National Academy of Sciences, Proceedings of the National Academy of Sciences, 47(114), p. 12472-12477, 2017
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Significance The marginal stability of most natural proteins presents a challenge for the exploitation of natural and engineered enzymes in biotechnology and industrial biocatalysis. Protein stabilization can be time- and labor-intensive and often involves extensive amino acid substitutions, which may impair the activity and/or selectivity of the enzyme. Here, we describe a computational design method for enzyme stabilization that uses structure-based modeling to introduce covalent ‟staples” in a protein scaffold via a genetically encodable noncanonical amino acid. This method was applied to obtain stapled variants of a stereoselective cyclopropanation biocatalyst featuring greatly increased thermostability and robustness to high concentrations of organic cosolvents. This minimally invasive strategy for protein stabilization should be applicable to a variety of enzymes and proteins.