Soluble, oligomeric, and ligand-binding extracellular domain of the human alpha7 acetylcholine receptor expressed in yeast: replacement of the hydrophobic cysteine loop by the hydrophilic loop of the ACh-binding protein enhances protein solubility

The Journal of Biological Chemistry
Vassiliki AvramopoulouSocrates Tzartos

Abstract

The N-terminal extracellular domain (ECD; amino acids 1-208) of the neuronal nicotinic acetylcholine receptor (AChR) alpha7 subunit, the only human AChR subunit known to assemble as a homopentamer, was expressed as a glycosylated form in the yeast Pichia pastoris in order to obtain a native-like model of the extracellular part of an intact pentameric nicotinic AChR. This molecule, alpha7-ECD, although able to bind the specific ligand alpha-bungarotoxin, existed mainly in the form of microaggregates. Substitution of Cys-116 in the alpha7-ECD with serine led to a decrease in microaggregate size. A second mutant form, alpha7-ECD(C116S,Cys-loop), was generated in which, in addition to the C116S mutation, the hydrophobic Cys-loop (Cys(128)-Cys(142)) was replaced by the corresponding hydrophilic Cys-loop from the snail glial cell acetylcholine-binding protein. This second mutant protein was water-soluble, expressed at a moderate level (0.5 +/- 0.1 mg/liter), and had a size corresponding approximately to a pentamer as judged by gel filtration and electron microscopy studies. It also bound (125)I-alpha-bungarotoxin with relatively high affinity (K(d) = 57 nm), the binding being inhibited by unlabeled alpha-bungarotoxin, d-tubocurarine,...Continue Reading

References

Jan 1, 1993·Annual Review of Neuroscience·P B Sargent
Mar 1, 1995·Trends in Neurosciences·M O Ortells, G G Lunt
Feb 1, 1997·Trends in Neurosciences·S Wonnacott
May 1, 1997·Brain Research. Molecular Brain Research·V M GehleK Sumikawa
Nov 19, 1997·Progress in Neurobiology·C GottiF Clementi
Dec 13, 1997·Molecular Neurobiology·J Lindstrom
Dec 24, 1997·Brain Research. Brain Research Reviews·H R Arias
Feb 14, 1998·The Journal of Biological Chemistry·G B WellsJ Lindstrom
Jan 21, 2000·FEMS Microbiology Reviews·J L Cereghino, J M Cregg
Apr 20, 2000·European Journal of Pharmacology·F ClementiC Gotti
Dec 1, 2000·Molecular Biotechnology·J M CreggD R Higgins
Mar 15, 2001·Proceedings of the National Academy of Sciences of the United States of America·M FischerJ P Changeux
Feb 28, 2002·Proceedings of the National Academy of Sciences of the United States of America·Nicolas Le NovèreJean-Pierre Changeux
Jun 6, 2002·European Journal of Biochemistry·Victor I TsetlinChristoph Methfessel
Nov 19, 2002·Journal of Neurobiology·Steven M Sine
Nov 19, 2002·Journal of Neurobiology·Nicolas Le NovèreJean-Pierre Changeux
Jun 27, 2003·Nature·Atsuo MiyazawaNigel Unwin
Sep 19, 2003·Proceedings of the National Academy of Sciences of the United States of America·Yoav PaasJean-Pierre Changeux

❮ Previous
Next ❯

Citations

Nov 20, 2013·International Journal of Biological Macromolecules·Konstantinos LazaridisSocrates J Tzartos
Aug 25, 2010·Journal of the National Cancer Institute·Chia-Hwa LeeYuan-Soon Ho
May 30, 2008·Frontiers in Bioscience : a Journal and Virtual Library·Gregg B Wells
Jul 31, 2007·International Journal of Biological Macromolecules·Marios ZouridakisSocrates J Tzartos
Mar 26, 2009·IUBMB Life·Marios ZouridakisSocrates J Tzartos
Jul 27, 2007·The FEBS Journal·Dimitra KalamidaSocrates J Tzartos
Feb 2, 2008·Journal of Pharmaceutical Sciences·Saul R TrevinoC Nick Pace
Jan 11, 2011·Journal of Neurochemistry·Victor TsetlinIgor Kasheverov
Nov 26, 2009·Immunology Letters·Olena LykhmusMaryna Skok
Sep 22, 2005·The Journal of Biological Chemistry·Alexandra M PersonGregg B Wells
Nov 7, 2019·Nature Biotechnology·Irma QuerquesOrsolya Barabas
Nov 18, 2009·Journal of Molecular Biology·Hugues NuryMarc Delarue

❮ Previous
Next ❯

Related Concepts

Related Feeds

ASBMB Publications

The American Society for Biochemistry and Molecular Biology (ASBMB) includes the Journal of Biological Chemistry, Molecular & Cellular Proteomics, and the Journal of Lipid Research. Discover the latest research from ASBMB here.