HotSpot Wizard 2.0 is a web server for automated identification of hot spots and design of smart libraries for engineering proteins' stability, catalytic activity, substrate specificity and enantioselectivity. The server integrates sequence, structural and evolutionary information obtained from 3 databases and 20 computational tools. Users are guided through the processes of selecting hot spots using four different protein engineering strategies and optimizing the resulting library's size by narrowing down a set of substitutions at individual randomized positions. The only required input is a query protein structure. The results of the calculations are mapped onto the protein's structure and visualized with a JSmol applet. HotSpot Wizard lists annotated residues suitable for mutagenesis and can automatically design appropriate codons for each implemented strategy. Overall, HotSpot Wizard provides comprehensive annotations of protein structures and assists protein engineers with the rational design of site-specific mutations and focused libraries. It is freely available at http://loschmidt.chemi.muni.cz/hotspotwizard.

Loschmidt Laboratories Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX Masaryk University 625 00 Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX Masaryk University 625 00 Brno Czech Republic Department of Information Systems Faculty of Information Technology Brno University of Technology 612 66 Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX Masaryk University 625 00 Brno Czech Republic Department of Information Systems Faculty of Information Technology Brno University of Technology 612 66 Brno Czech Republic International Clinical Research Center St Anne's University Hospital Brno Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX Masaryk University 625 00 Brno Czech Republic International Clinical Research Center St Anne's University Hospital Brno Brno Czech Republic

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SoluProtMutDB: A manually curated database of protein solubility changes upon mutations

HotSpot Wizard 3.0: web server for automated design of mutations and smart libraries based on sequence input information

FireProt: web server for automated design of thermostable proteins