Showing papers by "Madeline A. Shea published in 1995"

Quantitative endoproteinase GluC footprinting of cooperative Ca2+ binding to calmodulin: proteolytic susceptibility of e31 and e87 indicates interdomain interactions

Abstract: Calmodulin is the primary eukaryotic intracellular calcium receptor. Cooperative calcium binding to two sites in each of two domains drives large conformational changes that enable it to activate target proteins. An understanding of the molecular mechanism of cooperativity requires determination of the conformational states populated by calmodulin, the intrinsic free energies of binding calcium to four sites, and the nature and degree of intradomain and interdomain interactions. To monitor residue-specific conformational changes within calmodulin as calcium binds, we have developed a new quantitative proteolytic footprinting method using endoproteinase GluC (EndoGluC). Under conditions of very limited proteolysis, cleavage occurred at only five of the sixteen positions possible in calmodulin. The relative abundance of fragments indicated that calcium induced changes in the susceptibility of individual peptide bonds. Quantitative susceptibility profiles were resolved for two positions: E31-L32, in site I in the N-terminal domain, and E87-A88, preceding site III in the C-terminal domain. In apocalmodulin, E87-A88 was susceptible to EndoGluC; calcium binding to sites III and IV caused monotonic protection from proteolysis. The response of E31-L32 was biphasic. In apocalmodulin, it was resistant to cleavage. Susceptibility was induced by calcium binding to sites III and IV, followed by protection induced by calcium binding to sites I and II. This indicated that calmodulin must adopt at least three distinguishable conformations and suggested that the two domains interact. Model-dependent equilibrium constants were resolved from the EndoGluC susceptibility profiles for E31 and E87; they indicated cooperative binding within each domain. Approaches taken to validate this proteolytic footprinting method are described.

Discontinuous equilibrium titrations of cooperative calcium binding to calmodulin monitored by 1-D 1H-nuclear magnetic resonance spectroscopy.

Abstract: 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 individual Ca(2+)-binding sites and other positions at partial degrees of saturation. This study is the first use of 1-D proton NMR to monitor the equilibrium Ca(2+)-binding properties of calmodulin. Protein concentrations required for NMR experiments (approximately 1 mM) are approximately 1000-fold greater than the Kd values for calcium binding to calmodulin, preventing a direct continuous equilibrium titration of calmodulin. Thus, dialysates of calmodulin in buffers of experimentally determined [Ca2+]free were prepared to conduct discontinuous equilibrium titrations at both 92 and 152 mM KCl. For the C-terminal domain, the normalized area of the delta-protons of Y138 defined calcium binding isotherms. For N-terminal domain resonances (F16C delta H, T26C alpha H, D64C alpha H, and F65C delta H), the calcium-dependent change in chemical shift defined isotherms. These are the first residue-specific studies to monitor the energetics of Ca2+ binding to the N-terminal domain in wild-type holo calmodulin. Calcium binding to both domains appeared cooperative and binding affinity decreased in higher KCl. Isotherms resolved from the side chain resonances of F16 and F65 had a lower median ligand activity and a slightly higher degree of cooperativity than isotherms resolved from the backbone resonances of D64 and T26. Salt-dependent changes in apparent intradomain cooperativity differed for the domains: at higher salt, delta Gc increased for the C-terminal domain while remaining constant or decreasing for the N-terminal domain.