Single Molecule Förster Resonance Energy Transfer Studies of the Fc Region of a Human Antibody

Abstract

The crystallizable fragment (Fc) region of IgG antibodies interacts with a variety of molecules in the immune system. The removal of the oligosaccharides bound to this region affects the ability of antibodies to generate an immune response. A comparison of the structures of the Fc region with and without the oligosaccharides present can indicate if a structural change is the basis for this effect. To carry out these studies, we have labeled the Fc region of a human IgG antibody with donor and acceptor dye molecules. Enzymatic removal of the oligosaccharides was used to generate an oligosaccharide-free antibody. Both ensemble and single molecule Forster resonance energy transfer (FRET) spectroscopy were used to examine the structures of the Fc region. All single molecule FRET measurements performed involved the examination of photon bursts from freely diffusing donor-acceptor labeled antibodies, from which a histogram of the conformations present was constructed.