Computationally designed libraries for rapid enzyme stabilization

Hein J. Wijma, Robert J. Floor, Peter A. Jekel, David Baker, Siewert J. Marrink, Dick B. Janssen*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

139 Citations (Scopus)
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Abstract

The ability to engineer enzymes and other proteins to any desired stability would have wide-ranging applications. Here, we demonstrate that computational design of a library with chemically diverse stabilizing mutations allows the engineering of drastically stabilized and fully functional variants of the mesostable enzyme limonene epoxide hydrolase. First, point mutations were selected if they significantly improved the predicted free energy of protein folding. Disulfide bonds were designed using sampling of backbone conformational space, which tripled the number of experimentally stabilizing disulfide bridges. Next, orthogonal in silico screening steps were used to remove chemically unreasonable mutations and mutations that are predicted to increase protein flexibility. The resulting library of 64 variants was experimentally screened, which revealed 21 (pairs of) stabilizing mutations located both in relatively rigid and in flexible areas of the enzyme. Finally, combining 1012 of these confirmed mutations resulted in multi-site mutants with an increase in apparent melting temperature from 50 to 85C, enhanced catalytic activity, preserved regioselectivity and a 250-fold longer half-life. The developed Framework for Rapid Enzyme Stabilization by Computational libraries (FRESCO) requires far less screening than conventional directed evolution.

Original languageEnglish
Pages (from-to)49-58
Number of pages10
JournalProtein Engineering, Design & Selection
Volume27
Issue number2
DOIs
Publication statusPublished - Feb-2014

Keywords

  • enzyme stability
  • in silico design
  • in silico screening
  • protein stability engineering
  • thermostability
  • ITERATIVE SATURATION MUTAGENESIS
  • MOLECULAR-DYNAMICS SIMULATIONS
  • ENGINEERED DISULFIDE BONDS
  • BACKBONE PROTEIN DESIGN
  • DIRECTED EVOLUTION
  • LIMONENE-1,2-EPOXIDE HYDROLASE
  • RATIONAL DESIGN
  • STABILITY
  • THERMOSTABILITY
  • BIOCATALYSIS

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