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Calcium binding to troponin C (TnC) is insufficient for full activation of myosin ATPase activity by actin-tropomyosin-troponin. Previous attempts to investigate full activation utilized ATP-free myosin or chemically modified myosin to stabilize the active state of regulated actin. We utilized the Δ14-TnT and the A8V-TnC mutants to stabilize the activated state at saturating Ca 2+ and to eliminate one of the inactive states at low Ca 2+ . The observed effects differed in solution studies and in the more ordered in vitro motility assay and in skinned cardiac muscle preparations. At saturating Ca 2+ , full activation with Δ14-TnT·A8V-TnC decreased the apparent K M for actin-activated ATPase activity compared to bare actin filaments. Rates of in vitro motility increased at both high and low Ca 2+ with Δ14-TnT; the maximum shortening speed at high Ca 2+ increased 1.8-fold. Cardiac muscle preparations exhibited increased Ca 2+ sensitivity and large increases in resting force with either Δ14-TnT or Δ14-TnT·A8V-TnC. We also observed a significant increase in the maximal rate of tension redevelopment. The results of full activation with Ca 2+ and Δ14-TnT·A8V-TnC confirmed and extended several earlier observations using other means of reaching full activation. Furthermore, at low Ca 2+ , elimination of the first inactive state led to partial activation. This work also confirms, in three distinct experimental systems, that troponin is able to stabilize the active state of actin-tropomyosin-troponin without the need for high-affinity myosin binding. The results are relevant to the reason for two inactive states and for the role of force producing myosin in regulation.

In cardiac muscle, troponin (Tn) and tropomyosin inhibit actin and myosin interactions through the steric blocking of myosin binding to F-actin. Ca 2+ binding to Tn C modulates this inhibition. Thin filaments become activated upon Ca 2+ binding, which enables strong binding of myosin with a concomitant release of ATP hydrolysis products and level arm swinging responsible for force generation. Despite this level of description, the current cross-bridge cycle model does not fully define the structural events that take place within Tn during combinatorial myosin and Ca 2+ interventions. Here, we studied conformational changes within Tn bound to F-actin and tropomyosin by fluorescence lifetime imaging combined with Förster resonance energy transfer. Fluorescent dye molecules covalently bound to the Tn C C-lobe and Tn I C-terminal domain report Ca 2+ - and myosin-induced activation of Tn. Reconstituted thin filaments were deposited on a myosin-coated surface similar to an in vitro motility assay setup without filament sliding involved. Under all the tested conditions, Ca 2+ was responsible for the most significant changes in Tn activation. Rigor myosin activated Tn at subsaturated Ca 2+ conditions but not to the degree seen in thin filaments with Ca 2+ . ATP-γ-S did not affect Tn activation significantly; however, blebbistatin induced significant activation at subsaturating Ca 2+ levels. The relation between the extent of Tn activation and its conformational flexibility suggests that active/inactive Tn states coexist in different proportions that depend on the combination of effectors. These results satisfy an allosteric activation model of the thin filament as a function of Ca 2+ and the myosin catalytic cycle state., (Published by Elsevier Inc.)

The Ca(2+)-sensitive regulatory switch of cardiac muscle is a paradigmatic example of protein assemblies that communicate ligand binding through allosteric change. The switch is a dimeric complex of troponin C (TnC), an allosteric sensor for Ca(2+), and troponin I (TnI), an allosteric reporter. Time-resolved equilibrium Förster resonance energy transfer (FRET) measurements suggest that the switch activates in two steps: a TnI-independent Ca(2+)-priming step followed by TnI-dependent opening. To resolve the mechanistic role of TnI in activation we performed stopped-flow FRET measurements of activation after rapid addition of a lacking component (Ca(2+) or TnI) and deactivation after rapid chelation of Ca(2+). Time-resolved measurements, stopped-flow measurements, and Ca(2+)-titration measurements were globally analyzed in terms of a new quantitative dynamic model of TnC-TnI allostery. The analysis provided a mesoscopic parameterization of distance changes, free energy changes, and transition rates among the accessible coarse-grained states of the system. The results reveal that 1), the Ca(2+)-induced priming step, which precedes opening, is the rate-limiting step in activation; 2), closing is the rate-limiting step in de-activation; 3), TnI induces opening; 4), there is an incompletely deactivated population when regulatory Ca(2+) is not bound, which generates an accessory pathway of activation; and 5), there is incomplete activation by Ca(2+)-when regulatory Ca(2+) is bound, a 3:2 mixture of dynamically interconverting open (active) and primed-closed (partially active) conformers is observed (15 degrees C). Temperature-dependent stopped-flow FRET experiments provide a near complete thermokinetic parameterization of opening: the enthalpy change (DeltaH = -33.4 kJ/mol), entropy change (DeltaS = -0.110 kJ/mol/K), heat capacity change (DeltaC(p) = -7.6 kJ/mol/K), the enthalpy of activation (delta(double dagger) = 10.6 kJ/mol) and the effective barrier crossing attempt frequency (nu(adj) = 1.8 x 10(4) s(-1)).

