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 nico tinic acetylcholine receptor (nAChR), we have developed methods for the hig h-level expression and purification of an important functional portion of t he N-terminal extracellular domain (ECD) of the alpha-subunit. Two soluble receptor fragments comprising residues 143-210 of the Torpedo californica a lpha-subunit were expressed in E. coli: alpha T68His(6), which contains a h istidine tag, and alpha T68M1, which includes the first transmembrane regio n, M1, of the alpha-subunit. Both proteins demonstrate saturable, high-affi nity alpha-bungarotoxin (Bgtx) binding with an apparent equilibrium K-D (3 nM) that is comparable to the affinities reported for preparations comprisi ng the entire alpha-subunit ECD. These results demonstrate that the ECD det erminants required for Bgtx recognition of the alpha-subunit are entirely s pecified by residues 143-210. The binding of small ligands was demonstrated in competition assays with I-125-Bgtx yielding K-I values of 58 and 105 mu M for d-tubocurarine and nicotine, respectively. Circular dichroism (CD) a nalysis of monomeric alpha T68His(6) protein revealed considerable secondar y structure. Furthermore, a cooperative, two-state folding transition was o bserved upon urea denaturation. To circumvent concentration-dependent aggre gation of the alpha T68His(6) protein at the millimolar concentrations need ed for NMR study, we utilized the M1 transmembrane domain to anchor the rec ombinant receptor fragment onto membrane-mimicking micelles. Monodispersed preparations of alpha T68M1 in dodecylphosphocholine micelles demonstrate h igh-affinity Bgtx binding and considerable secondary structure by CD. The s tructural features revealed in the CD profile appear to undergo a cooperati ve, two-state folding transition upon thermal denaturation. Initial NMR stu dies suggest that micellar preparations of the alpha T68M1 fragment are ame nable to further high-resolution heteronuclear NMR analysis.