Quantitative measurements of the cooperativity in an EF‐hand protein with sequential calcium binding

Abstract

Positive cooperativity, defined as an enhancement of the ligand affinity at one site as a consequence of binding the same type of ligand at another site, is a free energy coupling between binding sites. It can be present both in systems with sites having identical ligand affinities and in systems where the binding sites have different affinities. When the sites have widely different affinities such that they are filled with ligand in a sequential manner, it is often difficult to quantify or even detect the positive cooperativity, if it occurs. This study presents verification and quantitative measurements of the free energy coupling between the two calcium binding sites in a mutant form of calbindin Dyk. Wild-type calbindin Dgk binds two calcium ions with similar affinities and positive cooperativity-the free energy coupling, AAG, is around -8 kJ.

Structural Independence of the Two EF-hand Domains of Caltractin

“…Most resonances of apo-and fully Ca 2ϩ -loaded CRC-C are sharp, and the line widths are similar. However, peaks are broader in the range 0.25-2 molar equivalents of Ca 2ϩ , indicative of an exchange between chemical environments occurring in the NMR intermediate exchange regime (24). This was readily apparent in complementary two-dimensional 15 N, 1 H HSQC experiments because resonance lines broaden sufficiently to cause significant reductions in peak intensity (data not shown).…”

The Mode of Action of Centrin

“…Despite the clear limitations in the values of the binding constants, the NMR data reliably show that the two binding constants differ by 100-fold or more in both the absence and presence of K 19 , consistent with a model of two independent sequential binding sites. In this model, the macroscopic K d values can be regarded as the microscopic binding constants for each individual site (20). The weaker affinity in each case is assigned to site III based on the presence of Asp-122 in place of the highly conserved glutamate residue in position 12 of the canonical EF-hand calcium binding loop.…”

Characterization of the N‐terminal half‐saturated state of calbindin D9k: NMR studies of the N56A mutant

“…The measurement of calcium binding properties of the N56A mutant of calbindin Dgk is the subject of the accompanying paper (Linse & Chazin, 1995). A description of the Ca2+ titration is given here to set the stage for the detailed 'H NMR analysis.…”

“…In fact, the N56A mutation does not reduce the site I1 binding constant (Linse & Chazin, 1995) sufficiently to allow preparation of a pure (Ca2+li sample: at 1 molar equivalent of added Ca2+, significant amounts of all three states of the proSlow cis-rrans isomerization about the Gly 42-Pro 43 peptide bond gives rise to two discrete sets of signals in the NMR spectrum . In order to avoid this complication, the protein has been mutated at position 43 (e.& P43G, P43M).…”