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

Bednar D, Beerens K, Sebestova E, Bendl J, Khare S, Chaloupkova R, Prokop Z, Brezovsky J, Baker D, Damborsky J - PLoS Comput. Biol. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Masaryk University, Brno, Czech Republic; International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic.

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.

No MeSH data available.


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.
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pcbi.1004556.g003: 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.

Mentions: The combined mutant DhaA115 was characterized in detail, in terms of its thermostability in the presence of organic co-solvents, half-life at elevated temperature and temperature profile. The Tm determinations show that the mutations had stabilizing effects in the presence of three organic co-solvents comparable to those in the pure buffer (ΔTm: 20 to 26°C; Fig 3A). The enhanced thermostability was also reflected in a strongly improved half-life at 60°C (Fig 3B). After seven days incubation at 60°C, the mutant DhaA115 still retained about 50% of its initial activity, while the wild-type became inactivated within six hours. Two inactivation phases were observed for all DhaA variants: rapid initial inactivation followed by a slower decay of activity (Fig 3B). The wild-type lost around 80% of its activity in the first fast phase, while the mutations in DhaA115 reduced the loss during the first inactivation phase to only 30%. The rate of inactivation during the first phase was comparable for both wild-type and DhaA115, while the rate during the second phase was dramatically (100-fold) slower for the mutant. Similar effects were observed with a previously reported stable variant of DhaA63 (denoted Dhla8) [28] constructed using Gene Site-Saturation Mutagenesis (S3 Fig). The apparent optimal temperature shifted from 45°C for the wild-type enzyme to 65°C for the mutant DhaA115, and its specific activity with 1-iodohexane at optimum temperature was 28% higher, but the shape of the temperature profile remained largely unchanged (Fig 3C). Steady-state kinetic constants of the two enzymes determined at their respective suboptimal temperatures with 1-iodohexane revealed comparable catalytic properties (Table 2).


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

Bednar D, Beerens K, Sebestova E, Bendl J, Khare S, Chaloupkova R, Prokop Z, Brezovsky J, Baker D, Damborsky J - PLoS Comput. Biol. (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4631455&req=5

pcbi.1004556.g003: 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.
Mentions: The combined mutant DhaA115 was characterized in detail, in terms of its thermostability in the presence of organic co-solvents, half-life at elevated temperature and temperature profile. The Tm determinations show that the mutations had stabilizing effects in the presence of three organic co-solvents comparable to those in the pure buffer (ΔTm: 20 to 26°C; Fig 3A). The enhanced thermostability was also reflected in a strongly improved half-life at 60°C (Fig 3B). After seven days incubation at 60°C, the mutant DhaA115 still retained about 50% of its initial activity, while the wild-type became inactivated within six hours. Two inactivation phases were observed for all DhaA variants: rapid initial inactivation followed by a slower decay of activity (Fig 3B). The wild-type lost around 80% of its activity in the first fast phase, while the mutations in DhaA115 reduced the loss during the first inactivation phase to only 30%. The rate of inactivation during the first phase was comparable for both wild-type and DhaA115, while the rate during the second phase was dramatically (100-fold) slower for the mutant. Similar effects were observed with a previously reported stable variant of DhaA63 (denoted Dhla8) [28] constructed using Gene Site-Saturation Mutagenesis (S3 Fig). The apparent optimal temperature shifted from 45°C for the wild-type enzyme to 65°C for the mutant DhaA115, and its specific activity with 1-iodohexane at optimum temperature was 28% higher, but the shape of the temperature profile remained largely unchanged (Fig 3C). Steady-state kinetic constants of the two enzymes determined at their respective suboptimal temperatures with 1-iodohexane revealed comparable catalytic properties (Table 2).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX, Masaryk University, Brno, Czech Republic; International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic.

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.

No MeSH data available.