Expression and spectroscopic analysis of soluble nicotinic acetylcholine receptor fragments derived from the extracellular domain of the alpha-subunit.

To facilitate structural studies of the ligand binding region from the nicotinic acetylcholine receptor (nAChR), we have developed methods for the high-level expression and purification of an important functional portion of the N-terminal extracellular domain (ECD) of the alpha-subunit. Two soluble receptor fragments comprising residues 143-210 of the Torpedo californica alpha-subunit were expressed in E. coli: alphaT68His6, which contains a histidine tag, and alphaT68M1, which includes the first transmembrane region, M1, of the alpha-subunit. Both proteins demonstrate saturable, high-affinity alpha-bungarotoxin (Bgtx) binding with an apparent equilibrium KD (3 nM) that is comparable to the affinities reported for preparations comprising the entire alpha-subunit ECD. These results demonstrate that the ECD determinants required for Bgtx recognition of the alpha-subunit are entirely specified by residues 143-210. The binding of small ligands was demonstrated in competition assays with 125I-Bgtx yielding KI values of 58 and 105 microM for d-tubocurarine and nicotine, respectively. Circular dichroism (CD) analysis of monomeric alphaT68His6 protein revealed considerable secondary structure. Furthermore, a cooperative, two-state folding transition was observed upon urea denaturation. To circumvent concentration-dependent aggregation of the alphaT68His6 protein at the millimolar concentrations needed for NMR study, we utilized the M1 transmembrane domain to anchor the recombinant receptor fragment onto membrane-mimicking micelles. Monodispersed preparations of alphaT68M1 in dodecylphosphocholine micelles demonstrate high-affinity Bgtx binding and considerable secondary structure by CD. The structural features revealed in the CD profile appear to undergo a cooperative, two-state folding transition upon thermal denaturation. Initial NMR studies suggest that micellar preparations of the alphaT68M1 fragment are amenable to further high-resolution heteronuclear NMR analysis.