Comparison of Five Protein Engineering Strategies for Stabilizing an α/β-Hydrolase.

TitleComparison of Five Protein Engineering Strategies for Stabilizing an α/β-Hydrolase.
Publication TypeJournal Article
Year of Publication2017
AuthorsJones BJ, Lim HYee, Huang J, Kazlauskas RJ
Date Published2017 Dec 19
KeywordsAmino Acid Sequence, Base Sequence, Crystallography, X-Ray, Enzyme Stability, Hydrolases, Models, Molecular, Mutagenesis, Mutation, Point Mutation, Protein Engineering, Proteins

A review of the previous stabilization of α/β-hydrolase fold enzymes revealed many different strategies, but no comparison of strategies on the same enzyme. For this reason, we compared five strategies to identify stabilizing mutations in a model α/β-hydrolase fold enzyme, salicylic acid binding protein 2, to reversible denaturation by urea and to irreversible denaturation by heat. The five strategies included one location agnostic approach (random mutagenesis using error-prone polymerase chain reaction), two structure-based approaches [computational design (Rosetta, FoldX) and mutation of flexible regions], and two sequence-based approaches (addition of proline at locations where a more stable homologue has proline and mutation to consensus). All strategies identified stabilizing mutations, but the best balance of success rate, degree of stabilization, and ease of implementation was mutation to consensus. A web-based automated program that predicts substitutions needed to mutate to consensus is available at .

Alternate JournalBiochemistry
PubMed ID29087185
PubMed Central IDPMC5736438
Grant ListR01 GM102205 / GM / NIGMS NIH HHS / United States
T32 GM008347 / GM / NIGMS NIH HHS / United States