Histidine-Mediated Intramolecular Electrostatic Repulsion for Controlling pH-Dependent Protein-Protein Interaction

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Protein-protein interactions (PPIs) controllable by environmental triggers are valuable for biological and industrial applications.
• Engineering pH-dependent PPIs offers precise control over molecular interactions.

Purpose of the Study:

• To engineer a pH-dependent protein-protein interaction using intramolecular electrostatic repulsion.
• To enhance the pH sensitivity of Streptococcal protein G (SpG) binding to immunoglobulin G (IgG).

Main Methods:

• Employed a structure-guided histidine substitution approach to introduce electrostatic repulsion.
• Implemented a single point mutation on SpG, an IgG affinity ligand.
• Analyzed structural stability, binding function, and thermodynamic properties.

Main Results:

• A single point mutation significantly improved the pH sensitivity of SpG-IgG interactions.
• The engineered PPI was disrupted by electrostatic repulsion between histidine and nearby positive residues at acidic pH.
• Thermodynamic analysis revealed reduced exothermic binding heat under acidic conditions, with enthalpy-entropy compensation observed.

Conclusions:

• Engineered pH-sensitive SpG enables antibody purification under mildly acidic conditions.
• The intramolecular design strategy can be combined with conventional protein interface design.
• The proposed method offers new criteria for optimizing pH-dependent molecular interactions.