Site-Specific Measurement of Terbium and Calcium Binding to Calmodulin and Calmodulin Fragments

Abstract

Calcium controls a variety of cellular processes including muscle contraction, cellular motility, and synaptic function. A key player in many of these signaling events is the evolutionarily conserved Ca2+ sensor, calmodulin (CaM), a 148 amino acid protein consisting of four EF hand Ca2+-binding domains arranged as pairs in two globular domains (N- and C-lobe) connected by a flexible linker. Though extensive work has investigated Ca2+ binding to CaM, published Ca2+ association constants and cooperativity factors vary by several orders of magnitude. Surprisingly little of this variability is attributable to differences in experimental conditions. We have determined through simulations that macroscopic total binding curves are fundamentally insufficient to constrain a complete, quantitative model of Ca2+ binding to CaM. To improve estimates of Ca2+ affinity and cooperativity in binding to CaM we have conducted site-specific binding measurements at each binding site. To detect metal occupancy at individual sites, terbium (Tb3+) ions, which have been shown to effectively substitute for Ca2+ in activating CaM, are sensitized via energy transfer from a minimally invasive tryptophan residue inserted in each EF hand. Through competition experiments, both Ca2+ and Tb3+ association constants can be determined. We have obtained site-specific Tb3+ binding isotherms at all four sites in full CaM and measurements of Tb3+ binding and Ca2+ competition in an isolated, N-lobe CaM fragment. These data suggest distinct Ca2+ and Tb3+ affinities at each of the sites in the N-lobe CaM fragment and distinct Tb3+ affinities at all four sites in full CaM. The additional information obtained through site-specific binding measurements enhances estimates of Ca2+ affinity and cooperativity in binding to CaM. This technique is powerful in its applicability to a variety of EF-hand containing calcium-sensing proteins in isolation or in complex with signaling partners.