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

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.