Kinetic and molecular insight into immunoglobulin G binding to immobilized recombinant protein A of different orientations.

Mechanistic understanding of immunoglobulin G (IgG) binding to protein A is crucial for the design and development of high-performance protein A chromatography. In this work, the IgG binding domain (Z) of protein A from Staphylococcus aureus was genetically modified by introducing a cysteine residue at the N-terminus (Cys-Z) or a cysteine-lysine dipeptide at the C-terminus (Z-Cys), and the two ligands were used to unravel the IgG binding mechanism by means of binding kinetics and different single molecule measurements. Surface plasma resonance (SPR) measurement of the binding kinetics of mouse myeloma IgG2a (mIgG2a) to the two ligands indicated that oriented ligand immobilization significantly increased the association rate constant of mIgG2a, and Z-Cys had the highest binding affinity to mIgG2a among the three ligands (Cys-Z, Z-Cys and Z). This was attributed to the synergistic contribution of the high association rate constant and low dissociation rate constant to mIgG2a. Furthermore, quartz crystal microbalance with energy dissipation monitoring (QCM-D) measurement provided the maximum adsorption densities of IgGs on the Z-Cys-immobilized chip as zeta potentials of IgGs were nearly zero. The QCM-D investigation revealed that the adsorbed layer was dependent on ligand type and density, and IgG. Moreover, Z-Cys and Cys-Z induced IgG binding in flipped orientations, as evidenced by the antigen-antibody reaction. Finally, rectangular DNA origami tiles were introduced to analyze the molecular orientation of adsorbed IgG. Single-molecule imaging showed that mIgG2a was associated with flexible Z-Cys on the tiles predominantly in side-on and end-on orientations. The research has provided molecular insight into the binding mechanism of IgG molecules at liquid-solid interfaces and would help design new protein A-based ligands and high-capacity adsorbents.