Strcutural Evolution Of Protein A Molecules At Solid-liquid Interface And Its Effect On IgG Binding

The orientation of immunoglobulin G（IgG）at the solid-liquid surface is crucial for the development of high-capacity affinity adsorbent using a ligand from Staphylococcus protein A（SpA）and design of efficient immunodetection and biosensor technologies,which directly affect the sensitivity of the detection process.The molecular orientation of bound IgG in SpA-based system,which was extensively applied in biotechnology,was also dependent on molecular orientation and structure of SpA molecules at solid-liquid surface.In this paper,dextran coated Fe₃O₄ magnetic particles（Fe₃O₄@Dex）and maleimide functionalized 96-well plates were introduced to couple the mutants of Z domain from SpA（SpA-Z）by different methods to obtain multiple molecular orientations of SpA-Z at solid-liquid surface.Based on them,the molecular evolution of SpA molecules and its effect on IgG binding and the molecular orientation of bound IgG molecules were investigated in detail at various buffer conditions and at various solid-liquid interfaces.The research provided a guidance for the development of high-capacity SpA adsorbent and deign of high-sensitivity immune-detection reagents and biosensors.In this paper,Fe₃O₄@Dex MNPs were obtained by coating dextran on the surface of synthesized Fe₃O₄ according to the principle of embedding method.FT-IR results indicated that the characteristic peaks of Fe-O,O-H,C-H and other groups are present in MNPs.In addition,the results of VSM indicate that the saturation magnetization of the MNPs is reduced relative to Fe₃O₄,which was attributed to the partial magnetic covering of Fe₃O₄ by dextran and a decreasing content of Fe₃O₄in Fe₃O₄@Dex MNPs.The result confirmed that the surface of Fe₃O₄ was coated with dextran,and Fe₃O₄@Dex was successfully prepared.Based on previous studies,three mutants of SpA-Z domains were constructed respectively by introducing cysteine into the threeα-helixes of the SpA-Z domain:Z₁（N11C）,Z₂（A29C）,and CK tag at C-terminus of Z domain（Z₃）.Furthermore,three SpA-Z ligands constructed above were coupled to Fe₃O₄@Dex and maleimide-functionalized 96-well plates,respectively,to prepare affinity nanoparticles and affinity96-well plates coupled with SpA-Z ligands.The circular dichroism（CD）and fluorescence（FL）results of Fe₃O₄@Dex affinity nanoparticles indicated that p H had an important effect on the structure of the immobilized SpA-Z ligand.In the experimental p H range（4.5-9.0）,three immobilized SpA-Z ligands had the most severe structural damage at p H 4.5,and the characteristic peaks of the CD spectrum could not be observed.At the same time,the red shift of the fluorescence spectrum was the most significant.With an increase of p H,the helix content of immobilized ligands increased,and the degree of red shift also decreased accordingly.The helix contents of immobilized SpA-Z ligands at p H 9.0 basically exceeded 20%,and their maximum emission wavelength was basically consistent with the corresponding free SpA-Z ligands.The result indicated that molecular structure of immobilized SpA-Z ligands at solid-liquid interface was dominated by electrostatic interaction.As buffer p H was lower than p I of ligands,electrostatic attraction between ligand and solid surface led to a severe damage of structure of SpA-Z ligands.After the electrostatic attraction was transformed into electrostatic repulsion at buffer p H over p I of ligands,the molecular structure of immobilized SpA-Z ligand gradually transformed to its native structure,especially at p H 9.0.In addition,the isothermal titration calorimetric（ITC）results of IgG binding with free and immobilized SpA-Z ligands further indicated that the lower the helix content of the SpA-Z ligand,the lower its affinity for IgG.Compared to the free SpA-Z ligand,the IgG affinity with the affinity nanoparticles increased.This work further examined the IgG binding capabilities of the three Fe₃O₄@Dex affinity nanoparticles.The results showed that the Z₁ ligand immobilized affinity nanoparticle had the highest affinity with IgG,and its IgG binding ability was also the highest among the three affinity nanoparticles.In contrast,the Z₂ ligand immobilized affinity nanoparticle had the weakest affinity and the least amount of bound IgG.Based on this,the interaction between the bound IgG on the surface of the affinity nanoparticles and its antigen was further studied.The results showed that IgG bound to the Z₂ ligand immobilized affinity nanoparticle had the strongest binding ability to the antigen.At the same tiem,IgG molecule bound to the Z₁ ligand immobilized affinity nanoparticle had the weakest ability to bind to the antigen.The result reflected that the IgG bound to the former are more likely to interact with the antigen.The above results can be inferred that IgG molecules bound to Z₁ and Z₂ ligands immobilized affinity nanoparticles have different molecular orientations.This indicated that the helix I of the SpA-Z molecule was closer to the Fab fragment as the SpA-Z domain binds to the Fc fragment of IgG molecules.The same conclusion was also reflected on three SpA-Z ligand-immobilized 96-well plates.The research provided a useful guidance in the specific regulation to the molecular orientation of IgG at solid-liquid interfaces for the development of high-capacity SpA adsorbents and the design of high-sensitivity immunodetection reagents and biosensors.