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
]. 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.