CAVER Analyst 1.0: graphic tool for interactive visualization and analysis of tunnels and channels in protein structures

ABSTRACT Summary: The transport of ligands, ions or solvent molecules into proteins with buried binding sites or through the membrane is enabled by protein tunnels and channels. CAVER Analyst is a software tool for calculation, analysis and real-time visualization of access tunnels and channels in static and dynamic protein structures. It provides an intuitive graphic user interface for setting up the calculation and interactive exploration of identified tunnels/channels and their characteristics. Availability and Implementation: CAVER Analyst is a multi-platform software written in JAVA. Binaries and documentation are freely available for non-commercial use at http://www.caver.cz

FireProt : energy- and evolution-based computational design of thermostable multiple-point mutants

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 gamma-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (Delta T-m = 24 degrees C and 21 degrees 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

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