Circular dichroism and ultraviolet absorption difference spectroscopy have been used to study the binding of a series of divalent and trivalent cations to porcine intestinal calcium-binding protein (CaBP). When calcium is bound to the single high-affinity site on CaBP, the aromatic optical activity is greatly increased. Analysis of the circular dichroic spectra, obtained in the presence and absence of calcium, suggested that although changes in the optical activity of the single tyrosyl residue accounted for much of the overall change observed upon binding calcium, one or more of the five phenylalanyl residues was also perturbed. All the cations tested, with the exception of lead which gave rise to unique spectral effects, caused the same changes in optical activity between 300 and 250 nm. In the peptide absorption region, CaBP exhibited optical activity typical of an α-helical protein and no significant changes were observed in the presence of any of the cations tested. Cation-binding curves obtained from the circular dichroic data for the cations bound with high affinity (i.e., calcium, strontium, and the trivalent lanthanide ions) showed that the apparent number of binding sites was inversely related to the protein concentration. This phenomenon was accounted for by the concentration-dependent aggregation of CaBP observed in earlier studies. The binding data, obtained using circular dichroism, clearly indicated that the affinity of CaBP for the various cations was related to their ionic radius. Absorption difference spectra were observed when calcium was bound to CaBP. The features of these spectra confirmed that phenylalanyl as well as tyrosyl transitions were perturbed upon calcium binding. The extent to which the tyrosyl side chain was exposed to solvent was determined by solvent perturbation difference spectroscopy using perturbing agents of differing molecular radius. The apparent degree of exposure increased as the perturbant size decreased suggesting that the side chain was located in a cleft. Bound calcium did not change the degree of exposure. These data, together with complementary data obtained with bovine CaBP, were discussed in terms of the geometry of the cation-binding site.
Thermodynamics of cation binding to Nereis sarcoplasmic calcium-binding protein. Direct binding studies, microcalorimetry and conformational changes
