BINDING OF CA2+ TO CALBINDIN D-9K - STRUCTURAL STABILITY AND FUNCTION AT HIGH-SALT CONCENTRATION

Calcium binding constants of wild-type calbindin D-9k and mutant forms with one, two, and three neutralized negative charges in the vicinity of the Ca2+ binding sites are determined at varying KCl concentrations from 2 mM to 1 M. The results indicate that the added salt does not cause significant structural changes in calbindin D-9k and, along with site-directed mutagenesis, can be used as a well-controlled means for modulating electrostatic interactions. The lack of structural changes at high salt concentrations is also supported by two-dimensional H-1 NMR data. High salt concentrations are observed to substantially reduce the cooperativity of calcium binding to calbindin D-9k. This suggests that the cooperativity is strongly dependent on electrostatic interactions. The data have been used to test a dielectric continuum model for protein electrostatics using a macroscopic dielectric constant of water throughout the system. Excellent agreement between experiment and Monte Carlo simulations is observed for the whole set of data covering changes in the binding constant of more than 6 orders of magnitude. A simplified theoretical treatment using the Kirkwood-Tanford formula, based on the Debye-Huckel approximation, yields an almost equally good agreement with the experiment.