Anion solvation enhanced by positive supercharging mutations preserves thermal stability of an antibody in a wide pH range.

Proteins function through interactions with other molecules. In protein engineering, scientists often engineer proteins by mutating their amino acid sequences on the protein surface to improve various physicochemical properties. "Supercharging" is a method to design proteins by mutating surface residues with charged amino acids. Previous studies demonstrated that supercharging mutations conferred better thermal resistance, solubility, and cell penetration to proteins. Likewise, antibodies recognize antigens through the antigen-binding site on the surface. The genetic and structural diversity of antibodies leads to high specificity and affinity toward antigens, enabling antibodies to be versatile tools in various applications. When assessing therapeutic antibodies, surface charge is an important factor to consider because the isoelectric point plays a role in protein clearance inside the body. In this study, we explored how supercharging mutations affect physicochemical properties of antibodies. Starting from a crystal structure of an antibody with the net charge of -4, we computationally designed a supercharged variant possessing the net charge of +10. The positive-supercharged antibody exhibited marginal improvement in thermal stability, but the secondary structure and the binding affinity to the antigen (net charge of +8) were preserved. We also used physicochemical measurements and molecular dynamics simulations to analyze the effects of supercharging mutations in sodium phosphate buffer with different pH and ion concentrations, which revealed preferential solvation of phosphate ions to the supercharged surface relative to the wild-type surface. These results suggest that supercharging would be a useful approach to preserving thermal stability of antibodies in a wide range of pH, which may enable further diversification of antibody repertoires beyond natural evolution.

How the protonation state of a phosphorylated amino acid governs molecular recognition: insights from classical molecular dynamics simulations.

Physicochemical properties of proteins are controlled mainly by post-translational modifications such as amino acid phosphorylation. Although molecular dynamics simulations have been shown to be a valuable tool for studying the effects of phosphorylation on protein structure and dynamics, most of the previous studies assumed that the phosphate group was in the unprotonated ( PO32- ) state, even though the protonation state could in fact vary at physiological pH. In this study, we performed molecular dynamics simulations of four different protein-phosphorylated peptide complexes both in the PO32- and PO3 H- states. Our simulations delineate different dynamics and energetics between the two states, suggesting importance of the protonation state of a phosphorylated amino acid in molecular recognition.

Roles of the disulfide bond between the variable and the constant domains of rabbit immunoglobulin kappa chains in thermal stability and affinity.

Rabbit antibodies show unique structural characteristics in that kappa chains have an inter-domain disulfide bond between the variable and constant domains. Here we characterized this disulfide bond from physicochemical viewpoints both in stability and affinity. It was revealed that the disulfide bond contributed to the thermal stability of the antibody, but the affinity and mechanism of antigen recognition was not altered by the mutation. The present result expands the understanding of how rabbit antibodies with kappa light chains gain affinity under characteristic mechanism to gain thermal stability, and would give suggestions for the methods to artificially stabilize antibody molecules.

A Chemically Inducible Helper Module for Detecting Protein-Protein Interactions with Tunable Sensitivity Based on KIPPIS.

As protein-protein interactions (PPIs) play essential roles in regulating their functional consequences in cells, methods to detect PPIs in living cells are desired for correct understanding of intracellular PPIs and pharmaceutical development therefrom. Here we demonstrate a c-kit-based PPI screening (KIPPIS) system in combination with a chemically inducible helper module for detecting PPIs in living mammalian cells. In this system, a mutant of FK506-binding protein 12 (FKBPF36 V) is fused with a protein of interest and the intracellular domain of a receptor tyrosine kinase c-kit. Constitutive expression of two fusion proteins with interacting proteins of interest in interleukin-3 (IL-3)-dependent cells results in dimerization and subsequent activation of the c-kit intracellular domains, which allows cell proliferation in a culture medium devoid of IL-3. A helper ligand, a small synthetic chemical that homodimerizes FKBPF36 V, assists the formation of stable complexes of the fusion proteins and serves as a tuner for sensitivity of the system. Using this system, two model PPIs were successfully detected on the basis of cell proliferation, which was featured by the helper-ligand- and PPI-dependent phosphorylation of the Src family kinases, a hallmark of the c-kit signaling. Notably, the inclusion of the helper module enabled PPI detection with tunable sensitivity. The helper-assisted KIPPIS allows us to configure various affinity thresholds by changing the concentration of the helper ligand, which may be applied to select affinity-matured variants using the advantage of cell proliferation.