The effect of temperature on some calcium-binding properties of troponin C and calmodulin

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Abstract
Some Ca-binding properties of [rabbit] skeletal and [beef] cardiac troponin C (TnC) were measured as functions of temperature employing several physical and spectroscopic techniques. The degree of exposure of the tyrosine residues in brain calmodulin was also determined by a new approach. Circular dichroism thermal unfolding profiles were established for the 3 cases: metal-free protein, high-affinity sites filled, and fully saturated. Some thermodynamic parameters were calculated for these reversible melting processes. The Ca-binding parameters n and K, where n is the fraction of the total conformational change and K is the apparent association constant, for both skeletal and cardiac TnC, did not vary significantly over the temperature range 10-38.degree. C, but at 50.degree. C differences became apparent, especially in the case of the skeletal protein. The technique of thermal perturbation difference spectroscopy was applied to determine the degree of exposure of aromatic chromophores for the TnC and calmodulin in the absence and presence of Ca. For skeletal TnC and calmodulin the results were in good agreement with previous observations, but the reduced degree of exposure of the tyrosine residues in cardiac TnC, in the absence of Ca2+, was contrary to the earlier work. Ca-induced difference absorption spectra were measured for the TnC over the temperature range 10-70.degree. C. Cardiac TnC showed greater heat stability than its skeletal counterpart, in terms of the rate and the amount of change of the difference spectral maxima.