Edited by David A. Baker, University of Washington, Seattle, WA, and approved October 6, 2017 (received for review May 29, 2017)
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.
Thermostabilization represents a critical and often obligatory step toward enhancing the robustness of enzymes for organic synthesis and other applications. While directed evolution methods have provided valuable tools for this purpose, these protocols are laborious and time-consuming and typically require the accumulation of several mutations, potentially at the expense of catalytic function. Here, we report a minimally invasive strategy for enzyme stabilization that relies on the installation of genetically encoded, nonreducible covalent staples in a target protein scaffold using computational design. This methodology enables the rapid development of myoglobin-based cyclopropanation biocatalysts featuring dramatically enhanced thermostability (ΔTm = +18.0 °C and ΔT50 = +16.0 °C) as well as increased stability against chemical denaturation [ΔCm (GndHCl) = 0.53 M], without altering their catalytic efficiency and stereoselectivity properties. In addition, the stabilized variants offer superior performance and selectivity compared with the parent enzyme in the presence of a high concentration of organic cosolvents, enabling the more efficient cyclopropanation of a water-insoluble substrate. This work introduces and validates an approach for protein stabilization which should be applicable to a variety of other proteins and enzymes.
Author contributions: E.J.M., S.D.K., and R.F. designed research; E.J.M., D.Z., and W.A.H. performed research; E.J.M., D.Z., W.A.H., S.D.K., and R.F. analyzed data; and S.D.K. and R.F. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1708907114/-/DCSupplemental.
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