Reductive methylation and pKa determination of the lysine side chains in calbindin D9k.
The Lys residues in the 75-residue Ca(2+)-binding protein calbindin D9k were reductively methylated with 13C-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 Ca(2+)- and apo-forms of the protein, the 13C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (1H, 13C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. The pKa values of the individual Lys residues in Ca(2+)-calbindin D9k and apo-calbindin D9k were obtained by combining pH titration experiments and (1H, 13C)-HMQC NMR spectroscopy. Each Lys residue in the Ca(2+)- and apo-forms of calbindin D9k 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 D9k has a very similar structure compared to Ca(2+)-calbindin D9k, the removal of two Ca2+ ions from the protein leads to an increase of the pKa values of the Lys residues.
Department of Biological Sciences, University of Calgary, Alberta, Canada.
M
M
Zhang
E
E
Thulin
H J
HJ
Vogel
D064026:Calbindins; D006863:Hydrogen-Ion Concentration; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D064030:S100 Calcium Binding Protein G / Q000737:chemistry*
D064026:Calbindins; D064030:S100 Calcium Binding Protein G; D008239:Lysine