Insertion of transmembrane 5-lipoxygenase activating protein (FLAP) into nanodiscs towards structure function studies of complex formation with soluble proteins

Leukotrienes are specialized lipid molecules derived from arachidonic acid that have severe pathological roles in inflammatory diseases like asthma, allergy and in the formation of cardiovascular diseases. Since these diseases can be fatal if not treated, it is significant to study leukotriene products and how they form. The formation of leukotrienes from arachidonic acid is a multi step process. This event is aided by different enzymes. Two such enzymes are 5–lipoxygenase (5-LO) and 5-lipoxygenase activating protein (FLAP). FLAP is an integral membrane protein and belongs to the MAPEG super family (Membrane Associated proteins in Eicosanoid and Glutathione metabolism). In the leukotriene synthesis pathway, the only function of FLAP is to increase the oxygenation reaction by assisting the transfer of arachidonic acid from nuclear membrane to 5-LO. In fact, FLAP doesn’t seem to have any enzymatic/mechanistic activity on its own. Many biological questions in this pathway are unanswered. Is there any functional activity of FLAP? Is there any physical contact between FLAP and 5-LO in the leukotriene formation? To address these questions, we introduced a novel method of studying the pathway by constructing nanodiscs that are small soluble pieces of membrane of defined sizes. The aim of the project is to develop a protocol for generation of nanodiscs that contain FLAP (so called reconstitution of FLAP) and to verify the presence of FLAP in the nanodiscs by practical techniques such as blue native PAGE and Western Blot. Images from transmission electron microscopy were less conclusive but improvement can be done in future by use of antibodies to visualize FLAP nanodiscs. Ultimately, reconstitution of FLAP was successfully completed for further structural and functional studies. To conclude, this project paves way for the studies of complex formation of integral membrane proteins with soluble proteins and further applications on pharmaceutical front