The principal task of the Ca(2+) activation of striated muscle is the release of the troponin I (TnI) inhibitory region (TnI-I) from actin. TnI-I release facilitates the repositioning of tropomyosin across the actin surface and the formation of strong, force generating, actin-myosin cross-bridges. Full activation of the Ca(2+) regulatory switch (CRS) requires two switching steps in cTnI: binding of the TnI regulatory region to hydrophobic sites in the N-domain of Ca(2+)-bound troponin C and release of the adjacent TnI-I from actin. Using Förster resonance energy transfer, we have examined the requirements for full activation of the cardiac CRS. In the presence of actin, both Ca(2+) and strong cross-bridges are required for full activation. Actin desensitizes the CRS to Ca(2+) and produces cooperativity in the Ca(2+) activation of the CRS. Strong cross-bridges eliminate cooperativity and re-sensitize the CRS to Ca(2+). We propose a kinetic scheme and a structural model to account for these findings.

Upon Ca2+ activation of cardiac muscle, several structural changes occur in the troponin subunits. These changes include the opening of the cardiac troponin C (cTnC) N-domain, the change of secondary structure of the inhibitory region of cardiac troponin I (cTnI), and the change in the separation between these two proteins in the cTnC-cTnI interface. We have used Förster resonance energy transfer in Ca2+ titration and stopped-flow experiments to delineate these transitions using a reconstituted cardiac troponin. Energy transfer results were quantified to yield time-dependent profiles of changes in intersite distances during Ca2+ dissociation. The closing of the cTnC N-domain induced by release of regulatory Ca2+ from cTnC occurs in one step (t1/2 approximately 5 ms), and this transition is not affected by Ca2+ release from the C-domain. The other two transitions triggered by Ca2+ dissociation are biphasic with the fast phase (t1/2 approximately 5 ms) correlated with Ca2+ release from the cTnC N-domain. These transitions are slower than the release of bound regulatory Ca2+ (t1/2 3.6 ms) and are coupled to one another in a cooperative manner in restoring their conformations in the deactivated state. The kinetic results define the magnitudes of structural changes relevant in Ca2+ switching between activation and deactivation of cardiac muscle contraction.

Straightforward interpretation of Förster resonance energy transfer (FRET) data in terms of the distance from donor-labeled troponon-tropomyosin (TnTm) to acceptor-labeled actin is complicated by the potential for energy transfer to acceptors on neighboring actin monomers (cross-transfer). Calculations indicate that cross-transfer can account for a substantial percentage of the total transfer efficiency. In some cases, this renders isolated FRET data uninterpretable. To overcome these limitations, we have developed an analysis method that incorporates cross-transfer and can, in principle, define the most probable (in the "least-squares" sense) position of a TnTm residue on the actin filament. The technique analyzes data from four or more FRET experiments using acceptors attached to different residues on actin. We have used this method to specify the coordinates of skeletal troponin I (sTnI) residue 133 relative to the actin filament under Mg(2+) and Ca(2+)-saturating conditions. Ca(2+)-activation causes the C terminus of the regulatory domain of TnI to move away from the actin surface by 6.3A, laterally along the actin surface toward actin subdomain 3 by 22.0A, and azimuthally toward the actin inner domain by 13.2A. This information is used to construct a low-resolution structural model of thin filament activation.

