Ca2+-binding stoichiometry of calbindin D-28k as assessed by spectroscopic analyses of synthetic peptide fragments

Calbindin D-28k is an intracellular Ca2+-binding protein noted for its abundance and specific distribution in mammalian brain and sensory neurons. This protein contains six putative Ca2+-binding sites, referred to as EF-hands. Due to the presence of the large number of putative sites, previous studies have been unsuccessful in definitively establishing the stoichiometry of Ca2+ binding. We describe a synthetic approach to identify the number of Ca2+-binding sites in which 6 33-residue peptides, designated EF1-EF6, corresponding to the 6 EF-hand sequences of calbindin D-28k, were made. The response of each peptide to Ca2+ addition was assessed by H-1 NMR spectroscopy, circular dichroism (CD) spectroscopy, and agarose gel electrophoresis. The Ca2+ binding by CD experiments was performed at two peptide concentrations, 20 and 200 mu M, and the NMR studies at peptide concentrations ranging from 20 to 100 mu M. The CD and H-1 NMR data show that five of the six peptides bind Ca2+ as isolated peptides, namely, EF1, EF3, EF4, EF5, and EF6. The EF6 peptide appears to bind Ca2+ with lower affinity than the other four functional sites. In contrast, EF2 does not appear to bind Ca2+ under any of the spectroscopic conditions tested. The data suggest that at least five of the six putative sites in the native protein bind Ca2+, although their relative affinities cannot be deduced from studies of the isolated peptides.