Direct evidence for deprotonation of a lysine side chain buried in the hydrophobic core of a protein

Yuki Takayama, Carlos A. Castañeda, Michael Chimenti, Bertrand García-Moreno, Junji Iwahara

Research output: Contribution to journalArticle

40 Scopus citations


We report direct evidence for deprotonation of a lysine side chain buried in the hydrophobic core of a protein, demonstrating heteronuclear 1H-15N NMR data on the Lys-66 side chain amine (Nζ) group in the Δ-PHS/V66K variant of staphylococcal nuclease. Previous crystallographic study has shown that the Lys-66 Nζ group is completely buried in the hydrophobic core. On the basis of double and triple resonance experiments, we found that the 1Hζ and 15Nζ chemical shifts at pH 8.0 and 6 °C for the buried lysine are 0.81 and 23.3 ppm, respectively, which are too abnormal to correspond to the protonated (NH3 +) state. Further investigations using a model system suggested that the abnormal 1H and 15N chemical shifts represent the deprotonated (NH2) state of the Lys-66 Nζ group. More straightforward evidence for the deprotonation was obtained with 2D F1-1H-coupled 1H-15N heteronuclear correlation experiments. Observed 15N multiplets clearly indicated that the spin system for the Lys-66 Nζ group is AX2 (NH2) rather than AX3 (NH3 +). Interestingly, although the amine group is buried in the hydrophobic core, the hydrogen exchange between water and the Lys-66 Nζ group was found to be relatively rapid (93 s-1 at -1 °C), which suggests the presence of a dynamic process such as local unfolding or water penetration. The partial self-decoupling effect on 15Nζ multiplets due to the rapid hydrogen exchange is also discussed.

Original languageEnglish (US)
Pages (from-to)6714-6715
Number of pages2
JournalJournal of the American Chemical Society
Issue number21
StatePublished - May 28 2008


ASJC Scopus subject areas

  • Chemistry(all)

Cite this