title: Direct evidence for deprotonation of a lysine side chain buried in the hydrophobic core of a protein.
abstract: 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 (Nzeta) group in the delta-PHS/V66K variant of staphylococcal nuclease. Previous crystallographic study has shown that the Lys-66 Nzeta group is completely buried in the hydrophobic core. On the basis of double and triple resonance experiments, we found that the 1Hzeta and 15Nzeta 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 (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 Nzeta 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 Nzeta 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 Nzeta 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 15Nzeta multiplets due to the rapid hydrogen exchange is also discussed.
authors.affiliation: Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0647, USA.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Takayama
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Castañeda
authors.forename: Michael
authors.initials: M
authors.lastname: Chimenti
authors.forename: Bertrand
authors.initials: B
authors.lastname: García-Moreno
authors.forename: Junji
authors.initials: J
authors.lastname: Iwahara
mesh_terms: D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008836:Micrococcal Nuclease / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011522:Protons
chemical_list: D011522:Protons; D008836:Micrococcal Nuclease; D008239:Lysine