Uncovering the Mode of Action of Lipid II Flippase: A New Antibiotic Target. (360G-Wellcome-110270_Z_15_Z)

Understanding the cellular process by which lipid-anchored molecules reverse their polarity to traverse the cell membrane is a fundamental mechanistic question that remains unsolved. I propose to study the lipid II flippase in E. coli, which transports lipid II across the inner-membrane and into the periplasm where peptidoglycan biosynthesis occurs. There has been controversy surrounding the true identity of the E. coli lipid II flippase and very little is known about how this enzyme works. Ther efore, I aim to meet the following goals during my tenure of this fellowship; (1) Identify the true lipid II flippase using photoaffinity labelling. Synthetic lipid II precursors containing photoaffinity and fluorescent labels will allow direct detection of the flippase. (2) Clone, express and purify active lipid II flippase and obtain crystals of this enzyme. Efforts will then be directed towards solving the crystal structure of lipid II flippase. (3) Develop an in vitro assay using pr oteoliposomes to assay flippase substrate specificity. A library of lipid II analogues with modifications on the lipid, peptide and carbohydrate portions will be synthesized and screened. (4) Generate a library of flippase mutants with cysteines at key locations to probe the flipping mechanism.