Assessment of Nicotinic Acetylcholine Receptor Detergent Complexes Purity and Stability for Structural Studies

Abstract

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 study we performed an in-depth analysis of the purity of six nAChR-DCs prepared lipid analogue detergents: Foscholine (12,14,16) and Lysofoscholine (12,14,16) using microfluidic capillary gel electrophoresis (Angilent Bioanalizer 2100). This method provides a detailed analysis of the contaminants present in the nAChR-DCs. In addition, we evaluated the state of aggregation of these nAChR-DCs. The present study provides a roadmap for the preparation of high quality nAChR-DCs for structural studies. 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.