Previous resonance energy transfer studies suggested that murine immunoglobulin E (IgE) is bent near the junction of its Fc and Fab segments when bound to its high-affinity receptor (Fc epsilon RI) on RBL cells. To examine further the conformations of IgE, both bound to this receptor and in solution, a mutant recombinant IgE (epsilon/C gamma 3*) was prepared that has a cysteine replacing a serine near the C-terminal ends of the heavy chain. The introduced cysteine residues provide a means for specific modification of IgE, and the sulfhydryl groups were selectively labeled with fluorescein-5-maleimide (FM-epsilon/C gamma 3*). This IgE also binds a 5-(dimethylamino)naphthalene-1-sulfonyl (DNS) group in the antigen-binding sites. Resonance energy transfer experiments carried out on receptor-bound FM-epsilon/C gamma 3* yielded a distance of 53 A between fluorescein near the C-terminal end of the Fc segment and amphipathic acceptor probes at the membrane surface. The average distance between this C-terminal fluorescein and acceptor eosin-DNS in the antigen-binding sites at the N-terminal ends of the Fab segments was found to be 69 A. These results combine with those from previous structural studies to provide an unprecedented detailed description of the bent geometry of IgE bound to its receptor on the membrane. Energy transfer measured for FM-epsilon/C gamma 3* in solution between fluorescein near the C-terminal end of the Fc segment and eosin-DNS at the N-terminal ends of the Fab segments indicates that the average distance between these probes is about 71 A.(ABSTRACT TRUNCATED AT 250 WORDS)

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 A for IgE in solution and 69 A 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 A and greater than or equal to 85 A 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 A, hw = 24 A for IgE in solution; rho = 71 A, hw = 12 A for IgE bound to Fc epsilon RI; and rho = 100 A, hw = 88 A for IgG in solution.(ABSTRACT TRUNCATED AT 400 WORDS)

No abstract

No abstract

No abstract