Structural investigations of nicotinic acetylcholine receptor and its intracellular domain

Abstract

In order to study the conformation of the nicotinic acetylcholine receptor (nAChR), we follow two routes: (a) crystallization of the intact nAChR isolated from Torpedo electric organ and (b) heterologous expression of the intracellular domain of nAChR. (a) In order to produce nAChR solutions that meet known prerequirements for protein crystallization (a monodisperse solution of mixed nAChR‐detergent‐micelles without contamination by other proteins) we will adapt the purification protocol, focusing on the removal of the associated protein rapsyn and deglycosylation. (b) We found that the Torpedo delta subunit intracellular loop can be expressed in E. coli relatively easily, whereas we obtained no expression of the a7 subunit loop, possibly due to its high cysteine content. Therefore our purification attempts will focus on the delta subunit. The purified loop domain will be analyzed using NMR spectroscopy.