Direct Evidence for Deprotonation of a Lysine Side Chain Buried in the Hydrophobic Core of a Protein

详细信息    查看全文

• 作者：Yuki Takayama ; Carlos A. Castañ ; eda ; Michael Chimenti ; Bertrand Garcí ; a-Moreno ; Junji Iwahara
• 刊名：Journal of the American Chemical Society
• 出版年：2008
• 出版时间：May 28, 2008
• 年：2008
• 卷：130
• 期：21
• 页码：6714 - 6715
• 全文大小：167K
• 年卷期：v.130,no.21(May 28, 2008)
• ISSN：1520-5126

文摘

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.