DISCONTINUOUS EQUILIBRIUM TITRATIONS OF COOPERATIVE CALCIUM-BINDING TO CALMODULIN MONITORED BY 1-D H-1-NUCLEAR MAGNETIC-RESONANCE SPECTROSCOPY

Calmodulin binds up to four calcium ions cooperatively in response to cellular signaling events. To understand the functional energetics of calcium activation of calmodulin, it is important to monitor individua l Ca2+-binding sites and other positions at partial degrees of saturat ion. This study is the first use of 1-D proton NMR to monitor the equi librium Ca2+-binding properties of calmodulin. Protein concentrations required for NMR experiments (similar to 1 mM) are similar to 1000-fol d greater than the Kd values for calcium binding to calmodulin, preven ting a direct continuous equilibrium titration of calmodulin. Thus, di alysates of calmodulin in buffers of experimentally determined [Ca2+]( free) were prepared to conduct discontinuous equilibrium titrations at both 92 and 152 mM KCI, For the C-terminal domain, the normalized are a of the 8-protons of Y138 defined calcium binding isotherms. For N-te rminal domain resonances (F16(C delta H), T26(C alpha H), D64(C alpha H), and F65(C delta H)), the calcium-dependent change in chemical shif t defined isotherms. These are the first residue-specific studies to m onitor the energetics of Ca2+ binding to the N-terminal domain in wild -type hole calmodulin. Calcium binding to both domains appeared cooper ative and binding affinity decreased in higher KCl. Isotherms resolved from the side chain resonances of F16 and F65 had a lower median liga nd activity and a slightly higher degree of cooperativity than isother ms resolved from the backbone resonances of D64 and T26, Salt-dependen t changes in apparent intradomain cooperativity differed for the domai ns: at higher salt, Delta G(c) increased for the C-terminal domain whi le remaining constant or decreasing for the N-terminal domain.