Single molecule studies of antibody-antigen interaction strength versus intra-molecular antigen stability

J Mol Biol. 2005 Apr 1;347(3):597-606. doi: 10.1016/j.jmb.2005.01.042.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies* / chemistry
  • Antibodies* / genetics
  • Antibodies* / immunology
  • Antigen-Antibody Reactions
  • Antigens* / chemistry
  • Antigens* / genetics
  • Antigens* / immunology
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Bacteriorhodopsins / chemistry
  • Binding Sites, Antibody
  • Epitopes
  • Halobacterium salinarum / chemistry
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / immunology
  • Microscopy, Atomic Force
  • Protein Conformation
  • Protein Denaturation
  • Purple Membrane / chemistry
  • Purple Membrane / immunology
  • Spectrum Analysis / methods

Substances

  • Antibodies
  • Antigens
  • Bacterial Proteins
  • Epitopes
  • Membrane Proteins
  • Bacteriorhodopsins