Abstract
A pH titration study of calbindin D 9kwas performed using heteronuclear 1H- 13C two-dimensional NMR spectroscopy. The protein was produced with carbon-13 label in the side-chain of lysine residues, next to the titrating group. The site-specific p K avalues of these lysine residues, ranging from 10.1 to 12.1, were obtained from the analysis of pH-dependent chemical shifts of 13C and 1H resonances. Ionization constants for both the Ca 2 +-free (apo) and Ca 2 +-loaded forms of the protein were determined. The proton uptake by lysine residues in the apo form was shifted up to 1.7 units towards high pH as compared to that for the model compound. The binding of calcium affected the p K avalues of all lysine residues. The largest reduction of one p Kunit was observed for Lys55, which is also the closest to the calcium binding sites. A threefold increase in protein concentration, from 0.5 to 1.5 mM, reduced the p K avalues by 0.1 to 0.4 p Kunit in agreement with the screening concept of ionic interactions. All the observed p K ashifts were site-specific, depending on the local electrostatic environment and were reproduced in Monte Carlo simulations based on the three-dimensional structure of calbindin D 9kand a dielectric continuum model for the electrostatic interactions.
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