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Scientific article
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English

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

Published inScientific reports, vol. 9, no. 1
Publication date2019-09-10
First online date2019-09-10
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.

eng
Affiliation Not a UNIGE publication
Funding
  • Deutsche Forschungsgemeinschaft - [BO 3000/5-1]
  • NIGMS NIH HHS - [R01 GM124310]
  • National Institute for Health Research (NIHR) - [NIGMS R01GM124310-01]
Citation (ISO format)
TEUCHER, Markus et al. A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels. In: Scientific reports, 2019, vol. 9, n° 1. doi: 10.1038/s41598-019-49370-z
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Article (Published version)
Identifiers
ISSN of the journal2045-2322
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