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

Abstract

Interactions between the erythrocyte membrane and its skeleton are mediated primarily by binding of cytoskeletal components to a conformationally sensitive structure, the cytoplasmic domain of band 3 (cdb3). To e-mine the nanosecond segmental motions of cdb3, band 3 was labeled selectively by fluorescein maleimide at Cys-201 near the proposed hinge in cdb3 about which pH-dependent conformational changes occur. Time-resolved anisotropy of labeled cdb3 in Isolated form and in stripped erythrocyte membranes was measured by parallel-acquisition frequency-domain microfluorimetry. Samples had a single-component fluorescein lifetime of -4 ns. Multifrequency phase and modulation data (5-200 MHz) fitted well to a segmental motion model containing two correlation times (1c and 7) and two limiting anisotropies (r1. and r2.). Measurements in protease-cleaved and denatured samples indicated that Tjc (100-150 ps) corresponded to rapid rotation of bound fluorescein and rac (2-5 ns) corresponded to segmental motion of cdb3. Both motions were hindered as quantified by nonzero rl and ra.. The strong pH dependence of segmental motion correlated with that of cdb3 conformation measured by intrinsic tryptophan fluorescence. Significant changes in cdb3 segmental motion occurred upon interactions with the small ligands 2,3-bisphosphoglycerate and calcium and several glycolytic enzymes known to bind to the N terminus of band 3. These time-resolved fluorescence measurements of the nanosecond segmental dynamics of a labeled membrane protein provide evidence for the sensitivity of cdb3 conformation to ligand binding and suggest long-range structural communication through cdb3.The best known function ofband 3, the major integral protein of erythrocyte membranes, is the electroneutral exchange of anions across the plasma membrane (1). However, band 3 has several additional functions, many of which are mediated by interaction of its cytoplasmic domain with various soluble factors and enzymes (2). For example, the association between the cytoplasmic domain of band 3 (cdb3) and ankyrin provides the primary attachment site for the spectrin-based membrane skeleton (3, 4). Other components that interact with cdb3 include band 4.1, band 4.2, hemoglobin, and several glycolytic enzymes (2-4). To accommodate the binding of multiple substrates, cdb3 has an extended asymmetric structure (2). Whereas the binding sites for hemoglobin and glycolytic enzymes are restricted to the extreme N terminus of cdb3 (2), ankyrin and band 4.1 appear to interact also with distal regions of cdb3 (5-7). Several experiments suggest the existence of a flexible "hinge" near the middle of cdb3 (2, 8); the hinge may permit flexion of cdb3 to facilitate ligand binding to noncontiguous band 3 sequences.There is increasing evidence that band 3 has a dynamic structure. Band 3 appears to exist in situ as an equilibrium mixture of dimers and tetramers held together by noncovalent interactions (1, 9). In addition, cdb3 is thought to be poised in a facile equilibrium among th...

Development of a novel neodymium compound for in vivo fluorescence imaging

“…A lifetime of 2.3 μs was consequently obtained as reasonable for a Nd complex, as reported previously (10,17). In view of the ns-order lifetime of fluorescein [~4 ns (25)], 4AMF-DOTA(Nd) can be considered a potential NIR fluore-scent probe able to be detected and imaged separately from fluorescein, scattered or reflected excitation light and other organic fluorophores by the TRF technique.…”

Role of erythrocyte in regulating local O2delivery mediated by hemoglobin oxygenation

“…The binding sites for all these molecules, including deoxyhemoglobin, occur near the N-terminus of CDB3 (30). There is some debate in the literature about the degree of overlap among these binding sites but it has become clear that certainly PFK and deoxyhemoglobin, sharing very similar topology including the key central cavity, do compete at CDB3 (24,38). This competition implies a link between ATP production (glycolysis) and the oxygenation state of the RBC (via the relative concentration of deoxyhemoglobin present) (13,33).…”

Metabolic Effects of Endogenous and Exogenous Heterotropic Hemoglobin Modulators on Anion Transport: The Case of Pig Erythrocytes

“…Another important event that occurs during the transition HOS-LOS of the RBC is the change of the intracellular concentration of free 2,3-DPG. In this respect, it may be worthwhile to outline that the intraerythrocytic concentration of this organic phosphate in pig can be up to about 10 mM and that 2,3-DPG interacts with the erythrocyte membrane and with B3 in particular [1] [2] [18] [36]. Furthermore, 2,3-DPG causes an increase in the lateral mobility of integral membrane proteins [37] and, at concentrations which are normally present in erythrocytes, can weaken associations in the shell [38].…”