Role of amino acid residues in left-handed helical conformation for the conformational stability of a protein

Our previous study of six non-Gly to Gly/Ala mutant human lysozymes in a le ft-handed helical region showed that only one non-Gly residue at a rigid si te had unfavorable strain energy as compared with Gly at the same position (Takano et al., Proteins 2001; 44:233-243). To further examine the role of left-handed residues in the conformational stability of a protein, we const ructed ten Gly to Ala mutant human lysozymes. Most Gly residues in human ly sozyme are located in the left-handed helix region. The thermodynamic param eters for denaturation and crystal structures were determined by differenti al scanning calorimetry and Xray analysis, respectively. The difference in denaturation Gibbs energy (Delta DeltaG) for the ten Gly to Ala mutants ran ged from + 1.9 to -7.5 kJ/mol, indicating that the effect of the mutation d epends on the environment of the residue. We confirm that Gly in a left-han ded region is more favorable at rigid sites than non-Gly, but there is litt le difference in energetic cost between Gly and non-Gly at flexible sites. The present results indicate that dihedral angles in the backbone conformat ion and also the flexibility at the position should he considered for analy ses of protein stability, and protein structural determination, prediction, and design. (C) 2001 Wiley-Liss, Inc.