Structure-function relationships in EF-hand calcium-binding proteins. Protein engineering and biophysical studies of calbindin D9k

摘要: Genes encoding the minor A component of bovine calbindins D/sub 9k/-the smallest protein known with a pair EF-hand calcium-binding sites-with amino acid substitutions and/or deletions have been synthesized and expressed in Escherichia coli characterized different biophysical techniques. The mutations are confined to N-terminal Ca/sup 2 +/-binding site constitute Pro-20 ..-->.. Gly (M1), Asn-21 deleted (M2), (M3), Tyr-13 Phe (M4). /sup 1/H, 43/Ca, 113/Cd NMR studies show that structural changes induced primarily localized modified region, hardly any effects on C-terminal site. +/ exchange rate for from 3 s/sup -1/ wild-type (M0) M4 5000 M2 M3, whereas there is no detectable variation macroscopic +/binding constants obtained equilibration presence fluorescent chelator or by using +/-selective electrode. Microcalorimetric data enthalpy negative all sites except M3.more » binding entropy strongly positive cases. Through observed 1/H spectra during +/-titrations authors could obtain ratios between M0 M4. There evidence (from 113/ Cd NMR) site-site interactions also M1, M2, but magnitude ..delta delta..G not be determined because sequential binding.« less