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,...
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2019-01-01
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| Online Access: | https://www.mdpi.com/1996-1944/12/3/349 |
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| author | Haihong Huang Baosheng Ge Shuai Zhang Jiqiang Li Chenghao Sun Tongtao Yue Fang Huang |
| author_facet | Haihong Huang Baosheng Ge Shuai Zhang Jiqiang Li Chenghao Sun Tongtao Yue Fang Huang |
| author_sort | Haihong Huang |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-04-13T10:04:07Z |
| format | Article |
| id | doaj.art-5249f0abecd0455d92c2a79fd392a21b |
| institution | Directory Open Access Journal |
| issn | 1996-1944 |
| language | English |
| last_indexed | 2024-04-13T10:04:07Z |
| publishDate | 2019-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Materials |
| spelling | doaj.art-5249f0abecd0455d92c2a79fd392a21b2022-12-22T02:51:10ZengMDPI AGMaterials1996-19442019-01-0112334910.3390/ma12030349ma12030349Using Fluorescence Quenching Titration to Determine the Orientation of a Model Transmembrane Protein in Mimic MembranesHaihong Huang0Baosheng Ge1Shuai Zhang2Jiqiang Li3Chenghao Sun4Tongtao Yue5Fang Huang6State Key Laboratory of Heavy Oil Processing, China University of Petroleum (East China), Qingdao 266580, ChinaCenter for Bioengineering and Biotechnology, College of Chemical Engineering, China University of Petroleum (East China), Qingdao 266580, ChinaCenter for Bioengineering and Biotechnology, College of Chemical Engineering, China University of Petroleum (East China), Qingdao 266580, ChinaCenter for Bioengineering and Biotechnology, College of Chemical Engineering, China University of Petroleum (East China), Qingdao 266580, ChinaCenter for Bioengineering and Biotechnology, College of Chemical Engineering, China University of Petroleum (East China), Qingdao 266580, ChinaState Key Laboratory of Heavy Oil Processing, China University of Petroleum (East China), Qingdao 266580, ChinaState Key Laboratory of Heavy Oil Processing, China University of Petroleum (East China), Qingdao 266580, ChinaAfter 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.https://www.mdpi.com/1996-1944/12/3/349transmembrane proteinmimic membraneinsertionorientationfluorescence quenching titration |
| spellingShingle | Haihong Huang Baosheng Ge Shuai Zhang Jiqiang Li Chenghao Sun Tongtao Yue Fang Huang Using Fluorescence Quenching Titration to Determine the Orientation of a Model Transmembrane Protein in Mimic Membranes Materials transmembrane protein mimic membrane insertion orientation fluorescence quenching titration |
| 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 | transmembrane protein mimic membrane insertion orientation fluorescence quenching titration |
| url | https://www.mdpi.com/1996-1944/12/3/349 |
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