Single-Molecule Studies On Adhesion At Protein/Peptide Interfaces

Protein/peptide interface adhesion is involved in many applications such as biochemistry and materials science.It includes the interface adhesion between proteins/peptides and materials and the interface adhesion between pathogens and host cells.A variety of techniques have been developed to characterize the interfacial adhesion between proteins/peptides and materials,but there are no reports on the mechanical adhesion force,kinetics during the binding process and the influence of molecular structure on the binding ability of these solid-bound proteins/peptides.Understanding the mechanism of protein/peptide interface adhesion at the molecular level could make it possible to produce specific biological/inorganic interfaces and apply them to potentially nanoscale materials.The study of the adhesion between pathogens and host cells is of great significance to explore the mechanism of force-dependent protein interaction and binding and to design potential targets against viral and bacterial infections.Single molecular force spectroscopy(SMFS)based on atomic force microscopy(AFM)is the most widely used single molecular force spectroscopy technology in the field of measurement,with mature theoretical development.It has been widely used to study the mechanical properties of biological macromolecules and materials.In this paper,AFM based SMFS technology and molecular dynamics simulation were used to study the mechanical adhesion force and adhesion mechanism of protein/peptide interface adhesion.The main contents of the study are as follows.(1)Single-molecule study on the interaction between solid bound peptide SB7,LCI and silica substrate or polymer material substrate.The binding forces of SB7,LCI and different types of substrates were studied by SMFS based on AFM,genetic engineering,selection of appropriate fingerprint and design of reasonable tensile system.At the same time,molecular dynamics simulation(MD)and tensile dynamics simulation(SMD)were used to study the molecular mechanism of binding.The results show that the dissociation forces of polypeptide SB7 from the silica surface are ～ 220 p N and ～ 610 p N,which are higher than the unfolding forces of most mechanically stable proteins and the unbinding forces of most stable protein-protein interactions.The electrostatic interaction of the positively charged arginine residue with the silica surface dominates the binding of SB7 to silica.The adhesion of LCI to polystyrene(PS),polypropylene(PP)and polyethylene terephephate(PET)was distributed between 50 and 80 p N.Phenylalanine no.12,tyrosine no.30 and tryptophan no.37 on LCI predominate the interaction between LCI and substrate,and Van der Waals force mediates the interaction between LCI and substrate during the whole stretching process.This result provides a molecular characterization and explanation for the study of the binding effect of solid-bound peptides and solid surfaces,indicating that SB7 and LCI are ideal fusion labels for silicon-based,polymer surface immobilization or peptide-based bonding materials.(2)Single molecule study of adhesion between solid binding protein Si-tag and silica material.The adhesion properties and molecular mechanism between silica binding protein Si-tag and silica substrate were systematically studied by SMFS technology based on AFM,combined with genetic engineering and protein engineering.The application of Si-tag in single molecule modification was studied by using single molecule magnetic tweezers(MT)technology.Studies have shown that the adhesion force between Si-tag and glass substrate can reach 400 ～ 600 p N,which is higher than the unfolding force of most proteins and the adhesion force between solid bound peptide and silica substrate,and can be stably maintained in this range,even at high salt concentration and high ambient temperature,still maintain strong adsorption with silica.By using the strong adsorption of Si-tag and probe as well as glass substrate,Si-tag can be connected to the target protein molecule as a tag,and it can be fixed on the probe or substrate in one step,simplifying the traditional method of fixing protein molecule by chemical reaction between functional groups.(3)Single molecule/single cell study on the interface adhesion between wild type and mutant of pathogen SARS-Co V-2 spike protein(S protein)and host cell surface receptor angiotensin converting enzyme 2(ACE2).The interface adhesion of S protein and ACE2 receptor was studied by using AFM-based SMFS and single-cell force spectroscopy SCFS,and the binding mechanism of the two was studied by MD and SMD.The results show that the Delta variant S protein has the highest unbinding ability and binding probability for ACE2,both at the single molecule level and the single cell level.This stronger adhesive interaction between Delta variant and ACE2 may contribute to the stronger cell invasion ability and high infectivity of Delta variant.MD and SMD results showed that the E484 A and Y505 H mutations of the ACE2-RBD complex of Omicron BA.1 reduced its stability,while the S477 N and N501 Y mutations increased its stability compared with the Delta variant.Finally,neutralizing antibodies produced by immunizing mice with wild-type S protein effectively inhibited the binding of wild-type,Delta and Omicron(BA.1 and BA.5)S proteins to ACE2.The results provide new insights into the molecular mechanism of the adhesive interaction between S protein and ACE2 and suggest that effective monoclonal antibodies against different variants can be prepared using wild-type S protein.In summary,this paper mainly studies the protein/peptide interface adhesion at the single molecule level,supplemented by computer simulation to study the molecular mechanism of binding.It provides experimental demonstration and theoretical basis for the selection of fusion label for solid surface display.At the same time,a new insight was proposed on the effect of mutant amino acids on the adhesion of the binding interface between pathogen and host cell surface receptors.