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 (1)H-(15)N NMR data on the Lys-66 side chain amine (N zeta) group in the Delta-PHS/V66K variant of staphylococcal nuclease. Previous crystallographic study has shown that the Lys-66 N zeta group is completely buried in the hydrophobic core. On the basis of double and triple resonance experiments, we found that the (1)H zeta and (15)N zeta chemical shifts at pH 8.0 and 6 degrees C for the buried lysine are 0.81 and 23.3 ppm, respectively, which are too abnormal to correspond to the protonated (NH(3)(+)) state. Further investigations using a model system suggested that the abnormal (1)H and (15)N chemical shifts represent the deprotonated (NH(2)) state of the Lys-66 N zeta group. More straightforward evidence for the deprotonation was obtained with 2D F1-(1)H-coupled (1)H-(15)N heteronuclear correlation experiments. Observed (15)N multiplets clearly indicated that the spin system for the Lys-66 N zeta group is AX(2) (NH(2)) rather than AX(3) (NH(3)(+)) Interestingly, although the amine group is buried in the hydrophobic core, the hydrogen exchange between water and the Lys-66 N zeta group was found to be relatively rapid (93 s(-1) at -1 degrees C), which suggests the presence of a dynamic process such as local unfolding or water penetration. The partial self-decoupling effect on (15)N zeta multiplets due to the rapid hydrogen exchange is also discussed.