In a staphylococcal nuclease mutant the side-chain of a lysine replacing valine 66 is fully buried in the hydrophobic core

The crystal structure of the staphylococcal nuclease mutant V66K, in which valine 66 is replaced by lysine, has been solved at 1·97 Å resolution. Unlike lysine residues in previously reported protein structures, this residue appears to bury its side-chain in the hydrophobic core without salt bridging, hydrogen bonding or other forms of electrostatic stabilization. Solution studies of the free energy of denaturation. ΔG_H2O, show marked pH dependence and clearly indicate that the lysine residue must be deprotonated in the folded state. V66K is highly unstable at neutral pH but only modestly less stable than the wild-type protein at high pH. The pH dependence of stability for V66K, in combination with similar measurements for the wild-type protein, allowed determination of the pK_a values of the lysine in both the denatured and native forms. The ε{lunate}-amine of this residue has a pK_a value in the denatured state of 10·2, but in the native state it must be 6·4 or lower. The ε{lunate}-amine is thus deprotonated in the folded molecule. These values enabled an estimation of the ε{lunate}-amine's relative change in free energy of solvation between solvent and the protein interior at 5·1 kcal/mol or greater. This implies that the value of the dielectric constant of the protein interior must be less than 12·8. Lysine is usually found with the methylene groups of its side-chain partly buried but is nevertheless considered a hydrophilic surface residue. It would appear that the high pK_a value of lysine, which gives it a positive charge at physiological pH, is the primary reason for its almost exclusive confinement to the surface proteins. When deprotonated, this amino acid type can be fully incorporated into the hydrophobic core.