Segmental dynamics of the cytoplasmic domain of erythrocyte band 3 determined by time-resolved fluorescence anisotropy: sensitivity to pH and ligand binding.

摘要

Interactions between the erythrocyte membrane andits skeleton are mediated primarily by binding of cytoskeletal components to aconformationally sensitive structure, the cytoplasmic domain of band 3 (cdb3).To examine the nanosecond segmental motions of cdb3, band 3 was labeledselectively by fluorescein maleimide at Cys-201 near the proposed hinge in cdb3about which pH-dependent conformational changes occur. Time-resolved anisotropyof labeled cdb3 in isolated form and in stripped erythrocyte membranes wasmeasured by parallel-acquisition frequency-domain microfluorimetry. Samples hada single-component fluorescein lifetime of approximately 4 ns. Multifrequencyphase and modulation data (5-200 MHz) fitted well to a segmental motion modelcontaining two correlation times (tau 1c and tau 2c) and two limitinganisotropies (r1infinity and r2infinity). Measurements in protease-cleaved anddenatured samples indicated that tau 1c (100-150 ps) corresponded to rapidrotation of bound fluorescein and tau 2c (2-5 ns) corresponded to segmentalmotion of cdb3. Both motions were hindered as quantified by nonzero r1infinityand r2infinity. The strong pH dependence of segmental motion correlated withthat of cdb3 conformation measured by intrinsic tryptophan fluorescence.Significant changes in cdb3 segmental motion occurred upon interactions with thesmall ligands 2,3-bisphosphoglycerate and calcium and several glycolytic enzymesknown to bind to the N terminus of band 3. These time-resolved fluorescencemeasurements of the nanosecond segmental dynamics of a labeled membrane proteinprovide evidence for the sensitivity of cdb3 conformation to ligand binding andsuggest long-range structural communication through cdb3.