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Journal of Protein Chemistry

, Volume 13, Issue 6, pp 527–535 | Cite as

Reductive methylation andpKa determination of the lysine side chains in calbindin D9k

  • Mingjie Zhang
  • Eva Thulin
  • Hans J. Vogel
Article

Abstract

The Lys residues in the 75-residue Ca2+-binding protein calbindin D9k were reductively methylated with13C-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 apo-forms of the protein, the13C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (1H,13C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca2+-calbindin D9k and apo-calbindin D9k were obtained by combiningpH titration experiments and (1H,13C)-HMQC NMR spectroscopy. Each Lys residue in the Ca2+- and apo-forms of calbindin D9k has a uniquepKa value. The LyspKa 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 Ca2+-calbindin D9k, the removal of two Ca2+ ions from the protein leads to an increase of thepKa values of the Lys residues.

Key words

Calbindin D9k lysine reductive methylation pKa NMR 

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Copyright information

© Plenum Publishing Corporation 1994

Authors and Affiliations

  • Mingjie Zhang
    • 1
  • Eva Thulin
    • 2
  • Hans J. Vogel
    • 1
  1. 1.Department of Biological SciencesUniversity of CalgaryAlbertaCanada
  2. 2.Department of Physical Chemistry 2, Chemical CenterUniversity of LundLundSweden

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