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

Area of Science:

• Protein Engineering
• Computational Biology
• Bioinformatics

Background:

• Protein engineering relies on identifying key residues (hotspots) for modification.
• Previous tools required protein structures, limiting usability due to data scarcity.
• Accurate prediction of mutation effects on stability is crucial for protein design.

Purpose of the Study:

• To enhance the HotSpot Wizard web server for automated hotspot identification in protein design.
• To enable sequence-based input for protein structure modeling and analysis.
• To incorporate a stability prediction module for guiding mutagenesis strategies.

Main Methods:

• Utilizing protein sequences as input, generating structures via homology modeling (8 repositories) or *ab initio* methods (Modeller, I-Tasser).
• Implementing quality assessment for predicted structures using WHAT_CHECK, PROCHECK, and MolProbity.
• Integrating Rosetta and FoldX for thermodynamic stability estimation of potential mutations.

Main Results:

• HotSpot Wizard 3.0 accepts protein sequences, overcoming the structure limitation of previous versions.
• Automated quality control for homology models and warnings for redesigning unreliable regions.
• A new module predicts mutation-induced stability changes, filtering destabilizing variants.

Conclusions:

• HotSpot Wizard 3.0 significantly improves accessibility and utility for protein engineering by accepting sequence data.
• The integrated stability prediction module enhances the reliability of suggested mutations for experimental validation.
• This updated tool facilitates more efficient and effective semi-rational protein design.