In the development of force during Sr2+ activation, phenotypically cardiac muscle is more sensitive than fast-twitch skeletal muscle, and TnC is central in this mechanism. The uncertainty has remained, however, whether such functional manifestations in situ relied critically on protein-protein interactions in the fiber or whether the Sr2+ sensitivities were governed intrinsically within the TnC molecule. To resolve this, we substituted a tryptophan for phenylalanine-26 in both rabbit sTnC (sTnC.W26) and in a chimera (c1/s.W26) where the 41 N-terminal amino acid residues were of bovine cTnC and the remaining 42-160 residues of rabbit sTnC. The metal ion dependent fluorescence emissions of the constructs could be examined in solution isolated from the protein-protein interactions found in situ. The Sr2+ sensitivities of these proteins differed by 0.55 +/- 0.02 pSr unit, but Ca2+ sensitivities were indistinguishable, as in the fiber. In another mutant, where the 27VLGA30 cluster was replaced with D-AD to enable site 1 to coordinate metal ion binding despite closely preserved cardiac structure, the Sr(2+)-sensitivity response was transformed into the skeletal-type. The Hill coefficients were also characteristically distinct for the various constructs. The findings indicate that cardiac N-terminal 41 residues define TnC performance in solution similar to that in situ. Moreover, the study provides unambiguous evidence that TnC isoforms intrinsically dominate the phenotype in the switching mechanisms for both cardiac and skeletal contractilities.

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To probe attitudinal features of the Ca(2+)-deficient site (site I) in the Ca2+ switch of cardiac troponin C (cTnC), we have examined steady-state fluorescence emission and polarization of a Trp26 inserted in a recombinant cardiac TnC (cTnC3.W) and compared these with the properties of the Ca(2+)-competent site I in skeletal TnC (sTnC4.W). The Ca(2+)-induced fluorescence emission in cTnC3.W was a fraction (25-30%) of that in sTnC4.W, in agreement with previous observations on the Ca(2+)-deficient site incorporated in a cardiac/skeletal chimera c1/s.W [Gulati, J. & Rao, V. G. (1994) Biochemistry 33, 9052-9056]. Thus, the fractional quantum yield reflected intrinsic properties of the cardiac metal ion-deficient site I. Conversely, in sTnC-1.W, where the skeletal site I also was made Ca(2+)-deficient by D27-->A substitution, the Ca(2+)-induced quantum yield was lower than that in cTnC3.W. Nevertheless, similar steady-state fluorescence polarizations for Ca(2+)-saturated sTnC4.W and cTnC3.W indicated indistinguishable final conformations in the two activated TnC isoforms. In EGTA, the polarization parameter (PEGTA) of sTnC4.W is greater than that of cardiac TnC, and the cardiac PEGTA value is closer to the activated PCa. Comparison of the chimera c1/s.W with sTnC-1.W indicated that the differences in conformation of the site I Trp for the EGTA-treated cardiac/skeletal isoforms were due to the structural disparities in this region. This contention was further supported by examination of the chimera CBc1/s.W, where the cardiac EF-hand was altered by 27VLGA30-->DAD substitution. Polarization of the relaxed form was similar to that for sTnC4.W. These findings suggest that the relaxed conformation of the cardiac Ca2+ switch is more favorably predisposed to activation than the skeletal switch.

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Intrinsic tyrosines, as monitored by fluorescence spectroscopy, are sensitive reporters of local, Ca2'-induced conformational changes in troponin C (TnC). Rabbit skeletal TnC contains two tyrosines (YlO in the N-helix, and Y109 in site 3 in the C-terminal domain) in distinct microenviromnents: their individual contributions to total fluorescence intensity are elucidated here utilizing bacterially synthesized rabbit skeletal TnC (sTnC4) and a genetically engineered variant, termed 109YF, lacking one of the tyrosines (Y109 replaced with F109). The steady-state fluorescence emission spectra following excitation at 280 nm were recorded in EGTA (Cat'-free) and Ca*'-saturated (pCa4) solutions. For the wild-type sTnC4, pCa4 causes a significant (46%) increase in the peak fluorescence intensity over the value in EGTA. For the mutant 109YF, the EGTA fluorescence is only marginally affected (74% of the wild-type Fao&, but interestingly the Ca2' effect is completely suppressed (AF = Fe, -&.xA = 2% of the wild-type value). These results indicate that the two tyrosines make disparate contributions to the fluorescence spectrum of wild-type sTnC, both in the presence and absence of Ca'+; whereas Y 10 in the N-helix is dominant in Ca*'-free solution, Y 109 is the sole contributor to the Ca*+ effect. Furthermore, to explain the biphasic fluorescence response of Y 109 obtained during Ca*+ titrations, the hndings yield the most unequivocal evidence that Ca*+-induced conformational changes in the trigger sites operating the contractile switch modify properties of the C-terminal sites in TnC pari passu.