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
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
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
_version_	1718384774236602368

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

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