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Title: Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers

Abstract

Here, the structure of the p7 viroporin, an oligomeric membrane protein ion channel involved in the assembly and release of the hepatitis C virus, was determined from proteins expressed and inserted directly into supported model lipid membranes using cell-free protein expression. Cell-free protein expression allowed (i) high protein concentration in the membrane, (ii) control of the protein’s isotopic constitution, and (iii) control over the lipid environment available to the protein. Here, we used cell-free protein synthesis to directly incorporate the hepatitis C virus (HCV) p7 protein into supported lipid bilayers formed from physiologically relevant lipids (POPC or asolectin) for both direct structural measurements using neutron reflectivity (NR) and conductance measurements using electrical impedance spectroscopy (EIS). We report that HCV p7 from genotype 1a strain H77 adopts a conical shape within lipid bilayers and forms a viroporin upon oligomerization, confirmed by EIS conductance measurements. This combination of techniques represents a novel approach to the study of membrane proteins and, through the use of selective deuteration of particular amino acids to enhance neutron scattering contrast, has the promise to become a powerful tool for characterizing the protein conformation in physiologically relevant environments and for the development of biosensor applications.

Authors:
 [1];  [2];  [2]; ORCiD logo [3]
  1. Synthelis SAS, La Tronche (France); Univ. Grenoble Alpes, Grenoble (France)
  2. Univ. Grenoble Alpes, Grenoble (France)
  3. Institut Laue-Langevin, Grenoble (France); Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Publication Date:
Research Org.:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1364574
Report Number(s):
LA-UR-16-27372
Journal ID: ISSN 2045-2322
Grant/Contract Number:  
AC52-06NA25396
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 7; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; biological physics; biomaterials; membrane structure and assembly

Citation Formats

Soranzo, Thomas, Martin, Donald K., Lenormand, Jean -Luc, and Watkins, Erik Benjamin. Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers. United States: N. p., 2017. Web. doi:10.1038/s41598-017-03472-8.
Soranzo, Thomas, Martin, Donald K., Lenormand, Jean -Luc, & Watkins, Erik Benjamin. Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers. United States. doi:10.1038/s41598-017-03472-8.
Soranzo, Thomas, Martin, Donald K., Lenormand, Jean -Luc, and Watkins, Erik Benjamin. Tue . "Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers". United States. doi:10.1038/s41598-017-03472-8. https://www.osti.gov/servlets/purl/1364574.
@article{osti_1364574,
title = {Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers},
author = {Soranzo, Thomas and Martin, Donald K. and Lenormand, Jean -Luc and Watkins, Erik Benjamin},
abstractNote = {Here, the structure of the p7 viroporin, an oligomeric membrane protein ion channel involved in the assembly and release of the hepatitis C virus, was determined from proteins expressed and inserted directly into supported model lipid membranes using cell-free protein expression. Cell-free protein expression allowed (i) high protein concentration in the membrane, (ii) control of the protein’s isotopic constitution, and (iii) control over the lipid environment available to the protein. Here, we used cell-free protein synthesis to directly incorporate the hepatitis C virus (HCV) p7 protein into supported lipid bilayers formed from physiologically relevant lipids (POPC or asolectin) for both direct structural measurements using neutron reflectivity (NR) and conductance measurements using electrical impedance spectroscopy (EIS). We report that HCV p7 from genotype 1a strain H77 adopts a conical shape within lipid bilayers and forms a viroporin upon oligomerization, confirmed by EIS conductance measurements. This combination of techniques represents a novel approach to the study of membrane proteins and, through the use of selective deuteration of particular amino acids to enhance neutron scattering contrast, has the promise to become a powerful tool for characterizing the protein conformation in physiologically relevant environments and for the development of biosensor applications.},
doi = {10.1038/s41598-017-03472-8},
journal = {Scientific Reports},
number = 1,
volume = 7,
place = {United States},
year = {Tue Jun 13 00:00:00 EDT 2017},
month = {Tue Jun 13 00:00:00 EDT 2017}
}

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Works referenced in this record:

Polymer-supported membranes as models of the cell surface
journal, September 2005

  • Tanaka, Motomu; Sackmann, Erich
  • Nature, Vol. 437, Issue 7059, p. 656-663
  • DOI: 10.1038/nature04164