Biological function and site IICa2+-induced opening of the regulatory domain of skeletal troponin C are impaired by invariant site I or II Glu mutations

To investigate the roles of site I and II invariant Glu residues 41 and 77 in the functional properties and calcium-induced structural opening of skel etal muscle troponin C (TnC) regulatory domain, we have replaced them by Al a in intact F29W TnC and in wild-type and F29W N domains (TnC residues 1-90 ). Reconstitution of intact E41A/F29W and E77A/F29W mutants into TnC-deplet ed muscle skinned fibers showed that Ca2+-induced tension is greatly reduce d compared with the F29W control. Circular dichroism measurements of wild-t ype N domain as a function of pCa (= -log[Ca2+]) demonstrated that similar to 90% of the total change in molar ellipticity at 222 nm ([theta](222 nm)) could be assigned to site II Ca2+ binding. With E41A, E77A, and cardiac Tn C N domains this [theta](222 nm) change attributable to site II was reduced to less than or equal to 40% of that seen with wild type, consistent with their structures remaining closed in +Ca2+. Furthermore, the Ca2+-induced c hanges in fluorescence, near UV CD, and UV difference spectra observed with intact F29W are largely abolished with E41A/F29W and E77A/F29W TnCs. Taken together, the data indicate that the major structural change in N domain, including the closed to open transition, is triggered by site II Ca2+ bindi ng, all interpretation relevant to the energetics of the skeletal muscle Tn C and cardiac TnC systems.