Single Molecule Fluorescence Studies of Antibody Structure

Abstract

The structures of antibodies have been examined using a variety of techniques. Forster resonance energy transfer (FRET) is one technique well-suited for the study of these highly flexible proteins. We will present results from single molecule FRET studies of the distribution of distances between the two-antigen binding sites of an IgG antibody. The antibody studied is the catalytic aldolase antibody 38C2 (Wagner, J.; Lerner, R. A.; Barbas, C. F. (1995) Science270, 1797). This antibody was chosen because it is known to covalently bind a diketone hapten. By linking donor and acceptor dye molecules to the diketone hapten, the antigen-binding sites were selectively labeled for FRET studies. The FRET measurements involved the examination of photon bursts from freely diffusing donor-acceptor labeled antibodies, from which a histogram of the conformations present was constructed.