MOLECULAR MOBILITY OF THE CA2-DEFICIENT EF-HAND OF CARDIAC TROPONIN-CAS REVEALED BY FLUORESCENCE POLARIZATION OF GENETICALLY INSERTED TRYPTOPHAN()

To probe attitudinal features of the Ca2+-deficient site (site I) in t he Ca2+ switch of cardiac troponin C (cTnC), we have examined steady-s tate fluorescence emission and polarization of a Trp26 inserted in a r ecombinant cardiac TnC (cTnC3.W) and compared these with the propertie s of the Ca2+-competent site I in skeletal TnC (sTnC4.W). The Ca2+-ind uced fluorescence emission in cTnC3.W was a fraction (25-30%) of that in sTnC4.W, in agreement with previous observations on the Ca2+-defici ent site incorporated in a cardiac/skeletal chimera c1/s.W [Gulati, J. and Rao, V. G. (1994) Biochemistry 33, 9052-9056]. Thus, the fraction al quantum yield reflected intrinsic properties of the cardiac metal i on-deficient site I. Conversely, in sTnC-1.W, where the skeletal site I also was made Ca2+-deficient by D-27-->A substitution, the Ca2+-indu ced quantum yield was lower than that in cTnC3.W. Nevertheless, simila r steady-state fluorescence polarizations for Ca2+-saturated sTnC4.W a nd cTnC3.W indicated indistinguishable final conformations in the two activated TnC isoforms. In EGTA, the polarization parameter (P-EGTA) O f sTnC4.W is greater than that of cardiac TnC, and the cardiac P-EGTA value is closer to the activated P-Ca. 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 E F-hand was altered by (27)VLGA(30)-->DAD substitution. Polarization of the relaxed form was similar to that for sTnC4.W. These findings sugg est that the relaxed conformation of the cardiac Ca2+ switch is more f avorably predisposed to activation than the skeletal switch.