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

Abstract

We prepared five mutant lysozymes in which glycines whose dihedral angles are located in the region of the left-handed helix, Gly49, Gly67, Gly71, Gly102 and Gly117, were mutated to an alanine residue. From analyses of their thermal stabilities using differential scanning calorimetry, most of them were more destabilized than the native lysozyme, except for the G102A mutant, which has a stability similar to that of the native lysozyme at pH 2.7. As for the destabilized mutant lysozymes, their X-ray crystallographic analyses showed that their global structures did not change but that the local structures changed slightly. By examining the dihedral angles at the mutation sites based on X-ray crystallographic results, it was found that the dihedral angles at these mutation sites tended to adopt favorable values in a Ramachandran plot and that the extent and direction of their shifts from the original value had similar tendencies. Therefore, the change in dihedral angles may be the cause of the slight local structural changes around the mutation site. On the other hand, regarding the mutation of G102A, the global structure was almost identical with that of the native structure but the local structure was drastically changed. Therefore, it was suggested that the drastic local conformational change might be effective in releasing the unfavorable interaction of the native state at the mutation site.

Hydration of the peptide backbone largely defines the thermodynamic propensity scale of residues at the C′ position of the C-capping box of α-helices

“…It is conceivable that the dihedral angles of the CЈ residue in the G35V variant are moving closer to the allowable ␣ L region, leading to the changes in the resonances of the adjacent in-sequence residues. Such changes have been previously observed for T4 lysozyme (20), hen egg lysozyme (19), and rop (21). Overall it is apparent that there is no significant difference between the most stable and the least stable position 35 ubiquitin variant and thus we can assume that the structures of the remaining position 35 ubiquitin variants (with G35P to a lesser degree) are also structurally similar to the WT.…”

“…1, all three residues in the G35V variant show changes largely along the 1 H-axes. The changes appear to be consistent with a small rearrangement of the peptide backbone that alters the relative position of the amide protons (19). The dihedral angles for the CЈ position of ubiquitin in the WT protein are ϭ 81.2°and ϭ 5.3°, well outside the allowable positive dihedral angles for any nonglycine residue.…”

Calorimetric Analysis of Thermodynamic Stability and Aggregation for Apo and Holo Amyotrophic Lateral Sclerosis-associated Gly-93 Mutants of Superoxide Dismutase

“…Further study is clearly still required to understand the molecular basis for changes in stability caused by apparently small changes such as residue substitution or metal binding. We note, however, that because Gly-93 is relatively rigid in SOD (6,62), mutations may be expected to be more strongly destabilizing compared with mutations at more mobile positions (68).…”

“…However, metal binding has similar effects on t m and ⌬G in mutants and pseudo-WT (supplemental Table SI) and so the mutations do not substantially weaken metal binding. In other proteins, comparable mutations involving glycines resulted in similar changes in stability, because of either enthalpy or entropy changes (65)(66)(67)(68). Further study is clearly still required to understand the molecular basis for changes in stability caused by apparently small changes such as residue substitution or metal binding.…”

A Protein’s Conformational Stability Is an Immunologically Dominant Factor: Evidence That Free-Energy Barriers for Protein Unfolding Limit the Immunogenicity of Foreign Proteins

“…Differential scanning calorimetry (DSC) measurements and data analyses were carried out using a VP-DSC (GE Healthcare UK) system equipped with a Gateway personal computer, as described in the literature (26). The scan rate was 1.0˚C/min.…”