title: Single molecule studies of antibody-antigen interaction strength versus intra-molecular antigen stability.
abstract: We investigated molecular recognition of antibodies to membrane-antigens and extraction of the antigens out of membranes at the single molecule level. Using dynamic force microscopy imaging and enzyme immunoassay, binding of anti-sendai antibodies to sendai-epitopes genetically fused into bacteriorhodopsin molecules from purple membranes were detected under physiological conditions. The antibody/antigen interaction strength of 70-170 pN at loading rates of 2-50 nN/second yielded a barrier width of x = 0.12 nm and a kinetic off-rate (corresponding to the barrier height) of k(off) = 6s(-1), respectively. Bacteriorhodopsin unfolding revealed a characteristic intra-molecular force pattern, in which wild-type and sendai-bacteriorhodopsin molecules were clearly distinguishable in their length distributions, originating from the additional 13 amino acid residues epitope in sendai purple membranes. The inter-molecular antibody/antigen unbinding force was significantly lower than the force required to mechanically extract the binding epitope-containing helix pair out of the membrane and unfold it (126 pN compared to 204 pN at the same loading rate), meeting the expectation that inter-molecular unbinding forces are weaker than intra-molecular unfolding forces responsible for stabilizing native conformations of proteins.
authors.affiliation: Institute for Biophysics, University of Linz, Altenbergerstr. 69, A-4040 Linz, Austria.
authors.forename: Ferry
authors.initials: F
authors.lastname: Kienberger
authors.forename: Gerald
authors.initials: G
authors.lastname: Kada
authors.forename: Harald
authors.initials: H
authors.lastname: Mueller
authors.forename: Peter
authors.initials: P
authors.lastname: Hinterdorfer
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001436:Bacteriorhodopsins / Q000737:chemistry; D001666:Binding Sites, Antibody; D000939:Epitopes; D019617:Halobacterium salinarum / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D018625:Microscopy, Atomic Force; D011487:Protein Conformation; D011489:Protein Denaturation; D018717:Purple Membrane / Q000737:chemistry / Q000276:immunology; D013057:Spectrum Analysis / Q000379:methods
chemical_list: D000906:Antibodies; D000941:Antigens; D001426:Bacterial Proteins; D000939:Epitopes; D008565:Membrane Proteins; D001436:Bacteriorhodopsins