Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane

Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has...

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Autores principales:	Navid Khangholi, Marc Finkler, Ralf Seemann, Albrecht Ott, Jean-Baptiste Fleury
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	Archaerhodopsin-3 lipid bilayer microfluidics cell-free gene expression Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/e07a8d2c3c534885b082c162dfd0bdec
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id	oai:doaj.org-article:e07a8d2c3c534885b082c162dfd0bdec
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spelling	oai:doaj.org-article:e07a8d2c3c534885b082c162dfd0bdec2021-11-11T17:24:02ZPhotoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane10.3390/ijms2221119811422-00671661-6596https://doaj.org/article/e07a8d2c3c534885b082c162dfd0bdec2021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/21/11981https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely investigated in recent decades, the electrophysiological properties of pore-forming Archaerhodopsin (Arch), as studied in vitro, have remained largely unknown. Here, we formed unsupported bilayers between two channels of a microfluidic chip which enabled the simultaneous optical and electrical assessment of the bilayer in real time. Using a cell-free expression system, we recombinantly produced a GFP (green fluorescent protein) labelled as a variant of Arch-3. The label enabled us to follow the synthesis of Arch-3 and its incorporation into the bilayer by fluorescence microscopy when excited by blue light. Applying a green laser for excitation, we studied the electrophysiological properties of Arch-3 in the bilayer. The current signal obtained during excitation revealed distinct steps upwards and downwards, which we interpreted as the opening or closing of Arch-3 pores. From these steps, we estimated the pore radius to be 0.3 nm. In the cell-free extract, proteins can be modified simply by changing the DNA. In the future, this will enable us to study the photoelectrical properties of modified transmembrane protein constructs with ease. Our work, thus, represents a first step in studying signaling cascades in conjunction with coupled receptor proteins.Navid KhangholiMarc FinklerRalf SeemannAlbrecht OttJean-Baptiste FleuryMDPI AGarticleArchaerhodopsin-3lipid bilayermicrofluidicscell-free gene expressionBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 11981, p 11981 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Archaerhodopsin-3 lipid bilayer microfluidics cell-free gene expression Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	Archaerhodopsin-3 lipid bilayer microfluidics cell-free gene expression Biology (General) QH301-705.5 Chemistry QD1-999 Navid Khangholi Marc Finkler Ralf Seemann Albrecht Ott Jean-Baptiste Fleury Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane
description	Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely investigated in recent decades, the electrophysiological properties of pore-forming Archaerhodopsin (Arch), as studied in vitro, have remained largely unknown. Here, we formed unsupported bilayers between two channels of a microfluidic chip which enabled the simultaneous optical and electrical assessment of the bilayer in real time. Using a cell-free expression system, we recombinantly produced a GFP (green fluorescent protein) labelled as a variant of Arch-3. The label enabled us to follow the synthesis of Arch-3 and its incorporation into the bilayer by fluorescence microscopy when excited by blue light. Applying a green laser for excitation, we studied the electrophysiological properties of Arch-3 in the bilayer. The current signal obtained during excitation revealed distinct steps upwards and downwards, which we interpreted as the opening or closing of Arch-3 pores. From these steps, we estimated the pore radius to be 0.3 nm. In the cell-free extract, proteins can be modified simply by changing the DNA. In the future, this will enable us to study the photoelectrical properties of modified transmembrane protein constructs with ease. Our work, thus, represents a first step in studying signaling cascades in conjunction with coupled receptor proteins.
format	article
author	Navid Khangholi Marc Finkler Ralf Seemann Albrecht Ott Jean-Baptiste Fleury
author_facet	Navid Khangholi Marc Finkler Ralf Seemann Albrecht Ott Jean-Baptiste Fleury
author_sort	Navid Khangholi
title	Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane
title_short	Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane
title_full	Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane
title_fullStr	Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane
title_full_unstemmed	Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane
title_sort	photoactivation of cell-free expressed archaerhodopsin-3 in a model cell membrane
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/e07a8d2c3c534885b082c162dfd0bdec
work_keys_str_mv	AT navidkhangholi photoactivationofcellfreeexpressedarchaerhodopsin3inamodelcellmembrane AT marcfinkler photoactivationofcellfreeexpressedarchaerhodopsin3inamodelcellmembrane AT ralfseemann photoactivationofcellfreeexpressedarchaerhodopsin3inamodelcellmembrane AT albrechtott photoactivationofcellfreeexpressedarchaerhodopsin3inamodelcellmembrane AT jeanbaptistefleury photoactivationofcellfreeexpressedarchaerhodopsin3inamodelcellmembrane
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Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane

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