DYNAMIC CONFORMATIONS COMPARED FOR IGE AND IGG1 IN SOLUTION AND BOUND TO RECEPTORS

Dynamic conformations of two distinct immunoglobulin (Ig) isotypes, murine IgE and human IgG1, were examined with fluorescence resonance energy transfer measurements. The IgE mutant epsilon/C-gamma-3* and the IgG1 mutant gamma/C-gamma-3* each bind [5-(dimethylamino)naphthalen-1-yl]sulfonyl (DNS) in two identical antigen binding sites at the amino (N)-terminal ends of the Ig in the Fab segments. Eosin-DNS bound in these Fab sites served as the acceptor probe in these studies. Both Ig have a carboxy (C)-terminal domain (C-gamma-3*) which contains genetically introduced cysteine residues. Modification of these cysteine sulfhydryls with fluorescein maleimide provided donor probes near the C-terminal ends of the Ig in the Fc segment. Energy transfer between the C-terminal and N-terminal ends was compared for these two Ig in solution and when they were found to their respective high-affinity receptors on plasma membranes: IgE-Fc-epsilon-RI on RBL cell membranes and IgG1-Fc-gamma-RI on U937 cell membranes. Previous energy-transfer measurements with these probes yielded an average end-to-end distance of 71 angstrom for IgE in solution and 69 angstrom for IgE bound to Fc-epsilon-RI, indicating that in both situations IgE is bent such that the axes of the Fab segments and the axis of the Fc segment do not form a planar Y-shape [Zheng, Shopes, Holowka, & Baird (1991) Biochemistry 30, 9125]. In the current study we found the average end-to-end distance for IgG1 in solution is 75 angstrom and greater-than-or-equal-to 85 angstrom for IgG1 bound to Fc-gamma-RI, suggesting an average bend conformation for IgG1 as well. The contributions of segmental flexibility to the average distances were assessed directly by measuring the efficiency of energy transfer as a function of variations in donor quantum yield caused by a collisional quencher and using these data to extract a Gaussian distribution of end-to-end distances. The distribution average (rho) and half-width (hw) were determined to be as follows: rho = 75 angstrom, hw = 24 angstrom for IgE in solution; rho = 71 angstrom, hw = 12 angstrom for IgE bound to Fc-epsilon-RI; and rho = 100 angstrom, hw = 88 angstrom for IgG in solution. All of the results obtained are consistent with the view that IgE bound to Fc-epsilon-RI is bent with limited motion between N-terminal and C-terminal ends and that the structure is similar, although somewhat less rigid, for IgE in solution. In contrast, IgG in solution appears to be much more flexible, and this flexibility can account for the short average end-to-end distance measured.