A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels

Abstract Bax is a Bcl-2 protein crucial for apoptosis initiation and execution, whose active conformation is only partially understood. Dipolar EPR spectroscopy has proven to be a valuable tool to determine coarse-grained models of membrane-embedded Bcl-2 proteins. Here we show how the combination o...

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Autores principales:	Markus Teucher, Hui Zhang, Verian Bader, Konstanze F. Winklhofer, Ana J. García-Sáez, Andrzej Rajca, Stephanie Bleicken, Enrica Bordignon
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2019
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/daf6f5f4ac01421a922c80047164d90d
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spelling	oai:doaj.org-article:daf6f5f4ac01421a922c80047164d90d2021-12-02T16:07:52ZA new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels10.1038/s41598-019-49370-z2045-2322https://doaj.org/article/daf6f5f4ac01421a922c80047164d90d2019-09-01T00:00:00Zhttps://doi.org/10.1038/s41598-019-49370-zhttps://doaj.org/toc/2045-2322Abstract Bax is a Bcl-2 protein crucial for apoptosis initiation and execution, whose active conformation is only partially understood. Dipolar EPR spectroscopy has proven to be a valuable tool to determine coarse-grained models of membrane-embedded Bcl-2 proteins. Here we show how the combination of spectroscopically distinguishable nitroxide and gadolinium spin labels and Double Electron-Electron Resonance can help to gain new insights into the quaternary structure of active, membrane-embedded Bax oligomers. We show that attaching labels bulkier than the conventional MTSL may affect Bax fold and activity, depending on the protein/label combination. However, we identified a suitable pair of spectroscopically distinguishable labels, which allows to study complex distance networks in the oligomers that could not be disentangled before. Additionally, we compared the stability of the different spin-labeled protein variants in E. coli and HeLa cell extracts. We found that the gem-diethyl nitroxide-labeled Bax variants were reasonably stable in HeLa cell extracts. However, when transferred into human cells, Bax was found to be mislocalized, thus preventing its characterization in a physiological environment. The successful use of spectroscopically distinguishable labels on membrane-embedded Bax-oligomers opens an exciting new path towards structure determination of membrane-embedded homo- or hetero-oligomeric Bcl-2 proteins via EPR.Markus TeucherHui ZhangVerian BaderKonstanze F. WinklhoferAna J. García-SáezAndrzej RajcaStephanie BleickenEnrica BordignonNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 9, Iss 1, Pp 1-15 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Markus Teucher Hui Zhang Verian Bader Konstanze F. Winklhofer Ana J. García-Sáez Andrzej Rajca Stephanie Bleicken Enrica Bordignon A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels
description	Abstract Bax is a Bcl-2 protein crucial for apoptosis initiation and execution, whose active conformation is only partially understood. Dipolar EPR spectroscopy has proven to be a valuable tool to determine coarse-grained models of membrane-embedded Bcl-2 proteins. Here we show how the combination of spectroscopically distinguishable nitroxide and gadolinium spin labels and Double Electron-Electron Resonance can help to gain new insights into the quaternary structure of active, membrane-embedded Bax oligomers. We show that attaching labels bulkier than the conventional MTSL may affect Bax fold and activity, depending on the protein/label combination. However, we identified a suitable pair of spectroscopically distinguishable labels, which allows to study complex distance networks in the oligomers that could not be disentangled before. Additionally, we compared the stability of the different spin-labeled protein variants in E. coli and HeLa cell extracts. We found that the gem-diethyl nitroxide-labeled Bax variants were reasonably stable in HeLa cell extracts. However, when transferred into human cells, Bax was found to be mislocalized, thus preventing its characterization in a physiological environment. The successful use of spectroscopically distinguishable labels on membrane-embedded Bax-oligomers opens an exciting new path towards structure determination of membrane-embedded homo- or hetero-oligomeric Bcl-2 proteins via EPR.
format	article
author	Markus Teucher Hui Zhang Verian Bader Konstanze F. Winklhofer Ana J. García-Sáez Andrzej Rajca Stephanie Bleicken Enrica Bordignon
author_facet	Markus Teucher Hui Zhang Verian Bader Konstanze F. Winklhofer Ana J. García-Sáez Andrzej Rajca Stephanie Bleicken Enrica Bordignon
author_sort	Markus Teucher
title	A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels
title_short	A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels
title_full	A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels
title_fullStr	A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels
title_full_unstemmed	A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels
title_sort	new perspective on membrane-embedded bax oligomers using deer and bioresistant orthogonal spin labels
publisher	Nature Portfolio
publishDate	2019
url	https://doaj.org/article/daf6f5f4ac01421a922c80047164d90d
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A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels

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