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 ne urons. This protein contains six putative Ca2+-binding sites, referred to as EF-hands. Due to the presence of the large number of putative s ites, previous studies have been unsuccessful in definitively establis hing the stoichiometry of Ca2+ binding. We describe a synthetic approa ch 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+ a ddition 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 1 00 mu M. The CD and H-1 NMR data show that five of the six peptides bi nd Ca2+ as isolated peptides, namely, EF1, EF3, EF4, EF5, and EF6. The EF6 peptide appears to bind Ca2+ with lower affinity than the other f our functional sites. In contrast, EF2 does not appear to bind Ca2+ un der 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 o f the isolated peptides.