INTERACTION OF TROPONIN-I AND TROPONIN-C - F-19 NMR-STUDIES OF THE BINDING OF THE INHIBITORY TROPONIN-I PEPTIDE TO TURKEY SKELETAL TROPONIN-C

We have used F-19 nuclear magnetic resonance spectroscopy to study the interaction of the inhibitory region of troponin (TnI) with apo- and calcium(Il)-saturated turkey skeletal troponin C (TnC), using the synthetic TnI analogue N-alpha-acetyl[19FPhe106]TnI(104-115)amide. Dissociation constants of K(d) = (3.7 +/- 3.1) x 10(-5) M for the apo interaction and K(d) = (4.8 +/- 1.8) x 10(-5) M for the calcium(II)-saturated interaction were obtained using a 1:1 binding model of peptide to protein. The F-19 NMR chemical shifts for the F-phenylalanine of the bound peptide are different from the apo- and calcium-saturated protein, indicating a different environment for the bound peptide. The possibility of 2:1 binding of the peptide to Ca(Il)-saturated TnC was tested by calculating the fit of the experimental titration data to a series of theoretical binding curves in which the dissociation constants for the two hypothetical binding sites were varied. We obtained the best fit for 0.056 mM less-than-or-equal-to K(d1) less-than-or-equal-to 0.071 mM and 0.5 mM less-than-or-equal-to K(d)2 less-than-or-equal-to 2.0 mM. These results allow the possibility of a second peptide binding site on calcium(II)-saturated TnC with an affinity 10- to 20-fold weaker than that of the first site.