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Using Fluorescence Quenching Titration to Determine the Orientation of a Model Transmembrane Protein in Mimic Membranes
After synthesis of transmembrane proteins (TMPs), they are transferred and inserted into plasma membranes to play biological functions. Crucially, orientation of TMPs in membranes determines whether they have biological activities. In cellular environments, a number of cofactors, such as translocon,...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384929/ https://www.ncbi.nlm.nih.gov/pubmed/30678051 http://dx.doi.org/10.3390/ma12030349 |
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author | Huang, Haihong Ge, Baosheng Zhang, Shuai Li, Jiqiang Sun, Chenghao Yue, Tongtao Huang, Fang |
author_facet | Huang, Haihong Ge, Baosheng Zhang, Shuai Li, Jiqiang Sun, Chenghao Yue, Tongtao Huang, Fang |
author_sort | Huang, Haihong |
collection | PubMed |
description | After synthesis of transmembrane proteins (TMPs), they are transferred and inserted into plasma membranes to play biological functions. Crucially, orientation of TMPs in membranes determines whether they have biological activities. In cellular environments, a number of cofactors, such as translocon, can assist TMPs to be inserted into membranes in defined orientations. During in vitro reconstitution of TMPs with mimic membranes, both insertion and orientation of TMPs are primarily determined by interactions with the membrane. Yet the knowledge is limited, hindering the in vitro applications of TMPs. Here, we take Bacteriorhodopsin (bR) as a model TMP, using fluorescence quenching titration experiment to identify orientation of bR in mimic membranes, examining effects of a number of factors, including lipid composition, pH value, ionic strength and membrane curvature. The most effective determinant is the lipid type, which modulates insertion and orientation of bR in membranes by changing the membrane surface charge and the membrane fluidity. Both the pH value and the ionic strength play secondary roles by tuning the nature of the electrostatic interaction. The membrane curvature was found to have a minor effect on orientation of bR in membranes. By comparing orientations of bR in folded and unfolded states, no obvious change was observed, informing that nascent proteins could be inserted into membranes in defined orientations before folding into the native state inside the membrane. |
format | Online Article Text |
id | pubmed-6384929 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-63849292019-02-23 Using Fluorescence Quenching Titration to Determine the Orientation of a Model Transmembrane Protein in Mimic Membranes Huang, Haihong Ge, Baosheng Zhang, Shuai Li, Jiqiang Sun, Chenghao Yue, Tongtao Huang, Fang Materials (Basel) Article After synthesis of transmembrane proteins (TMPs), they are transferred and inserted into plasma membranes to play biological functions. Crucially, orientation of TMPs in membranes determines whether they have biological activities. In cellular environments, a number of cofactors, such as translocon, can assist TMPs to be inserted into membranes in defined orientations. During in vitro reconstitution of TMPs with mimic membranes, both insertion and orientation of TMPs are primarily determined by interactions with the membrane. Yet the knowledge is limited, hindering the in vitro applications of TMPs. Here, we take Bacteriorhodopsin (bR) as a model TMP, using fluorescence quenching titration experiment to identify orientation of bR in mimic membranes, examining effects of a number of factors, including lipid composition, pH value, ionic strength and membrane curvature. The most effective determinant is the lipid type, which modulates insertion and orientation of bR in membranes by changing the membrane surface charge and the membrane fluidity. Both the pH value and the ionic strength play secondary roles by tuning the nature of the electrostatic interaction. The membrane curvature was found to have a minor effect on orientation of bR in membranes. By comparing orientations of bR in folded and unfolded states, no obvious change was observed, informing that nascent proteins could be inserted into membranes in defined orientations before folding into the native state inside the membrane. MDPI 2019-01-23 /pmc/articles/PMC6384929/ /pubmed/30678051 http://dx.doi.org/10.3390/ma12030349 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Huang, Haihong Ge, Baosheng Zhang, Shuai Li, Jiqiang Sun, Chenghao Yue, Tongtao Huang, Fang Using Fluorescence Quenching Titration to Determine the Orientation of a Model Transmembrane Protein in Mimic Membranes |
title | Using Fluorescence Quenching Titration to Determine the Orientation of a Model Transmembrane Protein in Mimic Membranes |
title_full | Using Fluorescence Quenching Titration to Determine the Orientation of a Model Transmembrane Protein in Mimic Membranes |
title_fullStr | Using Fluorescence Quenching Titration to Determine the Orientation of a Model Transmembrane Protein in Mimic Membranes |
title_full_unstemmed | Using Fluorescence Quenching Titration to Determine the Orientation of a Model Transmembrane Protein in Mimic Membranes |
title_short | Using Fluorescence Quenching Titration to Determine the Orientation of a Model Transmembrane Protein in Mimic Membranes |
title_sort | using fluorescence quenching titration to determine the orientation of a model transmembrane protein in mimic membranes |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384929/ https://www.ncbi.nlm.nih.gov/pubmed/30678051 http://dx.doi.org/10.3390/ma12030349 |
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