Electron microscopic evidence for the assembly of soluble pentameric extracellular domains of the nicotinic acetylcholine receptor.
Exploitation of soluble extracellular domains (ECDs) of the nicotinic acetylcholine receptor may provide a route to crystallographic studies aimed at exploring the structure and function of the intact receptor. The first step towards this goal is to manufacture and isolate soluble fragments that fold and assemble to form a functionally relevant complex. The baculovirus insect cell expression system was used to co-express soluble ECDs of all four muscle-type nicotinic acetylcholine receptor subunits (alpha, beta, gamma & delta-ECD) from Torpedo. Protein complexes were purified using either the conformationally sensitive monoclonal antibody mAb35, specific for a folded alpha subunit, or a NiNTA affinity resin, specific for a polyhistidine tag engineered on the delta-ECD. Western blotting with subunit specific antibodies confirmed the co-expression of each ECD and furthermore, indicated that the alpha, beta and gamma-ECDs were being co-purified with the polyhistidine-tagged delta-ECD. Chemical cross-linking was used to show that these co-purified proteins had indeed interacted specifically to form soluble oligomeric complexes. A low-resolution, three-dimensional image of these purified complexes, composed only of ECDs, was obtained by electron microscopy. They were shown to resemble the extracellular vestibule of the native receptor, having the same pseudo-pentameric symmetry, size and shape. Expression of incomplete sets of the four nicotinic acetylcholine receptor ECDs did not yield detectable complexes.
Tierney
M L
ML
Division of Biochemistry & Molecular Biology, Faculty of Science, Australian National University, Canberra 0200, Australia. Louise.Tierney@anu.edu.du
Unwin
N
N
D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002038:Bungarotoxins / Q000378:metabolism; D002460:Cell Line; D003432:Cross-Linking Reagents; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D012995:Solubility; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism / Q000821:virology; D014101:Torpedo / Q000235:genetics
D000911:Antibodies, Monoclonal; D002038:Bungarotoxins; D003432:Cross-Linking Reagents; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins