The molecular mechanism of active nucleoside transport across biological membranes. (360G-Wellcome-091322_B_10_Z)

Nucleoside transporters are essential for the salvage synthesis of nucleotides by many cell types, and for the efficient uptake of hydrophilic nucleoside drugs. By regulating the extracellular concentrations of adenosine and other nucleosides, they also modulate numerous physiological processes, ranging from cardiovascular activity to lipolysis. Elucidating their molecular mechanisms is therefore not only crucial for a fuller understanding of nucleoside biology, but should also underpin the deve lopment of novel therapeutic strategies for human disease. Towards this end, the key goals of the proposed research are: [1] To elucidate the structures of bacterial representatives of the ubiquitous concentrative nucleoside transporter (CNT) family, in particular NupC from Escherichia coli. 2-D and 3-D crystallography of NupC will exploit the availability of large amounts of wild-type and conformationally locked mutant proteins. [2] In parallel to investigate helix arrangements and dynamics in NupC by non-crystallographic approaches, including Forster resonance energy transfer (FRET) and thiol cross-linking. [3] To perform complementary investigations on homologous sodium- and proton-linked CNTs from humans to elucidate the mechanisms underlying nucleoside selectivity, cation-coupling, and conformational changes. [4] To attempt the production of human CNTs in amounts sufficient for structural investigations, exploiting recent advances in high-throughput methods for screening euk aryote membrane protein expression.