title: Expression and spectroscopic analysis of soluble nicotinic acetylcholine receptor fragments derived from the extracellular domain of the alpha-subunit.
abstract: 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.
authors.affiliation: Department of Molecular Pharmacology, Physiology, and Biotechnology, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Grant
authors.forename: L N
authors.initials: LN
authors.lastname: Gentile
authors.forename: Q L
authors.initials: QL
authors.lastname: Shi
authors.forename: M
authors.initials: M
authors.lastname: Pellegrini
authors.forename: E
authors.initials: E
authors.lastname: Hawrot
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002038:Bungarotoxins / Q000378:metabolism; D018678:Cholinergic Agents / Q000378:metabolism; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D005110:Extracellular Space / Q000737:chemistry / Q000378:metabolism; D007457:Iodine Radioisotopes / Q000378:metabolism; D007834:Lasers; D008024:Ligands; D008027:Light; D008823:Micelles; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012542:Scattering, Radiation; D012995:Solubility; D014101:Torpedo
chemical_list: D002038:Bungarotoxins; D018678:Cholinergic Agents; D007457:Iodine Radioisotopes; D008024:Ligands; D008823:Micelles; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins