CALCIUM-BINDING TO THE REGULATORY N-DOMAIN OF SKELETAL-MUSCLE TROPONIN-C OCCURS IN A STEPWISE MANNER

Ca2+ binding to a recombinant regulatory N-domain (residues 1-90) of c hicken troponin C (NTnC) has been investigated with the use of heteron uclear multidimensional NMR spectroscopy. The protein has been cloned in pET3a vector and expressed in minimal media in Escherichia coli to allow uniform N-15 and C-13 labeling. The NMR spectra have been resolv ed and completely assigned [Gagne et al. (1994) Protein Sci. 3, 1961-1 974]. Ca2+ titration monitored by 2D {H-1, N-15}-HMQC NMR spectral cha nges revealed that Ca2+ binding to sites I and II of NTnC is a stepwis e process and that chemical shift changes occur throughout the N-domai n upon the binding of each Ca2+. The Ca2+ dissociation constants for t he binding of the first and second Ca2+ were determined to be 0.8 mu M less than or equal to K-dl less than or equal to 3 mu M and 5 mu M le ss than or equal to K-d2 less than or equal to 23 mu M, respectively. This mechanism is believed to represent that of the N-domain in intact TnC since we have shown earlier that the properties of the N-domain ( 1-90) were identical to those of the N-domain in intact TnC [Li et al. (1994) Biochemistry, 33, 917-925]. In contrast, however, our previous Ca2+ fluorescence and far-UV CD studies on F29W NTnC and F29W TnC ind icated cooperative Ca2+ binding to sites I/II and no detectable differ ences in their affinities. To rationalize these observations, a direct comparison was made of the Ca2+ titration of NTnC and F29W NTnC as mo nitored by far-UV CD spectroscopy. Unlike F29W NTnC, NTnC gave a bipha sic curve with binding constants in reasonable agreement with the NMR data. Although the far-UV CD spectra of NTnC and the F29W NTnC domain were the same in the absence of Ca2+, the Ca2+-induced negative ellipt icity increase for NTnC is significantly smaller than for F29W NTnC. T hese observations indicate that the F29W mutation has perturbed the Ca 2+ binding properties of the N-domain and its CD spectroscopic propert ies in the Ca2+-saturated state.