MEASUREMENT AND MODELING OF SEQUENCE-SPECIFIC PK(A) VALUES OF LYSINE RESIDUES IN CALBINDIN D-9K

A pH titration study of calbindin D-9k was performed using heteronucle ar H-1-C-13 two-dimensional NMR spectroscopy. The protein was produced with carbon-13 label in the side-chain of lysine residues, next to th e titrating group. The site-specific pK(a) values of these lysine resi dues, ranging from 10.1 to 12.1, were obtained from the analysis of pH -dependent chemical shifts of C-13 and H-1 resonances. Ionization cons tants for both the Ca2+-free (ape) and Ca2+-loaded forms of the protei n were determined. The proton uptake by lysine residues in the apo for m was shifted up to 1.7 units towards high pH as compared to that for the model compound. The binding of calcium affected the pK(a) values o f all lysine residues. The largest reduction of one pK unit was observ ed 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, red uced the pK(a) values by 0.1 to 0.4 pK unit in agreement with the scre ening concept of ionic interactions. All the observed pK(a) shifts wer e site-specific, depending on the local electrostatic environment and were reproduced in Monte Carlo simulations based on the three-dimensio nal structure of calbindin D-9k and a dielectric continuum model for t he electrostatic interactions. (C) 1996 Academic Press Limited