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Thermal Unfolding of a Mammalian Pentameric Ligand-gated Ion Channel Proceeds at Consecutive, Distinct Steps

Tol, Menno B. and Deluz, Cedric and Hassaine, Gherici and Graff, Alexandra and Stahlberg, Henning and Vogel, Horst. (2013) Thermal Unfolding of a Mammalian Pentameric Ligand-gated Ion Channel Proceeds at Consecutive, Distinct Steps. Journal of Biological Chemistry, 288 (8). pp. 5756-5769.

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Official URL: http://edoc.unibas.ch/dok/A6070479

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Abstract

Pentameric ligand-gated ion channels (LGICs) play an important role in fast synaptic signal transduction. Binding of agonists to the β-sheet structured extracellular domain opens an ion channel in the transmembrane -helical region of the LGIC. How the structurally distinct and distant domains are functionally coupled for such central transmembrane signaling processes remains an open question. To obtain detailed information about the stability of and the coupling between these different functional domains we analyzed the thermal unfolding of a homopentameric LGIC, the 5-hydroxytryptamine receptor (5-HT3R) (ligand binding, secondary structure, accessibility of Trp and Cys residues, aggregation), in plasma membranes as well as during detergent-extraction, purification and reconstitution into artificial lipid bilayers. We found a large loss in thermostability correlating with the loss of the lipid bilayer during membrane solubilization and purification. Thermal unfolding of 5-HT3R occurred in consecutive steps at distinct protein locations: First a loss of ligand binding is detected, followed by formation of different transient low oligomeric states of receptor pentamers, followed by partial unfolding of helical parts of the protein, which finally leads to the formation large receptor aggregates. Structural destabilization of the receptor in detergents could be partially reversed by reconstituting the receptor into lipid bilayers. Our results are important because they quantify the stability of LGICs during detergent extraction and purification, and can be used to stabilize these membrane proteins for structural and functional studies.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Stahlberg)
UniBasel Contributors:Stahlberg, Henning
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:American Society for Biochemistry and Molecular Biology
ISSN:0021-9258
e-ISSN:1083-351X
Note:Publication type according to Uni Basel Research Database: Journal article -- The final publication is available at American Society for Biochemistry and Molecular Biology, see DOI link.
Language:English
Identification Number:
Last Modified:19 Oct 2018 13:38
Deposited On:24 May 2013 08:58

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