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. 2015 Nov 3;11(11):e1004556.
doi: 10.1371/journal.pcbi.1004556. eCollection 2015 Nov.

FireProt: Energy- And Evolution-Based Computational Design of Thermostable Multiple-Point Mutants

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Free PMC article

FireProt: Energy- And Evolution-Based Computational Design of Thermostable Multiple-Point Mutants

David Bednar et al. PLoS Comput Biol. .
Free PMC article

Abstract

There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

Conflict of interest statement

JD and ZP are founders of the university spin-off company Enantis Ltd. All other authors have no competing interests.

Figures

Fig 1
Fig 1. Workflow of the FireProt method.
Individual steps involved in the energy- and evolution-based approaches
Fig 2
Fig 2. Location of stabilizing mutations in designed enzymes.
A) Locations of substitutions in energy-based, evolution-based and combined mutants of DhaA enzyme. Substitutions in the multiple-point mutant designed by the energy-based approach (DhaA112) are represented as orange spheres, while substitutions in multiple-point mutants designed by the evolution-based approach are represented as red (DhaA100), blue (DhaA101), green (DhaA102) and magenta (DhaA103) spheres. Mutations in the combined mutant (DhaA115) are colored in orange and blue in correspondence with their original mutants (DhaA112 and DhaA101). B) Locations of substitutions in energy-based, and evolution-based mutants of LinA enzyme. Substitutions in the multiple-point mutant designed by the energy-based approach (LinA01) are represented as orange spheres, while substitutions in multiple-point mutant designed by the evolution-based approach (LinA02) are represented as blue spheres.
Fig 3
Fig 3. Biochemical properties of DhaA wild-type and the final mutant DhaA115.
A) Melting temperatures of DhaA wild-type (blue) and DhaA115 (red) in the presence of indicated solvents. B) Half-life of DhaA wild-type (blue) and DhaA115 (red) determined at 60°C and pH 8.6 with the substrate 1-iodohexane. C) Temperature profiles of DhaA wild-type (blue) and DhaA115 (red) determined at pH 8.6 with the substrate 1-iodohexane.
Fig 4
Fig 4. Schematic comparison of protein stabilization methods.
Examples of representative methods with their characteristics and success rates are presented in S12 Table.

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Grant support

The work was supported by the Grant Agency of the Czech Republic (P503/12/0572; http://www.gacr.cz/) and the Czech Ministry of Education of the Czech Republic (LO1214 and LH14027; http://www.msmt.cz/). DBe received a Brno Ph.D. Talent Scholarship funded by the Brno City Municipality and his stay at Rutgers University was supported by European Regional Development Fund – project FNUSA-ICRC (CZ.1.05/1.1.00/02.0123; http://www.msmt.cz/), European Social Fund and the state budget of the Czech Republic and project ICRC Human Bridge – "Support of Study Stays of Czech Researchers Abroad III: Young Talent Incubator" (CZ.1.07/2.3.00/20.0239; http://www.msmt.cz/). KB and JBr were supported by the “Employment of Best Young Scientists for International Cooperation Empowerment” (CZ.1.07/2.3.00/30.0037) project co-financed by the European Social Fund (http://ec.europa.eu/esf/home.jsp) and the state budget of the Czech Republic (http://www.msmt.cz/). The work of JBe was supported by the Research and Application of Advanced Methods in ICT project (FIT-S-14-2299; http://www.fit.vutbr.cz/). MetaCentrum and CERIT-SC are acknowledged for providing access to computing facilities (LM2010005 and CZ.1.05/3.2.00/08.0144; http://www.msmt.cz/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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