REDUCTIVE METHYLATION AND PKA DETERMINATION OF THE LYSINE SIDE-CHAINS IN CALBINDIN D-9K

The Lys residues in the 75-residue Ca2+-binding protein calbindin D-9k were reductively methylated with C-13-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca2+- and ape-forms of the protein, the C-13-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (H-1, C-13)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. The pKa values of the individual Lys residues in Ca2+-calbindin D-9k and apo-calbindin D-9k were obtained by combining pH titration experiments and (H-1, C-13)-HMQC NMR spectroscopy. Each Lys residue in the Ca2+- and apo-forms of calbindin D-9k has a unique pKa value. The Lys pKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D-9k has a very similar structure compared to Ca2+-calbindin D-9k, the removal of two Ca2+ ions from the protein leads to an increase of the pKa values of the Lys residues.