Binding of Ca2+ to calbindin D9k: structural stability and function at high salt concentration.
Calcium binding constants of wild-type calbindin D9k and mutant forms with one, two, and three neutralized negative charges in the vicinity of the Ca2+ binding sites are determined at varying KCl concentrations from 2 mM to 1 M. The results indicate that the added salt does not cause significant structural changes in calbindin D9k and, along with site-directed mutagenesis, can be used as a well-controlled means for modulating electrostatic interactions. The lack of structural changes at high salt concentrations is also supported by two-dimensional 1H NMR data. High salt concentrations are observed to substantially reduce the cooperativity of calcium binding to calbindin D9k. This suggests that the cooperativity is strongly dependent on electrostatic interactions. The data have been used to test a dielectric continuum model for protein electrostatics using a macroscopic dielectric constant of water throughout the system. Excellent agreement between experiment and Monte Carlo simulations is observed for the whole set of data covering changes in the binding constant of more than 6 orders of magnitude. A simplified theoretical treatment using the Kirkwood-Tanford formula, based on the Debye-Hückel approximation, yields an almost equally good agreement with the experiment.
Kesvatera
T
T
Chemical Centre, Lund, Sweden.
Jönsson
B
B
Thulin
E
E
Linse
S
S
D001665:Binding Sites; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D004355:Drug Stability; D004563:Electrochemistry; D004926:Escherichia coli; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009010:Monte Carlo Method; D016297:Mutagenesis, Site-Directed; D011189:Potassium Chloride / Q000008:administration & dosage / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D064026:Calbindins; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D011189:Potassium Chloride; D002118:Calcium