Cathrine A. Southern

The effect of sugar removal on the structure of the Fc region of an IgG antibody as observed with single molecule Förster Resonance Energy Transfer.

The deglycosylation of immunoglobulin G (IgG) antibodies leads to a diminished immune response. This reduction in immune response is thought to arise from weakened binding of IgG antibodies to effector molecules as a result of a conformational change in the antibody. The nature of this structural alteration is uncertain due to the conflicting results obtained from different experimental methods. We have examined the impact of deglycosylation by the endoglycosidase PNGase F on the structure of the Fc region of a human IgG antibody using single molecule Förster Resonance Energy Transfer (FRET). The FRET efficiency histograms obtained indicate that the structure of the Fc region becomes more flexible upon deglycosylation. This is demonstrated by a change in the width of the energy transfer efficiency peak, which increases from 0.19 ± 0.02 to 0.6 ± 0.1 upon deglycosylation.

A comparison of Förster resonance energy transfer analysis approaches for Nanodrop fluorometry

Ensemble Förster resonance energy transfer (FRET) results can be analyzed in a variety of ways. Due to experimental artifacts, the results obtained from different analysis approaches are not always the same. To determine the optimal analysis approach to use for Nanodrop fluorometry, we have performed both ensemble and single-molecule FRET studies on oligomers of double-stranded DNA. We compared the single-molecule FRET results with those obtained using various ensemble FRET analysis approaches. This comparison shows that for Nanodrop fluorometry, analyzing the increase of the acceptor fluorescence is less likely to introduce errors in estimates of FRET efficiencies compared with analyzing the fluorescence intensity of the donor in the absence and presence of the acceptor.

Single molecule Förster resonance energy transfer studies of the effect of EndoS deglycosylation on the structure of IgG

The bacterial enzyme EndoS specifically cleaves glycans bound to immunoglobulin G (IgG) molecules. Because this deglycosylation procedure leads to a diminished immune response, this enzyme has potential applications as a therapeutic for autoimmune disorders. Although the diminished immune response is attributed to a structural change in the Fc region of IgG antibodies, the specific nature of this structural change is not known due to the variety of results obtained by different experimental approaches. In order to better understand how EndoS deglycosylation impacts the structure of the Fc region of IgG antibodies, we have conducted single molecule Förster resonance energy transfer (FRET) studies of dye-labeled, freely diffusing antibodies. A comparison of the FRET efficiency histograms obtained for glycosylated and EndoS deglycosylated antibodies indicates that the Fc region can take on a wider variety of structures upon deglycosylation. This is demonstrated by the presence of additional peaks in the FRET efficiency histogram for the deglycosylated case.

Single Molecule Fluorescence Studies of Antibody Structure

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.