Prev. Article Next Article Table of Contents

Article

Expression and Spectroscopic Analysis of Soluble Nicotinic Acetylcholine Receptor Fragments Derived from the Extracellular Domain of the α-Subunit^†

Marianne A. Grant, Lisa N. Gentile,^‡ Qing-Luo Shi, Maria Pellegrini, and Edward Hawrot*

Department of Molecular Pharmacology, Physiology, and Biotechnology, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

Biochemistry, 1999, 38 (33), pp 10730–10742

DOI: 10.1021/bi983007q

Publication Date (Web): July 27, 1999

†

This work was supported by Research Grants GM32629 and NS34348 (E.H.) from the National Institutes of Health. NMR instrumentation was provided for by grants 1S10-RR-08240 from the National Institutes of Health and DBI-N923282 from the National Science Foundation. M.A.G. and L.N.G. were supported in part by National Institutes of Health Predoctoral Training Grant GM07601. M.A.G. was a 1997 recipient of a Pre-Doctoral Fellowship from the Pharmaceutical Research and Manufacturers of America Foundation. This work was done in partial fulfillment of the requirements for a Ph.D. degree from Brown University (M.A.G.).

‡

Current address: Department of Chemistry, University of New England, Biddeford, ME.

Address correspondence to this author. Telephone: 401-863-1034. Fax: 401-863-1595. E-mail: Edward_Hawrot@brown.edu.

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 α-subunit. Two soluble receptor fragments comprising residues 143−210 of the Torpedo californica α-subunit were expressed in E. coli: αT68His₆, which contains a histidine tag, and αT68M1, which includes the first transmembrane region, M1, of the α-subunit. Both proteins demonstrate saturable, high-affinity α-bungarotoxin (Bgtx) binding with an apparent equilibrium K_D (3 nM) that is comparable to the affinities reported for preparations comprising the entire α-subunit ECD. These results demonstrate that the ECD determinants required for Bgtx recognition of the α-subunit are entirely specified by residues 143−210. The binding of small ligands was demonstrated in competition assays with ¹²⁵I-Bgtx yielding K_I values of 58 and 105 μM for d-tubocurarine and nicotine, respectively. Circular dichroism (CD) analysis of monomeric αT68His₆ protein revealed considerable secondary structure. Furthermore, a cooperative, two-state folding transition was observed upon urea denaturation. To circumvent concentration-dependent aggregation of the αT68His₆ 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 αT68M1 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 αT68M1 fragment are amenable to further high-resolution heteronuclear NMR analysis.