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.
- MeSH
- bodová mutace genetika fyziologie MeSH
- databáze genetické MeSH
- lyasy chemie genetika metabolismus MeSH
- molekulární modely MeSH
- počítačová simulace MeSH
- proteinové inženýrství metody MeSH
- stabilita enzymů genetika MeSH
- teplota MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- albuminurie diagnóza metabolismus patofyziologie MeSH
- bodová mutace fyziologie genetika MeSH
- cholesterol izolace a purifikace klasifikace metabolismus MeSH
- diabetes mellitus 1. typu etiologie genetika metabolismus MeSH
- diabetes mellitus 2. typu * etiologie metabolismus MeSH
- hyperinzulinismus diagnóza komplikace metabolismus MeSH
- inzulin fyziologie izolace a purifikace metabolismus MeSH
- inzulinová rezistence fyziologie genetika imunologie MeSH
- klinické zkoušky jako téma MeSH
- leptin * izolace a purifikace metabolismus MeSH
- lidé MeSH
- metaanalýza jako téma MeSH
- obezita * etiologie metabolismus MeSH
- pohlavní dimorfismus MeSH
- statistika jako téma MeSH
- těhotenství metabolismus účinky léků MeSH
- tukové buňky cytologie metabolismus účinky léků MeSH
- Check Tag
- lidé MeSH
- těhotenství metabolismus účinky léků MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
