Structural studies of membrane proteins in lipid bilayer

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DESCRIPTION (provided by applicant): We intend to pursue structural studies of recombinant, membrane-associated components of the E.coli ribose transporter by electron crystallography (aim I) and devise novel strategies for 2-D crystallization of integral membrane proteins in the lipid bilayer membrane (aim II). Our specific aims are described below. I. Structural studies of the components of the E. coli ribose transporter. Structural studies of the extremely hydrophobic component rbsC of the ribose transporter that encloses the solute transport pathway and a chimera of the nucleotide binding domain rbsA and rbsC will be pursued. Single particle image analysis will provide details of the quaternary organization and overall shape of the protein moieties at low resolution. Lipid reconstitution experiments will be carried out to generate 2-dimensional crystals that are appropriate for high-resolution electron crystallographic analysis. II. Novel strategies for crystallization of integral membrane proteins in the lipid bilayer. a) We intend to explore whether a membrane protein which readily crystallizes in a 2-dimensional array in the lipid bilayer can help in crystallizing or improving the crystallinity of another membrane protein when a chimera of the two are subjected to crystallization experiments. We will subject yeast-expressed and purified AQP1-AQP2 chimera, where AQP1 is the human, erythrocyte water channel and AQP2 is the homologous kidney collecting duct water channel, to lipid-reconstituted 2-D crystallization experiments. b) As an alternative to 2-D crystallization on planar membrane bilayers, we will extend the method of helical crystallization on unilamellar lipid tubules, hitherto carried out for soluble proteins, to the case of integral membrane proteins. Membrane proteins that function as transporters, channels and pumps mediate communication between the inside and outside of the cell, which is crucial for cell survival, and are therefore important pharmacological targets. The focus of our proposed research directed at investigation of the 3-dimensional structure in the lipid bilayer would potentially have impact on structure-based drug design.