Studying the transport paths of ligands, solvents, or ions in transmembrane proteins and proteins with buried binding sites is fundamental to the understanding of their biological function. A detailed analysis of the structural features influencing the transport paths is also important for engineering proteins for biomedical and biotechnological applications. CAVER Analyst 2.0 is a software tool for quantitative analysis and real-time visualization of tunnels and channels in static and dynamic structures. This version provides the users with many new functions, including advanced techniques for intuitive visual inspection of the spatiotemporal behavior of tunnels and channels. Novel integrated algorithms allow an efficient analysis and data reduction in large protein structures and molecular dynamic simulations. CAVER Analyst 2.0 is a multi-platform standalone Java-based application. Binaries and documentation are freely available at www.caver.cz. Supplementary data are available at Bioinformatics online.

HotSpot Wizard is a web server used for the automated identification of hotspots in semi-rational protein design to give improved protein stability, catalytic activity, substrate specificity and enantioselectivity. Since there are three orders of magnitude fewer protein structures than sequences in bioinformatic databases, the major limitation to the usability of previous versions was the requirement for the protein structure to be a compulsory input for the calculation. HotSpot Wizard 3.0 now accepts the protein sequence as input data. The protein structure for the query sequence is obtained either from eight repositories of homology models or is modeled using Modeller and I-Tasser. The quality of the models is then evaluated using three quality assessment tools-WHAT_CHECK, PROCHECK and MolProbity. During follow-up analyses, the system automatically warns the users whenever they attempt to redesign poorly predicted parts of their homology models. The second main limitation of HotSpot Wizard's predictions is that it identifies suitable positions for mutagenesis, but does not provide any reliable advice on particular substitutions. A new module for the estimation of thermodynamic stabilities using the Rosetta and FoldX suites has been introduced which prevents destabilizing mutations among pre-selected variants entering experimental testing. HotSpot Wizard is freely available at http://loschmidt.chemi.muni.cz/hotspotwizard.

Caver Web 1.0 is a web server for comprehensive analysis of protein tunnels and channels, and study of the ligands’ transport through these transport pathways. Caver Web is the first interactive tool allowing both the analyses within a single graphical user interface. The server is built on top of the abundantly used tunnel detection tool Caver 3.02 and CaverDock 1.0 enabling the study of the ligand transport. The program is easy-to-use as the only required inputs are a protein structure for a tunnel identification and a list of ligands for the transport analysis. The automated guidance procedures assist the users to set up the calculation in a way to obtain biologically relevant results. The identified tunnels, their properties, energy profiles and trajectories for ligands’ passages can be calculated and visualized. The tool is very fast (2–20 min per job) and is applicable even for virtual screening purposes. Its simple setup and comprehensive graphical user interface make the tool accessible for a broad scientific community. The server is freely available at https://loschmidt.chemi.muni.cz/caverweb.

Enzymes are efficient and specific catalysts for many essential reactions in biotechnological and pharmaceutical industries. Many times, the natural enzymes do not display the catalytic efficiency, stability or specificity required for these industrial processes. The current enzyme engineering methods offer solutions to this problem, but they mainly target the buried active site where the chemical reaction takes place. Despite being many times ignored, the tunnels and channels connecting the environment with the active site are equally important for the catalytic properties of enzymes. Changes in the enzymatic tunnels and channels affect enzyme activity, specificity, promiscuity, enantioselectivity and stability. This review provides an overview of the emerging field of enzyme access tunnel engineering with case studies describing design of all the aforementioned properties. The software tools for the analysis of geometry and function of the enzymatic tunnels and channels and for the rational design of tunnel modifications will also be discussed. The combination of new software tools and enzyme engineering strategies will provide enzymes with access tunnels and channels specifically tailored for individual industrial processes.

There is a continuous interest in increasing proteins stability to enhance their usability in numerous biomedical and biotechnological applications. A number of in silico tools for the prediction of the effect of mutations on protein stability have been developed recently. However, only single-point mutations with a small effect on protein stability are typically predicted with the existing tools and have to be followed by laborious protein expression, purification, and characterization. Here, we present FireProt, a web server for the automated design of multiple-point thermostable mutant proteins that combines structural and evolutionary information in its calculation core. FireProt utilizes sixteen tools and three protein engineering strategies for making reliable protein designs. The server is complemented with interactive, easy-to-use interface that allows users to directly analyze and optionally modify designed thermostable mutants. FireProt is freely available at http://loschmidt.chemi.muni.cz/fireprot.

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 (ΔT_m = 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. Proteins are increasingly used in numerous biotechnological applications. A key property determining proteins’ applicability is their stability under operating conditions. Natural proteins can be stabilized by modification of their structure. Methods of molecular biology allow introduction of modifications–mutations–to the protein structure at will, but it is not straightforward where to mutate and which amino acid to introduce for better stability. Computational methods can be used for prediction of stabilizing mutations using computers. Current computational methods predict libraries of single-point mutations, which need to be constructed individually, tested and recombined, resulting in non-trivial experimental effort. Here we present a robust computational strategy for predicting multiple-point mutants, providing extremely stabilized proteins with a minimal experimental effort.