The anion exchange protein (band 3) is a 100 k transmembrane protein in the human erythrocyte membrane which is composed of two distinct domains that serve two distinct functions. The integral membrane domain facilitates the exchange of HCO3- for Cl-, thereby providing an important physiological function related to CO2 transport from the distal tissues to the lungs. The cytoplasmic domain serves as a membrane binding site for peripheral proteins including those involved in bridging the cytoplasmic domain to the membrane skeleton, thereby providing a physical link between the membrane skeleton and the bilayer. This latter function is essential for maintaining the biconcave shape and unique viscoelastic properties of the erythrocyte. Studies in progress have long-term goals of characterization of structural features of band 3 which are important for its transport function and elucidation of dynamic properties of band 3 which reflect its interactions with other membrane proteins and with membrane lipids. These studies will provide important insights into functional and mechanical properties of band 3 in human erythrocytes and in the broader context, will further understanding of how structural and dynamic properties of cell membranes affect their functional properties. In the next grant period, electron paramagnetic resonance, fluorescence, and phosphorescence spectroscopies will be employed, in conjunction with new molecular probes which have been developed, to address the following specific questions: 1) How does the ordering of membrane lipids affect the transport function band 3? 2) What populations, in terms of rotational mobility, of band 3 exist in the erythrocyte and to what extent can these populations be assigned to known protein-protein interactions? A related question is what are the mechanical properties of the cytoplasmic domain of band 3 which links the membrane skeleton to the lipid bilayer and to what extent do these links exist in the intact cell? 3) What are the properties of specific protein-protein interactions involving the cytoplasmic domain of band 3 and the binding proteins ankyrin, protein 4.1, and protein 4.2 in a partially purified membrane system? and 4) What is the spatial arrangement between the functionally important anion binding site on band 3 and the interface between adjacent band 3 monomers? During the course of these studies, new molecular probes, spectroscopic methods, and data analysis methods will be developed which will be of utility in the biomedical research community.
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