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

• Published in: Journal of the American Chemical Society Vol. 126; no. 41; pp. 13194 - 13195
• Main Authors: Anil, Burcu, Song, Benben, Tang, Yuefeng, Raleigh, Daniel P
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 20-10-2004
• Subjects: Alanine - chemistry; Amino Acid Substitution; Biological and medical sciences; Biotechnology; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; Glycine - chemistry; Methods. Procedures. Technologies; Models, Molecular; Mutagenesis, Site-Directed; Protein Folding; Protein Structure, Tertiary; Ribosomal Proteins - chemistry; Structure-Activity Relationship; Synthetic digonucleotides and genes. Sequencing; Thermodynamics; Protein engineering; Chemical modification; Conformational dynamics; Genetic variant; Chemical stabilization; Chemical denaturation; Chemical substitution; Globular protein
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja047119i

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.