Discontinuous equilibrium titrations of cooperative calcium binding to calmodulin monitored by 1-D 1H-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 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.
Pedigo
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Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242-1109, USA.
Shea
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MA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D003460:Crystallization; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013816:Thermodynamics
D002147:Calmodulin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D011189:Potassium Chloride; D002118:Calcium