Computationally designed libraries for rapid enzyme stabilization

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

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

197 Citaten (Scopus)
1354 Downloads (Pure)


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.

Originele taal-2English
Pagina's (van-tot)49-58
Aantal pagina's10
TijdschriftProtein Engineering, Design & Selection
Nummer van het tijdschrift2
StatusPublished - feb.-2014

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