Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 114, Issue 47, Pages 12472-12477Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1708907114
Keywords
protein thermostabilization; computational protein design; myoglobin; Rosetta macromolecular modeling; noncanonical amino acids
Categories
Funding
- National Institute of Health [GM098628]
- National Science Foundation [MCB1716623, MCB1330760, CHE-0840410, CHE-0946653]
- NIH [T32GM118283]
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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 (Delta T-m = +18.0 degrees C and Delta T-50 = +16.0 degrees C) as well as increased stability against chemical denaturation [Delta C-m (GndHCI) = 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.
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