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A nanosecond fluorescence depolarization study on the segmental flexibility of receptor-bound immunoglobulin E
Insights
Time-resolved fluorescence anisotropy reveals immunoglobulin E (IgE) flexibility. Binding to its receptor restricts motion, but Fab segments retain rapid reorientation within limits.
Area of Science:
- Biophysics
- Molecular Biology
- Immunology
Background:
- Immunoglobulin E (IgE) plays a crucial role in allergic responses.
- The high-affinity IgE receptor (FcεRI) is central to IgE function.
- Understanding IgE-receptor dynamics is key to deciphering cellular signaling.
Purpose of the Study:
- To investigate the segmental flexibility of IgE when bound to its high-affinity receptor.
- To characterize the dynamics of IgE-receptor complexes on membrane surfaces.
- To elucidate the impact of ligand binding and solubilization on IgE dynamics.
Main Methods:
- Time-resolved fluorescence anisotropy measurements were employed.
- Experiments utilized membrane vesicles from rat basophilic leukemia cells.
- Analysis involved monitoring anisotropy decay over time.
Main Results:
- Membrane-bound IgE exhibits restricted segmental motion, with a fast component (15-35 ns) possibly due to Fab segment twisting.
- Intermolecular cross-linking abolished this segmental motion.
- Solubilization of IgE-receptor complexes revealed both fast and intermediate motion components.
Conclusions:
- IgE binding to its membrane receptor restricts global rotation and slower segmental flexibility.
- Rapid reorientation of Fab segments within a limited range is still possible.
- These dynamics are modulated by receptor binding, cross-linking, and solubilization.
Abstract:
Time-resolved fluorescence anisotropy measurements have been used to examine the segmental flexibility of anti-dansyl immunoglobulin E (IgE) bound to its high-affinity receptor on membrane vesicles from rat basophilic leukemia cells. Although IgE in this complex exhibits only a restricted angular range of segmental motion, much of this restricted motion occurs on a relatively rapid time scale. A fast component of motion with a rotational correlation time of 15-35 ns may correspond to the twisting of Fab segments about their major axis. Intermolecular cross-linking by a short bivalent ligand, N,N'-didansylcadaverine, results in complete loss of this segmental motion. Solubilization of monomeric IgE-receptor complexes using a zwitterionic detergent results in a time-dependent anisotropy decay that exhibits both a fast component and a slower component that is intermediate between the decay for soluble and membrane-bound forms of IgE at long times after excitation. These results are discussed in terms of a model in which binding of IgE to its membrane-bound receptor restricts not only its global rotation but also its slower modes of segmental flexibility as well, while allowing its Fab segments to undergo rapid reorientation within a limited angular range.
