Computationally designed libraries for rapid enzyme stabilization.

TitleComputationally designed libraries for rapid enzyme stabilization.
Publication TypeJournal Article
Year of Publication2014
AuthorsWijma, H. J., Floor R. J., Jekel P. A., Baker D., Marrink S. J., & Janssen D. B.
JournalProtein engineering, design & selection : PEDS
Volume27
Issue2
Pagination49-58
Date Published02/2014
ISSN1741-0134
KeywordsCollaborative Publication
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 10-12 of these confirmed mutations resulted in multi-site mutants with an increase in apparent melting temperature from 50 to 85°C, 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.

DOI10.1093/protein/gzt061
Custom1

http://www.ncbi.nlm.nih.gov/pubmed/24402331?dopt=Abstract

Alternate JournalProtein Eng. Des. Sel.
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