Characteristics of predicted multiple-point mutants of LinA.

<p>^a not applicable</p><p>^b activity determined with γ-hexachlorocyclohexane at 30°C and pH 8.6</p><p>^c initial γ-HCH concentration is given since it affects determined specific activity; ΔΔG–predicted change in Free Gibbs Energy; DSC–Differential Scanning Calorimetry; ND, not determined</p><p>Characteristics of predicted multiple-point mutants of LinA.</p

Biochemical properties of DhaA wild-type and the final mutant DhaA115.

<p>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.</p

Schematic comparison of protein stabilization methods.

<p>Examples of representative methods with their characteristics and success rates are presented in <b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004556#pcbi.1004556.s015" target="_blank">S12 Table</a></b>.</p

Characteristics of predicted multiple-point mutants of DhaA.

<p>^a not applicable</p><p>^b activity determined with 1-iodohexane at 37°C and pH 8.6; ΔΔG–predicted change in Free Gibbs Energy; DSC–Differential Scanning Calorimetry; DhaA115 combines mutations of DhaA101 and DhaA112</p><p>Characteristics of predicted multiple-point mutants of DhaA.</p

Location of stabilizing mutations in designed enzymes.

<p>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.</p

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.

<p><i>K</i>_0.5 –concentration of substrate at half maximal velocity, <i>k</i>_cat−catalytic constant, <i>n</i>–Hill coefficient <i>K</i>_si−substrate inhibition constant</p><p>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.</p

Site-Specific Analysis of Protein Hydration Based on Unnatural Amino Acid Fluorescence

Hydration of proteins profoundly affects their functions. We describe a simple and general method for site-specific analysis of protein hydration based on the in vivo incorporation of fluorescent unnatural amino acids and their analysis by steady-state fluorescence spectroscopy. Using this method, we investigate the hydration of functionally important regions of dehalogenases. The experimental results are compared to findings from molecular dynamics simulations