Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1994 Aug;13(6):527-35.
doi: 10.1007/BF01901534.

Reductive methylation and pKa determination of the lysine side chains in calbindin D9k

Affiliations

Reductive methylation and pKa determination of the lysine side chains in calbindin D9k

M Zhang et al. J Protein Chem. 1994 Aug.

Abstract

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.

PubMed Disclaimer

References

    1. J Biol Chem. 1986 Jul 5;261(19):8761-77 - PubMed
    1. Methods Enzymol. 1983;91:570-9 - PubMed
    1. Proteins. 1993 Mar;15(3):252-65 - PubMed
    1. Eur J Biochem. 1990 Jan 26;187(2):455-60 - PubMed
    1. Biochemistry. 1990 Jun 26;29(25):5925-34 - PubMed

Publication types