Overexpression and crystallization of amino acid transp*

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DESCRIPTION (provided by applicant): Amino acids are essential building blocks for proteins and many other molecules in the cell. In the central nervous system, they serve as major signaling molecules. Amino acid uptake and secretion across the cell membrane are mediated by a group of membrane proteins, called amino acid transporters. These transporters are involved in many important physiological processes, including: nutrient uptake at the absorptive epithelia in the small intestine, transport at the kidney proximal tubule for glucogeonesis and hepatic regeneration, supply of amino acids to the fetus by the mother at the placenta, and chemical neurotransmission at the synapse. Equally important, amino acid transporters are associated with numerous pathological conditions, such as cystinuria, lysinuria, schizophrenia, ischemia and cocaine addiction. To understand the molecular mechanism of amino acid transporters, three-dimensional structural information generated by crystallography is needed. Transporters from mammalian organisms are currently not suitable for crystallization, due to their low natural abundance and lack of appropriate overexpression systems. Bacterial amino acid transporters share sequence homology with their mammalian counterparts, and thus most likely have similar three-dimensional structures. We therefore plan to overexpress, purify and crystallize bacterial amino acid transporters from the amino acid/polyamine/choline transporter family. Proteins will be overexpressed in E. coli and purified to homogeneity. Crystallization strategy begins with screening homologues from different species and protein modification by genetic techniques. Crystal nucleation will be screened with various polyethylene glycols and pHs. Crystalline order will be improved by adjusting detergent micellar size and co-crystallization with inhibitors and heavy metal salts. Well-ordered crystals will enable us to determine the transporter structure at atomic resolution later. The structure will reveal the amino acid binding pocket and substrate translocation pathway, and will allow us to design experiments to elucidate the roles that critical residues play in the transport process. Such structural in formation will also shed light on the structure and molecular mechanism for mammalian amino acid transporters.