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

Protein-protein interactions that can be controlled by environmental triggers have immense potential in various biological and industrial applications. In the current study, we aimed to engineer a pH-dependent protein-protein interaction that employs intramolecular electrostatic repulsion through a structure-guided histidine substitution approach. We implemented this strategy on Streptococcal protein G, an affinity ligand for immunoglobulin G, and showed that even a single point mutation effectively improved the pH sensitivity of the binding interactions without adversely affecting its structural stability or its innate binding function. Depending on the pH of the environment, the protein-protein interaction was disrupted by the electrostatic repulsion between the substituted histidine and its neighboring positively charged residues. Structurally, the substituted histidine residue was located adjacent to a lysine residue that could form hydrogen bonds with immunoglobulin G. Thermodynamically, the introduced electrostatic repulsion was reflected in the significant loss of the exothermic heat of the binding under acidic conditions, whereas accompanying enthalpy-entropy compensation partly suppressed the improvement of the pH sensitivity. Thus, the engineered pH-sensitive protein G could enable antibody purification under mildly acidic conditions. This intramolecular design can be combined with conventional protein-protein interface design. Moreover, the method proposed here provides us with additional design criteria for optimization of pH-dependent molecular interactions.

Cellulose tris(3,5-dichlorophenylcarbamate) immobilised on silica: a novel chiral stationary phase for resolution of enantiomers.

CHIRALPAK IC is a new chiral stationary phase (CSP) made by immobilising cellulose tris(3,5-dichlorophenylcarbamate) on silica gel. The chiral selector is distinct from any other commercially available polysaccharide-based CSPs. Apart from its compatibility with the whole series of solvents; this CSP is able to operate under various chromatographic conditions and bring about new characteristics in enantiomeric recognition. It can afford many large and specific enantiomeric separations. It exhibits complementary properties with regard to the existing immobilised chiral packing materials of the same category.