Role of surface hydrophobic residues in the conformational stability of human lysozyme at three different positions

To evaluate the contribution of the amino acid residues on the surface of a protein to its stability, a series of hydrophobic mutant human lysozymes ( Val to Gly, Ala, Leu, Ile, Met, and Phe) modified at three different positi ons on the surface, which are located in the alpha -helix (Val 110), the be ta -sheet (Val 2), and the loop (Val 74), were constructed. Their thermodyn amic parameters of denaturation and crystal structures were examined by cal orimetry and by X-ray crystallography at 100 K, respectively. Differences i n the denaturation Gibbs energy change between the wild-type and the hydrop hobic mutant proteins ranged from 4.6 to -9.6 kJ/mol, 2.7 to -1.5 kJ/mol, a nd 3.6 to -0.2 kJ/mol at positions 2, 74, and 110, respectively. The identi cal substitution at different positions and different substitutions at the same position resulted in different degrees of stabilization. Changes in th e stability of the mutant proteins could be evaluated by a unique equation considering the conformational changes due to the substitutions [Funahashi et al. (1999) Protein Eng. 12, 841-850]. For this calculation, secondary st ructural propensities were newly considered. However, some mutant proteins were not adapted to the equation. The hydration structures around the mutat ion sites of the exceptional mutant proteins were affected due to the subst itutions. The stability changes in the exceptional mutant proteins could be explained by the formation or destruction of the hydration structures. The se results suggest that the hydration structure mediated via hydrogen bonds covering the protein surface plays an important role in the conformational stability of the protein.