2+ titration show negligible changes in the value of pCa50 in the cTn–cTm complex and a negligible decrease (5.69–5.60) in regulated thin filament. The Hill coefficients for cTn and cTn–cTm are 2.14 and 1.37, respectively, indicating that Ca2+ binding is considerably less cooperative in cTn–cTm than in cTn. Reconstitution of cTn–cTm into regulated thin filament with actin partially restores the Hill coefficient to 1.70. Overall, the Ca2+ sensitivity remains relatively unchanged in cTn, cTn–cTm, and cTn–cTm–A7. The Ca2+ binding, however, becomes considerably less cooperative in cTn–cTm and regulated thin filament than in unbound cTn. significant changes are seen in the Ca2+ titration curve for cTn–cTm–A7–S1(ADP). Strongly bound S1 significantly increases the pCa50 by 0.36 units (5.60–5.96) and reduces the Hill coefficient to 1.1. The gain in Ca2+ sensitivity is accompanied by a loss of binding cooperativity. cTm binds to the N-terminal segment of cTnT and anchors the trimeric troponin to the surface of cTm. The relatively high cooperativity observed with cTn may arise from Ca2+-induced inter-subunit cooperative interactions within the troponin complex. In the presence of cTm and actin, the N-terminal segment of cTnT is stabilized by cTm and the cTn–cTm may be constrained on the actin surface. This constraint may restrict cooperative interactions among the three components of cTn and reduce the apparent cooperativity observed with cTn–cTm. The small gain in cooperativity from cTn–cTm to cTn-cTm-A7 may reflect interaction between neighboring regulatory units on the actin filament. Binding of S1 to actin in the Mg2+ state may partially displace the inhibitory region of cTnI from actin. This partial disengagement of cTnI from actin may enhance the interaction between cTnI and cTnC, in favor of Ca2+ binding and a gain in the observed Ca2+ sensitivity. Thus, the cooperative interactions in troponin may be greatly reduced by the disengagement of cTnI from actin, resulting in leading to very small or no apparent cooperativity in Ca2+ binding.
The rate constants reported here are for decreases of the distance between Trp129 and Cys151 of the cTnI inhibitory region triggered by dissociation of bound Ca2+ from the regulatory site in the N-domain of cTnC. The return of the region from an extended conformation to a β-turn is reasonably fast (kf= 102 s−1) in cTn. This rate is reduced by a factor of 1.4 to 73 s−1 in the presence of bound cTm. This reduction may result from immobilization of the N-terminal segment of cTnT by cTm. During Ca2+ dissociation, the rate of this conformational transition may be accelerated if the inhibitory region is allowed to re-bind actin. This expectation is borne out as kf increases by a factor of ~1.7 from 73 to 122 s−1 when determined with regulated thin filament. Re-association with actin provides an additional driving force to enhance the kinetics of the conformational transition. This driving force appears considerably dampened in the presence of strongly bound S1. In the presence of bound Ca2+, strongly bound S1 needs to be displaced to accommodate re-association of the inhibitory region with actin. This displacement reduces the rate of the conformational transition by 39% from 122 to 88 s−1.
Phosphorylation of cTnI by PKA elicits a loss in Ca2+ sensitivity in cTn and the other three reconstituted systems. In all four systems, the fast transition rate for reversal of the conformation of the inhibitory region upon Ca2+ dissociation is enhanced by about the same extent (17–23%) if cTnI is phosphorylated. We do not know what phosphorylation-induced molecular events are involved in the observed rate enhancement. These results are consistent with what is known about the effect of β-adrenergic stimulation of myofilament: reduction in myofilament Ca2+ sensitivity and acceleration of myocardial relaxation [23], and increase in cross-bridge cycling rate and maximum shortening rate [24,25]. The present FRET results suggest that these physiological parameters may be modulated by the dynamic nature of the inhibitory region and their changes induced by β-stimulation are related to the enhanced rate of the conformational transition of this region of cTnI.
In summary, we report here FRET-based studies of the dynamic nature of the cTnI inhibitory region with reconstituted troponin, the cTn–cTm complex, and fully regulated cardiac thin filament preparations in the absence and presence of strongly bound myosin. These studies were carried out using FRET sensitized acceptor fluorescence to determine inter-site distances between Trp129 and Cys151 in the cTnI inhibitory region between pCa 7.5 and 3.8. Different levels of reconstitution from cTn to cTn–cTm–A7 and plus strongly bound S1 have Different effects on Ca2+ sensitivity and cooperativity of structural change, and on the kinetics of Ca2+ dissociation induced conformational transition of this region of cTnI. The Ca2+ sensitivity of the cTnI inhibitory structural change is not significantly affected by the presence of cTn–cTm interaction and actin filament, but the cooperativity of Ca2+ induced structural transition was decreased by the presence of cTn–cTm interaction and increased by the presence of actin. The rate of the structural transition is decreased in the cTn–cTm complex and increased with the presence of actin filament. These changes can be explained in terms of differences in intramolecular and intermolecular interactions imposed by Different levels of reconstitutions. S1 strongly bound to actin filament significantly increases Ca2+ sensitivity and slows down the kinetics of the structural transition of the inhibitory region of cTnI. These results suggest a feedback mechanism of modulation of cardiac thin filament regulation by strong cross-bridge interaction with actin. In contrast, PKA phosphorylation of cTnI decreases the Ca2+ sensitivity and accelerates the structural transition rate of the inhibitory region of cTnI in thin filament to a similar extent regardless of the levels of reconstitution. These changes of the inhibitory region from an extended conformation to a β-turn motif may be a common basis for some of the known physiological effects of β-adrenergic stimulation on cardiac myofilament.
