DOMAIN ORGANIZATION OF CALBINDIN D-28K AS DETERMINED FROM THE ASSOCIATION OF 6 SYNTHETIC EF-HAND FRAGMENTS

Calbindin D-28k is an intracellular Ca2+ binding protein containing si x subdomains of EF-hand type. The number and identity of the globular domains within this protein have been elucidated using six synthetic p eptide fragments, each corresponding to one EF-hand subdomain. All six peptides were mixed in equimolar amounts in the presence of 10 mM Ca2 + to allow for the reconstitution of domains. The mixture was compared to native calbindin D-28k and to the sum of the properties of the ind ividual peptides using circular dichroism (CD), fluorescence, and H-1 NMR spectroscopy, as well as gel filtration and ion-exchange chromatog raphy. It was anticipated that if the peptides associate to form nativ e-like domains, the properties would be similar to those of the intact protein, whereas if they did not interact, they would be the same as the properties of the isolated peptides. The results show that the pep tides in the mixture interact with one another. For example, the CD an d fluorescence spectra for the mixture are very similar to those of th e intact calbindin D-28k, suggesting that the mixed EF-hand fragments associate to form a native-like structure. To determine the number of domains and the subdomain composition of each domain in calbindin D-28 k, a variety of peptide combinations containing two to five EF-hand fr agments were studied. The spectral and chromatographic properties of a ll the mixtures containing less than six peptides were closer to the s um of the properties of the relevant individual peptides than to the m ixture of the six peptides, The results strongly suggest that all six EF hands are packed into one globular domain. The association of the p eptide fragments is observed to drive the folding of the individual su bdomains. For example, one of the fragments, EF2, which is largely uns tructured in isolation even in the presence of high concentrations of Ca2+, is considerably more structured in the presence of the other pep tides, as judged by CD difference spectroscopy. The CD data also sugge st that the packing between the individual subdomains is specific.