Assessment of Nicotinic Acetylcholine Receptor Detergent Complexes Purity and Stability for structural studies

The Torpedo californica (Tc) nicotinic acetylcholine receptor (nAChR) is a large (~290 kDa) integral membrane protein composed of four homologous subunits with a stoichiometry α2βγδ. The main obstacle towards the achieving of high resolution structure of the nAChR is the preparation of milligram amounts of pure, functional and stable nAChR-detergent complex (nAChR-DC). A crucial aspect in the preparation nAChR-DCs is the functionality, purity and aggregation state of the solubilized protein. The main contaminants present in the crude Torpedo californica (Tc) membranes are the peripheral nAChR- associated proteins, mainly the 43-kDa rapsyn protein and the V-type ATPase. In the present project we aim to: perform an in-depth analysis of the purity of six nAChR-DCs: Foscholine (12,14,16) and Lysofoscholine (12, 14, 16) using Microfuiidic Capillary Gel Electrophoresis (MCGE), evaluate the state of aggregation, functionality and stability of these nAChR-DC's after the removal of intrinsic proteins. The preparation of functional and stable membrane protein-DCs from natural source will provide the groundwork to validate forthcoming developing technologies for the crystallization of large membrane protein complexes.