Protein surface charges and calcium binding to individual sites in calbindin D9k: stopped-flow studies

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Abstract
The kinetics of calcium dissociation from two groups of site-specific mutants of calbindin D9k - a protein in the calmodulin superfamily with two Ca2+ sites and a tertiary structure closely similar to that of the globular domains of troponin C and calmodulin - have been studied by stopped-flow kinetic methods, using the fluorescent calcium chelator Quin 2, and by 43Ca NMR methods. The first group of mutants comprises all possible single, double, and triple neutralizations of three particular carboxylate groups (Glu-17, Asp-19, and Glu-26) that are located on the surface of the protein. These carboxylates are close to the two EF-hand calcium binding sites, but are not directly liganded to the Ca2+ ions. Conservative modification of these negative carboxylate side chains by conversion to the corresponding amides results in a marked reduction in the Ca2+ binding constants for both sites, as recently reported [Linse et al. (1988) Nature 335, 651-652]. The stopped-flow kinetic results show that this reduction in Ca2+ affinity derives primarily from a reduction in the Ca2+ association rate constant, Kon. The estimated maximum value of the association rate constant (kon(max)) for Ca2+ binding to the wild-type protein is ca. 109 M-1 s-1. In contrast, for the mutant protein with three charges neutralized the maximum association rate constant is estimated to be only 2 .times. 10- M-1 s-1. In the presence of 0.1 M KCl, the value of kon(max) is only slightly affected in the case of the triple mutant but is substantially reduced in the case of the wild-type protein (to ca. 2 .times. 107 M-1 s-1). The second group of mutants involves amino acid substitutions and/or deletions in the Ca2+ binding loop of the N-terminal site. Although this site has a high affinity for Ca2+, it is unusual in that it contains two extra amino acids compared with an archetypal EF hand (such as that found at the C-terminal site). The alterations are aimed at making this N-terminal loop sequence more like that of an archetypal EF hand. Within this group of mutants are found examples of decreased rates of association and/or increased rate of dissociation for Ca2+ at the N-terminal site. In contrast, the modifications of the N-terminal site are found to have no significant effect on the kinetics of the C-terminal site.