HotSpot Wizard

HotSpot Wizard automates identification and prioritization of protein sequence positions for semi-rational protein engineering by integrating sequence-based structure modeling, structural quality assessment, hotspot detection, and thermodynamic stability estimation to support design of mutation libraries.

Key Features:

• Input Flexibility: Accepts protein sequences as input and can operate without a pre-existing experimental structure.
• Structure Retrieval and Prediction: Retrieves homology models from eight repositories or generates models using Modeller and I-Tasser.
• Structure Quality Assessment: Evaluates predicted structure quality using WHAT_CHECK, PROCHECK, and MolProbity.
• Hotspot Identification: Identifies residue positions suitable for mutagenesis (hotspots) for targeted redesign.
• Thermodynamic Stability Estimation: Estimates thermodynamic stabilities and filters destabilizing mutations using Rosetta and FoldX.

Scientific Applications:

• Mutation Library Design: Supports design of smart mutation libraries for semi-rational protein engineering experiments.
• Protein Annotation and Redesign: Assists annotation of protein structures and selection of residues for structure-guided redesign.
• Enzyme Optimization: Enables engineering aimed at improving catalytic activity, substrate specificity, enantioselectivity, and stability.
• Drug Development and Synthetic Biology: Provides computational support for enzyme design applications relevant to drug development and synthetic biology.

Methodology:

Retrieves or generates homology models from eight repositories or using Modeller and I-Tasser, evaluates models with WHAT_CHECK, PROCHECK, and MolProbity, identifies mutagenesis hotspots, and estimates thermodynamic stability using Rosetta and FoldX.