Exploiting the Right Side of the Ramachandran Plot:  Substitution of Glycines by d-Alanine Can Significantly Increase Protein Stability

A major goal of protein engineering is the enhancement of protein stability. Here we demonstrate a rational method for enhancing the stability of globular proteins by targeting glycine residues which adopt conformations with Φ > 0. Replacement of such a glycine by d-alanine can lead to a significant increase in stability. The approach is tested at three sites in two model proteins. NMR and CD indicated that the substitutions do not alter the structure. Replacement of glycine-24 of the N-terminal domain of L9 (NTL9) with d-Ala results in an increase in stability of 1.3 kcal mol-1, while replacement of glycine-34 of NTL9 leads to an increase of 1.9 kcal mol-1. Replacement of glycine-331 of the UBA domain with d-Ala leads to an increase in stability of 0.6 kcal mol-1.