Structure-function relationships in EF-hand Ca2+-binding proteins. Protein engineering and biophysical studies of calbindin D/sub 9k/

Genes encoding the minor A component of bovine calbindins D/sub 9k/-the smallest protein known with a pair of EF-hand calcium-binding sites-with amino acid substitutions and/or deletions have been synthesized and expressed in Escherichia coli and characterized with different biophysical techniques. The mutations are confined to the N-terminal Ca2+-binding site and constitute Pro-20 → Gly (M1), Pro-20 → Gly and Asn-21 deleted (M2), Pro-20 deleted (M3), and Tyr-13 → Phe (M4). 1H, 43Ca, and 113Cd NMR studies show that the structural changes induced are primarily localized in the modified region, with hardly any effects on the C-terminal Ca2+-binding site. The Ca2+ exchange rate for the N-terminal site changes from 3 s-1 in the wild-type protein (M0) and M4 to 5000 s-1 in M2 and M3, whereas there is no detectable variation in the Ca2+ exchange from the C-terminal site. The macroscopic Ca2+binding constants have been obtained from equilibration in the presence of a fluorescent chelator or by using a Ca2+-selective electrode. Microcalorimetric data show that the enthalpy of Ca2+-binding is negative for all sites except the N-terminal site in M2 and M3. The binding entropy is strongly positive in all cases. Through the observed changes in the 1H NMR spectra during Ca2+-titrations the authors could obtain ratios between site binding constants in M0 and M4. There is evidence (from 113 Cd NMR) for site-site interactions also in M1, M2, and M3, but the magnitude of ΔΔG could not be determined because of sequential Ca2+ binding