Cardiac muscle activation is initiated by the binding of Ca(2+) to the single N-domain regulatory site of cardiac muscle troponin C (cTnC). Ca(2+) binding causes structural changes between cTnC and two critical regions of cardiac muscle troponin I (cTnI): the regulatory region (cTnI-R, residues 150-165) and the inhibitory region (cTnI-I, residues130-149). These changes are associated with a decreased cTnI affinity for actin and a heightened affinity for cTnC. Using Förster resonance energy transfer, we have measured three intra-cTnI distances in the deactivated (Mg(2+)-saturated) and Ca(2+)-activated (Ca(2+)-saturated) states in reconstituted binary (cTnC-cTnI) and ternary (cTnC-cTnI-cTnT) troponin complexes. Distance A (spanning cTnI-R) was unaltered by Ca(2+). Distances B (spanning both cTnI-R and cTnI-I) and C (from a residue flanking cTnI-I to a residue in the center of cTnI-R) exhibited Ca(2+)-induced increases of >8 A. These results compliment our previous determination of the distance between residues flanking cTnI-I alone. Together, the data suggest that Ca(2+) activation causes residues within cTnI-I to switch from a beta-turn/coil to an extended quasi-alpha-helical conformation as the actin-contacts are broken, whereas cTnI-R remains alpha-helical in both Mg(2+)- and Ca(2+)-saturated states. We have used the data to construct a structural model of the cTnI inhibitory and regulatory regions in the Mg(2+)- and Ca(2+)-saturated states.

The inhibitory region of troponin I (TnI) plays a central regulatory role in the contraction and relaxation cycle of skeletal and cardiac muscle through its Ca(2+)-dependent interaction with actin. Detailed structural information on the interface between TnC and this region of TnI has been long in dispute. We have used fluorescence resonance energy transfer (FRET) to investigate the global conformation of the inhibitory region of a full-length TnI mutant from cardiac muscle (cTnI) in the unbound state and in reconstituted complexes with the other cardiac troponin subunits. The mutant contained a single tryptophan residue at the position 129 which was used as an energy transfer donor, and a single cysteine residue at the position 152 labeled with IAEDANS as energy acceptor. The sequence between Trp129 and Cys152 in cTnI brackets the inhibitory region (residues 130-149), and the distance between the two sites was found to be 19.4 A in free cTnI. This distance was insensitive to reconstitution of cTnI with cardiac troponin T (cTnT), cTnC, or cTnC and cTnT in the absence of bound regulatory Ca(2+) in cTnC. An increase of 9 A in the Trp129-Cys152 separation was observed upon saturation of the Ca(2+) regulatory site of cTnC in the complexes. This large increase suggests an extended conformation of the inhibitory region in the interface between cTnC and cTnI in holo cardiac troponin. This extended conformation is different from a recent model of the Ca(2+)-saturated skeletal TnI-TnC complex in which the inhibitory region is modeled as a beta-turn. The observed Ca(2+)-induced conformational change may be a switch mechanism by which movement of the regulatory region of cTnI to the exposed hydrophobic patch of the open regulatory N-domain of cTnC pulls the inhibitory region away from actin upon Ca(2+) activation in cardiac muscle., (Copyright 2001 Academic Press.)

Calcium activation of fast striated muscle results from an opening of the regulatory N-terminal domain of fast skeletal troponin C (fsTnC), and a substantial exposure of a hydrophobic patch, essential for Ca(2+)-dependent interaction with fast skeletal troponin I (fsTnI). This interaction is obligatory to relieve the inhibition of strong, force-generating actin-myosin interactions. We have determined intersite distances in the N-terminal domain of cardiac TnC (cTnC) by fluorescence resonance energy transfer measurements and found negligible increases in these distances when the single regulatory site is saturated with Ca(2+). However, in the presence of bound cardiac TnI (cTnI), activator Ca(2+) induces significant increases in the distances and a substantial opening of the N-domain. This open conformation within the cTnC.cTnI complex has properties favorable for the Ca(2+)-induced interaction with an additional segment of cTnI. Thus, the binding of cTnI to cTnC is a prerequisite to achieve a Ca(2+)-induced open N-domain similar to that previously observed in fsTnC with no bound fsTnI. This role of cardiac TnI has not been previously recognized. Our results also indicate that structural information derived from a single protein may not be sufficient for inference of a structure/function relationship.