title: Expression and circular dichroism studies of the extracellular domain of the alpha subunit of the nicotinic acetylcholine receptor.
abstract: To provide material suitable for structural studies of the nicotinic acetylcholine receptor, we have expressed and purified the NH2-terminal extracellular domain of the mouse muscle alpha subunit. Several constructs were initially investigated using Xenopus oocytes as a convenient small scale expression system. A fusion protein (alpha210GPI) consisting of the 210 NH2-terminal amino acids of the alpha subunit and a glycosylphosphatidylinositol anchorage sequence conferred surface alpha-bungarotoxin binding in oocytes. Coexpression of alpha210GPI with an analogous construct made from the delta subunit showed no evidence of heterodimer formation. The alpha210GPI protein was chosen for large scale expression in transfected Chinese hamster ovary cells. The alpha210GPI protein was cleaved from these cells and purified on an immunoaffinity column. Gel and column chromatography show that the purified protein is processed as expected and exists as a monomer. The purified protein also retains the two distinct, conformation-specific binding sites expected for the correctly folded alpha subunit. Circular dichroism studies of alpha210GPI suggest that this region of the receptor includes considerable beta-sheet secondary structure, with a small proportion of alpha-helix.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: A P
authors.initials: AP
authors.lastname: West
authors.forename: P J
authors.initials: PJ
authors.lastname: Bjorkman
authors.forename: D A
authors.initials: DA
authors.lastname: Dougherty
authors.forename: H A
authors.initials: HA
authors.lastname: Lester
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002645:Chickens; D002942:Circular Dichroism; D006224:Cricetinae; D005110:Extracellular Space / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism; D014981:Xenopus
chemical_list: D011978:Receptors, Nicotinic