The objective of this research project is to determine the molecular structure of Band 3, the erythrocyte anion transporter.
The specific aims of this proposal are (1) to continue the development of 2-dimensional (2- D) crystallization methods for membrane proteins and (2) to obtain molecular structure information of Band 3 by electron crystallographic methods. We have successfully reconstituted Band 3 protein with phospholipids (DMPC) yielding 2-D crystalline arrays that give promising results for high resolution structural studies. We will continue our effort in improving the crystal quality of Band 3 protein; this effort will provide a greater understanding about 2-D crystallization of membrane proteins, which is crucial to the widespread use of electron crystallography for membrane protein structural determination. We will first determine the 3-dimensional (3-D) structure of Band 3, negatively stained with uranyl acetate, to provide information about the overall channel architecture. As a part of the overall goal of our research in understanding the functional mechanism of Band 3, we will also crystallize the Band 3 mutant found in hereditary ovalocytosis, which is a red blood cell condition characterized by a rigid, oval shaped erythrocyte that is resistant to invasion by malaria parasites. The goal here is to obtain the structural information about this """"""""defective"""""""" Band 3 in order to understand the underlying basis for the resistance to invasion of malaria parasites. Even at a rather low resolution of negatively stained samples, the expected large structural change of the """"""""defective"""""""" Band 3 based on biochemical and biophysical studies can be verified. We will devote major effort toward determining the detailed molecular structure of this protein embedded in glucose. Knowledge of the molecular structures of Band 3 will provide information about anion binding sites, the detailed structural design of the pathways for anions and the structural role of the cytoplasmic soluble portion of Band 3 in the transport function. The structural understanding will provide us with information about the regulation of the transport of ions across the membrane. The overall goal of our structural studies of Band 3 is to give a rigorous conceptual framework for the rational understanding of the molecular mechanism of membrane transport systems and the specificity for the transport of solutes and ions across the membrane, and the basis for understanding the underlying causes of ovalocytosis and its resistance to invasion of malaria parasites.
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