Conformations of IgE bound to its receptor Fc epsilon RI and in solution.
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)
Zheng
Y
Y
Department of Chemistry, Cornell University, Ithaca, New York 14853.
Shopes
B
B
Holowka
D
D
Baird
B
B
D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001666:Binding Sites, Antibody; D002462:Cell Membrane / Q000378:metabolism; D004735:Energy Transfer; D007073:Immunoglobulin E / Q000737:chemistry / Q000648:ultrastructure*; D066298:In Vitro Techniques; D051379:Mice; D011487:Protein Conformation; D011961:Receptors, Fc / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D017455:Receptors, IgE; D012995:Solubility
D000944:Antigens, Differentiation, B-Lymphocyte; D011961:Receptors, Fc; D017455:Receptors, IgE; D007073:Immunoglobulin E