Enzyme stabilization via computationally guided protein stapling

Proc Natl Acad Sci U S A. 2017 Nov 21;114(47):12472-12477. doi: 10.1073/pnas.1708907114. Epub 2017 Nov 6.


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.

Keywords: Rosetta macromolecular modeling; computational protein design; myoglobin; noncanonical amino acids; protein thermostabilization.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Validation Study

MeSH terms

  • Biocatalysis
  • Computational Biology
  • Enzyme Stability
  • Enzymes / chemistry*
  • Kinetics
  • Models, Chemical*
  • Models, Structural
  • Molecular Structure
  • Protein Engineering / methods*
  • Solubility
  • Temperature


  • Enzymes