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

Abstract 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 protei...

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Autores principales: Thomas Soranzo, Donald K. Martin, Jean-Luc Lenormand, Erik B. Watkins
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Publicado: Nature Portfolio 2017
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Acceso en línea:https://doaj.org/article/4f6443728bef49bf87f2da70bdcdf95f
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spelling oai:doaj.org-article:4f6443728bef49bf87f2da70bdcdf95f2021-12-02T16:07:01ZCoupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers10.1038/s41598-017-03472-82045-2322https://doaj.org/article/4f6443728bef49bf87f2da70bdcdf95f2017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-03472-8https://doaj.org/toc/2045-2322Abstract 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.Thomas SoranzoDonald K. MartinJean-Luc LenormandErik B. WatkinsNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-10 (2017)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
Thomas Soranzo
Donald K. Martin
Jean-Luc Lenormand
Erik B. Watkins
Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers
description Abstract 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.
format article
author Thomas Soranzo
Donald K. Martin
Jean-Luc Lenormand
Erik B. Watkins
author_facet Thomas Soranzo
Donald K. Martin
Jean-Luc Lenormand
Erik B. Watkins
author_sort Thomas Soranzo
title Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers
title_short Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers
title_full Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers
title_fullStr Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers
title_full_unstemmed Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers
title_sort coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers
publisher Nature Portfolio
publishDate 2017
url https://doaj.org/article/4f6443728bef49bf87f2da70bdcdf95f
work_keys_str_mv AT thomassoranzo couplingneutronreflectivitywithcellfreeproteinsynthesistoprobemembraneproteinstructureinsupportedbilayers
AT donaldkmartin couplingneutronreflectivitywithcellfreeproteinsynthesistoprobemembraneproteinstructureinsupportedbilayers
AT jeanluclenormand couplingneutronreflectivitywithcellfreeproteinsynthesistoprobemembraneproteinstructureinsupportedbilayers
AT erikbwatkins couplingneutronreflectivitywithcellfreeproteinsynthesistoprobemembraneproteinstructureinsupportedbilayers
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