Structural changes induced by calcium and magnesium in a high affinity calcium-binding protein from crayfish sarcoplasm.

Abstract

The sarcoplasmic calcium-binding protein (SCP) from crayfish has in its dimer form two calcium-specific and four Ca-Mg sites. The conformations of this protein in the calcium- (SCP2 *Cas), magnesium- (SCP2 -Mg4), and metal-free forms as well as in intermediary states have been studied by circular dichroism (CD) in the far and near-ultraviolet regions, tryptophan fluorescence, sulfhydryl reactivity, and trypsin susceptibility. SCP appears to contain eight a helical segments (per subunit), each consisting of about 10 amino acid residues. Three of these helices require eaz+ to be formed and are stable even in the presence of 8 M urea provided SCP is saturated by calcium. This is not the case for the remaining five helical segments. One of the three calcium-sensitive helices is unaffected by Mg+, and is formed upon ea2+ binding to the Ca-specific site. In the calcium-saturated SCP, 8 to 9 Phe residues out of 13 (per monomer) are immobilized in a helical segments. Two Phe side chains are present in the Ca-sensitive helix adjacent to the Ca-specific site. Some of the Tyr chromophores are located in the metal-sensitive helices flanking both the calcium-specific and the Ca-Mg sites, contrary to the Trp residues, which are not included in these a helical segments. Two thiols out of three (per monomer) display a marked decrease in reactivity when SCP binds Ca2+ or M8+, and are less accessible to 5,5’-dithiobis(2-nitrobenzoic acid) in the calciumthan in the magnesium-form of the protein. In the presence of physiological levels of Mg+, Ca2+ induces major structural changes (SCPz-Mg, -+ SCPZ*C~~) which involve the calcium-specific sites rather than the Ca-Mg sites. This contrasts sharply with the situation prevailing in vertebrate SCP (parvalbumin) as the latter has no calcium-specific sites and displays very limited conformational changes upon Ca-Mg exchange.