Molecular Mobility of the Ca2+-Deficient EF-Hand of Cardiac Troponin as Revealed by Fluorescence Polarization of Genetically Inserted Tryptophan

Abstract

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.

Structure of Cardiac Muscle Troponin C Unexpectedly Reveals a Closed Regulatory Domain

“…In this report, we show that, in contrast to predicted models (7)(8)(9), the analogous conformational change does not occur in cardiac TnC, and that this is the direct structural consequence of inactivating Ca 2ϩ -binding site I. In addition, a structural understanding of cardiac TnC has potential therapeutic value in the understanding of the mechanism of cardiac TnC-binding drugs known as "calcium-sensitizing drugs" (8,10).…”

Sequence mutations in teleost cardiac troponin C that are permissive of high Ca2+ affinity of site II

“…Fluorescent probes engineered into TnC through Phe to Trp mutation in site I have been used previously to study the Ca 2ϩ binding dynamics of this molecule (8,19,21,27,29,32,38). We have demonstrated the effectiveness of Trp at residue 27 in reporting Ca 2ϩ binding to site II in McTnC and ScTnC without significantly affecting the ␣-helical content of the protein, which reflects the general structure of the molecule, using far UV circular dichroism spectra (27).…”

Ca2+binding to cardiac troponin C: effects of temperature and pH on mammalian and salmonid isoforms

“…The engineering of fluorescent probes into TnC through phenylalanine-to-tryptophan mutation has been used previously to study the Ca 2ϩ binding dynamics of this molecule (12,24,25,28,31,34,41). The effectiveness of tryptophan at residue 27 in reporting Ca 2ϩ binding to site II in BcTnC and ScTnC without significantly affecting the tertiary structure of the molecule has been previously established (28).…”