Relationship between local structure and stability in hen egg white lysozyme mutant with alanine substituted for glycine

Kiyonari Masumoto, Tadashi Ueda1, Hiroyuki Motoshima handed helix (Ramachandran et al., 1963). Most amino acid residues, which take a left-handed helix conformation in a and Taiji Imoto protein, have similar dihedral angles. On the other hand, Graduate School of Pharmaceutical Sciences, Kyushu University, glycine residues are often present in the region with a dihedral 62 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan angle around φ 60° and ψ 60°, even though they are not 1To whom correspondence should be addressed. in a left-handed helix. Therefore, glycine residues are unique E-mail: ueda@phar.kyushu-u.ac.jp and more attention should be given to the mutation of glycine to other residues in a protein. Matthews (1987) reported that We prepared five mutant lysozymes in which glycines the loss of entropy in the denatured state of a protein by subwhose dihedral angles are located in the region of the leftstituting alanine for glycine was estimated to be 10.0 J/mol·K, handed helix, Gly49, Gly67, Gly71, Gly102 and Gly117, resulting in stabilization of a protein. On the other hand, were mutated to an alanine residue. From analyses of their substitution of alanine for glycine may induce an unfavorable thermal stabilities using differential scanning calorimetry, interaction in the native state of a protein owing to steric most of them were more destabilized than the native hindrance, resulting in destabilization of the protein. Therefore, lysozyme, except for the G102A mutant, which has a in order to improve the stability of a protein by such a stability similar to that of the native lysozyme at pH 2.7. mutation, we should investigate the conformational change As for the destabilized mutant lysozymes, their X-ray accompanying the mutation in detail. So far, although there crystallographic analyses showed that their global struchave been many examples of the mutation of glycine to alanine tures did not change but that the local structures changed in proteins, there have been no systematic analyses of the slightly. By examining the dihedral angles at the mutation mutation in a protein. Hen egg white lysozyme is the first sites based on X-ray crystallographic results, it was found enzyme whose tertiary structure has been elucidated (Blake that the dihedral angles at these mutation sites tended to et al., 1965). Moreover, many data exist on thermal denaturation adopt favorable values in a Ramachandran plot and that using differential scanning calorimetry (DSC). Therefore, we the extent and direction of their shifts from the original consider that it is a suitable protein with which to investigate value had similar tendencies. Therefore, the change in the relationship between structure and stability in mutant dihedral angles may be the cause of the slight local lysozymes where glycines are mutated to alanines. There are structural changes around the mutation site. On the other 12 glycines in hen egg white lysozyme. The dihedral angles hand, regarding the mutation of G102A, the global strucat eight of these 12 glycine residues are located in the region ture was almost identical with that of the native structure of the left-handed helix (Figure 1). Five of those glycines, but the local structure was drastically changed. Therefore, Gly49, Gly67, Gly71, Gly102 and Gly117, are located at the it was suggested that the drastic local conformational surface of the lysozyme molecule (Blake et al., 1965). In this change might be effective in releasing the unfavorable work, we prepared five one-point mutants of hen egg white interaction of the native state at the mutation site. lysozyme, whose glycines were mutated to alanines, and