SEGMENTAL DYNAMICS OF THE CYTOPLASMIC DOMAIN OF ERYTHROCYTE BAND-3 DETERMINED BY TIME-RESOLVED FLUORESCENCE ANISOTROPY - SENSITIVITY TO PH AND LIGAND-BINDING

Interactions between the erythrocyte membrane and its skeleton are med iated primarily by binding of cytoskeletal components to a conformatio nally sensitive structure, the cytoplasmic domain of band 3 (cdb3). To examine the nanosecond segmental motions of cdb3, band 3 was labeled selectively by fluorescein maleimide at Cys-201 near the proposed hing e in cdb3 about which pH-dependent conformational changes occur. Time- resolved anisotropy of labeled cdb3 in isolated form and in stripped e rythrocyte membranes was measured by parallel-acquisition frequency-do main microfluorimetry. Samples had a single-component fluorescein life time of approximate to 4 ns. Multifrequency phase and modulation data (5-200 MHz) fitted well to a segmental motion model containing two cor relation times (tau(1c) and tau(2c)) and two limiting anisotropies (r( 1 infinity) and r(2 infinity)). Measurements in protease-cleaved and d enatured samples indicated that tau(1c) (100-150 ps) corresponded to r apid rotation of bound fluorescein and tau(2c), (2-5 ns) corresponded to segmental motion of cdb3. Both motions were hindered as quantified by nonzero r(1 infinity) and r(2 infinity). The strong pH dependence o f segmental motion correlated with that of cdb3 conformation measured by intrinsic tryptophan fluorescence. Significant changes in cdb3 segm ental motion occurred upon interactions with the small ligands 2,3-bis phosphoglycerate and calcium and several glycolytic enzymes known to b ind to the N terminus of band 3. These time-resolved fluorescence meas urements of the nanosecond segmental dynamics of a labeled membrane pr otein provide evidence for the sensitivity of cdb3 conformation to lig and binding and suggest long-range structural communication through cd b3.