FORCES BETWEEN MACROSCOPIC SURFACES IN SOLUTIONS OF CALCIUM-BINDING PROTEINS

Forces have been measured between macroscopic sheets of mica exposed t o aqueous solutions of the calcium binding protein, calbindin D-9k. Th e study focuses on the nature of the long-range exponential double lay er force and on the adsorption behavior of calbindin on mica. These tw o features have been monitored as a function of molecular protein comp osition. We have varied the primary structure of calbindin by genetic substitution of specific amino acids. In particular, we report on the effects of neutralizing individual negative amino acid groups. Advanta ge is also taken of the protein's ability to bind calcium ions to redu ce its net negative charge further. At a fixed protein concentration, in the absence of added salt, the variation in net charge affects the decay length of the exponential force at large separations, in the way expected from classical continuum theory. In fact, after spectroscopi c analysis of the solutions we find that the measured decay length agr ees with the Debye length. The force at short separations is greatly a ffected by the presence of calcium bound to the protein. Specifically, when the protein's binding affinity is high, the presence of bound ca lcium provokes a very strong adhesive force between the protein-adsorb ed mica sheets. This we attribute to an ion correlation effect, which in the biological literature would be termed calcium bridging. In this and other cases the conformation of adsorbed caibindin is greatly inf luenced by calcium.