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

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. 10(9) 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 X 10(7) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

The Calmodulin Regulator Protein, PEP-19, Sensitizes ATP-induced Ca2+ Release

“…Interestingly, mutations that compromise the ability of PEP-19 to modulate Ca 2ϩ binding to CaM have proportional effects on both k on and k off (see Table 1), which suggests that a similar mechanism is responsible, at least in part, for modulating both parameters. A role for acidic residues in tuning the Ca 2ϩ k on but not k off for binding to Ca 2ϩ EF-hand proteins was demonstrated by Martin et al (32), who The second goal of this study was to determine whether PEP-19 modulates CaM-dependent signaling pathways that affect intercellular Ca 2ϩ homeostasis. We selected purinergic ATP-induced Ca 2ϩ release as a model system because this pathway involves multiple potential points of regulation by CaM.…”

Electrostatic distance geometry in a K+ channel vestibule.

“…To understand experiments of this kind, we define the "electrostatic compliance" between channel residue i and toxin residue i, a(i, (22)(23)(24)(25)(26)(27)(28) argue that 0.7 < y < 1.0 for nonpathological convex surface geometries. We think it likely that these conditions also apply to the solventexposed surface of the CTX-Shaker complex under study here.…”

Rapid Kinetic Techniques

“…For example, the dissociation of calcium from calcium binding proteins such as calmodulin and calbindin D 9k can be studied by rapid mixing with an excess of a fluorescent calcium chelator such as Quin 2 (Martin et al, 1990)), which forms a strongly fluorescing, high affinity 1:1 complex with calcium.…”