Acknowledgments
This work was supported in part by American Heart Association National Grant 0330170N (W.-J. D.), National Institutes of Health Grant HL80186 (W.-J. D.) and National Institutes of Health Grant HL52508 (H. C. C.).
Footnotes
1Abbreviations used: Tn, troponin; TnC, troponin C; TnI, troponin I; TnT, troponin T; c, cardiac muscle; PKA, Protein Kinase A; FRET, Förster resonance energy transfer; DTT, dithiothreitol; Mops, 3-(N-mopholino)propanesulfonic acid; EGTA, ethylene glycol-bis-(β-aminoethyl ether)-N,N,N,N′-tetraacetic acid; IAEDANS, 5-(iodoacetamidoethyl) aminonaphthelene-1-sulfonic acid.
1. Ebashi S, Endo M. Prog. Biophys. Mol. Biol. 1968;18:123–183. [PubMed]
2. Farah CS, Reinach FC. FASEB J. 1995;9:755–767. [PubMed]
3. Liao R, Wang CK, Cheung HC. Biochemistry. 1994;33:12729–12734. [PubMed]
4. Gordon AM, Homsher E, Regnier M. Physiol. Rev. 2000;80:853–924. [PubMed]
5. van Eerd JP, Takahashi K. Biochem. Biophys. Res. Commun. 1975;64:122–127. [PubMed]
6. Robertson SP, Johnson JD, Holroyde MJ, Kranias EG, Potter JD, Solaro RJ. J. Biol. Chem. 1982;257:260–263. [PubMed]
7. Solaro RJ, Moir AJ, Perry SV. Nature. 1976;262:615–617. [PubMed]
8. McKay RT, Tripet BP, Pearlstone JR, Smillie LB, Sykes BD. Biochemistry. 1999;38:5478–5489. [PubMed]
9. Tripet B, Van Eyk JE, Hodges RS. J. Mol. Biol. 1997;271:728–750. [PubMed]
10. Li MX, Spyracopoulos L, Sykes BD. Biochemistry. 1999;38:8289–8298. [PubMed]
11. Dong WJ, Robinson JM, Stagg S, Xing J, Cheung HC. J. Biol. Chem. 2003;278:8686–8692. [PubMed]
12. Dong WJ, Xing J, Robinson JM, Cheung HC. J. Mol. Biol. 2001;314:51–61. [PubMed]
13. Dong WJ, Xing J, Villain M, Hellinger M, Robinson JM, Chandra M, Solaro RJ, Umeda PK, Cheung HC. J. Biol. Chem. 1999;274:31382–31390. [PubMed]
14. Dong WJ, Chandra M, Xing J, Solaro RJ, Cheung HC. Biochemistry. 1997;36:6745–6753. [PubMed]
15. Smillie LB. Methods Enzymol. 1982;85:234–241. [PubMed]
16. Pardee JD, Spudich JA. Methods Enzymol. 1982;85:164–181. [PubMed]
17. Xing J, Cheung HC. Arch. Biochem. Biophys. 1994;313:229–234. [PubMed]
18. Robinson JM, Dong W-J, Xing J, Cheung HC. J. Mol. Biol. 2004;340:295–305. [PubMed]
19. Finley N, Abbott MB, Abusamhadneh E, Gaponenko V, Dong W, Gasmi-Seabrook G, Howarth JW, Rance M, Solaro RJ, Cheung HC, Rosevear PR. FEBS Lett. 1999;453:107–112. [PubMed]
20. Dong WJ, Xing J, Chandra M, Solaro J, Cheung HC. Proteins. 2000;41:438–447. [PubMed]
21. Fabiato A. Methods Enzymol. 1988;157:378–417. [PubMed]
22. Dong WJ, Robinson JM, Xing J, Cheung HC. J. Biol. Chem. 2003;278:42394–42402. [PubMed]
23. Solaro RJ. Modulation of cardiac myofilament activity by protein phosphorylation. In: Page EF, Harry A, Solaro RJ, editors. Handbook of Physiology. Section 2: The Cardiovascular System. Volume I: The Heart. Oxford University Press; Oxford: 2002. pp. 264–300.
24. Hoh JF, Rossmanith GH, Kwan LJ, Hamilton AM. Circ. Res. 1988;62:452–461. [PubMed]
25. Strang KT, Sweitzer NK, Greaser ML, Moss RL. Circ. Res. 1994;74:542–549. [PubMed]