Structural transition of the inhibitory region of troponin I within the regulated cardiac thin filament

Abstract

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 beta-turn/coil motif to an extended conformation upon Ca(2+) activation. During the relaxation process, the kinetics of the reversal of this conformation is coupled to the closing of the Ca(2+)-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. Ca(2+)-induced conformational changes in the segment of Trp129-Cys151 of cTnI were monitored by FRET sensitized acceptor (AEDANS) emission in Ca(2+) 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 Ca(2+) independent. The present results show that the rate of Ca(2+)-induced structural transition and Ca(2+) 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. Ca(2+) 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 Ca(2+) sensitivity and slowed down the kinetics of structural transition. In contrast, PKA phosphorylation of cTnI decreased the Ca(2+) 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 beta-adrenergic stimulation.

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: donor–acceptor sample containing cTnI(129W/151C)_AEDANS plus Mg²⁺; open circle: donor–acceptor sample containing cTnI(129W/151C)_AEDANS plus Ca²⁺; solid square: acceptor only sample containing cTnI(151C)_AEDANS plus Mg²⁺; open square: acceptor only sample containing cTnI(151C)_AEDANS plus Ca²⁺. The open squares and solid squares are superimposed on each other. Protein concentration was 1 μM, and excitation was at 295 nm for all samples.

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). In the absence of Ca²⁺: solid line, solid circle, and solid square. In the presence of Ca²⁺: dashed line, open circle, and open square. (B) Directly excited acceptor emission spectra of tryptophanless cTnI(151C)_AEDANS reconstituted into cTn–cTm complex (solid line), cTn–cTm–A₇ (open circle), and cTn–cTm–A₇ plus strongly bound S1 (open square). In the absence of Ca²⁺: solid line, solid circle, and solid square. In the presence of Ca²⁺: dashed line, open circle, and open square. Protein concentrations are were 1 μM, and excitation was at 295 nm.

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 from the Hill equation, and the fitted parameters are listed in Table 1. The titration data were obtained from FRET sensitized acceptor emission.

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 fluorescence, and the two black tracings were obtained from sensitized acceptor emission. (A) Wild-type cTnC was used in reconstitution. A single exponential function was used to fit the slow phase, yielding a rate constant of 1.24 s⁻¹ from donor signal (blue) and a rate constant of 1.11 s⁻¹ from sensitized acceptor signal (black). (B) Tracings were obtained as in (A), except that a cTnC mutant in which Ca²⁺ binding site II modified (D64V/D66A) was used for reconstitution. The Ca²⁺ binding site II was inactivated in this mutant. The single exponential fit yielded rate constant of 1.14 s⁻¹ from donor signal (blue) and 1.25 s⁻¹ from sensitized acceptor signal (black). Excitation wavelength was 295 nm, and protein concentration was 1 μM after mixing.

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 emission and the donor emission, respectively. The base lines were obtained by rapidly mixing a Ca²⁺-saturated protein solution with a similar solution in which proteins were omitted. Both tracings were fitted to a two-term exponential function. Donor tracing: k_f= 102 s⁻¹, a_f= 0.87; k_s 12.6 s⁻¹, a_s = 0.13. Sensitized acceptor tracing: k_f = 98 s⁻¹, a_f = 0.84; k_s = 14 s⁻¹, a_s=0.16. k_f and k_s are the rate constants from the fast and slow phase, respectively; a_f and a_s are the fractional signal changes associated with the fast and slow phases, respectively. (B) residuals plots of the fittings from a single-term (red) and a double-term (black) exponential fit of the sensitized acceptor emission.The fit with a double-term exponential function is more satisfactory than that from the single-term function.

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 cTnI. The error bars are SEM.