Stuctural studies on human erythrocyte BAND-3

The Human Erythrocyte Anion exchanger, AE1, also known as Band 3, allows bicarbonate to cross the red blood cell membrane in exchange for chloride. This is an essential process in allowing the bloodstream to transport large quantities of carbonate to the lungs. Band 3 is also associated with a number of human disease states. In order to understand the precise mechanism of action of the protein and reveal further details on these disease states it is necessary to obtain precise structural information for the human form of Band 3 membrane domain. The most suitable technique for this is X-ray crystallography. The structure determination of human membrane proteins is extremely challenging as evidenced by the fact that no X-ray structure has been solved. So far it has been possible to obtain highly pure Band 3 protein from human red blood cells which has yielded crystals. However the data obtained from the X-ray analysis of the crystals is not of sufficient quality to solve the structure. This application focuses on the optimisation of the Band 3 crystals in order to obtain high quality data suitable for structure determination.

The Human Erythrocyte Anion exchanger, AE1, also known as Band 3, acts as an anion transporter in red blood cells, allowing bicarbonate to cross the membrane in exchange for chloride. The protein can be cleaved into the two domains which maintain functional integrity. The C-terminal membrane domain is predicted to have fourteen transmembrane helices and is responsible for the anion transport activity. A very low resolution (20 A) 3D map of the dimeric membrane domain was obtained using electron microscopy more than a decade ago. However in order to reveal molecular detail on the structure-function relationships of the membrane domain of Band 3 it is necessary to obtain high resolution structural information for the protein using the technique of X-ray crystallography. The C-terminal domain of Band 3 has been isolated from human red blood cells and crystals obtained which diffract to 7 A. This application is focussed on improving the crystals in order to obtain high resolution diffraction data suitable for solving the structure. Sample quality will be improved in order to allow reproducibility of crystals. In addition, we will submit the Band 3 protein to our tried and tested optimisation strategy; screening in particular additives and detergents in order to obtain better diffracting crystals. We will also identify alternative crystal forms which may produce better diffracting crystals. These rational screening approaches have resulted in a number of membrane protein structures from our lab. However in some cases it may be necessary to use alternative methods such as extending the hydrophilic domain of the membrane protein in order to obtain well-diffracting crystals. We will use both monocolonal antibodies and artificial binding proteins to extend the hydrophilic domain of band 3. We will also establish expression system of Band 3 using Pichia pastoris for functional and structural studies.