Fig 1.
Workflow of the FireProt method.
Individual steps involved in the energy- and evolution-based approaches
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.
Table 1.
Characteristics of predicted multiple-point mutants of DhaA.
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.
Table 2.
Steady-state kinetic constants of DhaA wild-type and the final mutant Dha115 determined with 1-iodohexane at 37°C and 57°C, respectively, and pH 8.6.
Table 3.
Characteristics of predicted multiple-point mutants of LinA.
Fig 4.
Schematic comparison of protein stabilization methods.
Examples of representative methods with their characteristics and success rates are presented in S12 Table.