HotSpot Wizard 2.0: automated design of site-specific mutations and smart libraries in protein engineering

Abstract

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.

Figure 1.

Workflow of HotSpot Wizard.

Figure 2.

Some notable applications of the four protein engineering strategies implemented in the HotSpot Wizard web server.

Figure 3.

HotSpot Wizard's graphical user interface, showing results obtained for the haloalkane dehalogenase LinB (PDB ID: 1CV2). (A) The ‘Report’ panel shows the status of the calculations in the individual steps of the computational pipeline. (B) Results obtained using the four protein engineering strategies. (C) The ‘Residue features’ panel, which provides an overview of the identified hot spots. (D) The ‘Residues selected for mutagenesis’ panel, which presents a user-adjustable list of residues representing targets for mutagenesis. (E) The JSmol viewer allows interactive visualization of the protein and the identified tunnels and pockets. (F) The ‘Residue details’ pop-up window, which provides comprehensive information on the residue's annotations, organized under several tabs. (G) The ‘Library design’ panel, which shows the list of substitutions and appropriate codons for randomization of selected positions.