A metal binding in the polypeptide chain improves the folding efficiency of a denatured and reduced protein

In order to examine the effect of metal binding to the polypeptide chain on the aggregation of a protein in the refolding process, we prepared a mutant hen lysozyme possessing the same Ca 2 + binding site as in human α-lactalbumin by Escherichia coli expression system (Ser - 1 CaB lysozyme). In the presence of 2 mM CaCl 2 , the refolding yield of Ser - 1 CaB lysozyme at a low protein concentration (25 μg/mL) was similar to that of the wild-type lysozyme (80%), but that at high protein concentration (200 μg/mL) decreased (15%) due to aggregation comparing to that of the wild-type lysozyme (45%). However, the refolding yield of Ser - 1 CaB lysozyme in the presence of 100 mM CaCl 2 even at a protein concentration of 200 μg/mL was 80% and was higher than that of the wild-type lysozyme. From analysis of chemical shift changes of the cross peaks in the backbone region of total correlated spectroscopy (TOCSY) spectra of a decapeptide possessing the same calcium binding site as in Ser - 1 CaB lysozyme in the presence of various concentrations of Ca 2 + , it was suggested that the dissociation constant of Ca 2 + -peptide complex was estimated to be 20-36 mM. Moreover, the solubility of the denatured Ser - 1 CaB lysozyme in the presence of 100 mM CaCl 2 was higher than that in the presence of 2 mM CaCl 2 whereas the solubility of the denatured Ser - 1 lysozyme in the presence of 100 mM CaCl 2 was not higher than that in the presence of 2 mM CaCl 2 . Therefore, it was concluded that the reduced lysozyme possessing the Ca 2 + binding site was efficiently folded in the presence of high concentration of Ca 2 + (100 mM) even at high protein concentration due to depression of aggregation by the binding of Ca 2 + to the polypeptide chain in Ser - 1 CaB lysozyme.