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Häusler, E. ; Fredriksson, K. ; Goba, I. ; Peters, C.* ; Raltchev, K. ; Sperl, L.E. ; Steiner, A. ; Weinkauf, S.* ; Hagn, F.

Quantifying the insertion of membrane proteins into lipid bilayer nanodiscs using a fusion protein strategy.

Biochim. Biophys. Acta-Biomembr. 1862:183190 (2020)
Publ. Version/Full Text Postprint DOI PMC
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A membrane protein's oligomeric state modulates its functionality in various cellular processes. Since membrane proteins have to be solubilized in an appropriate membrane mimetic, the use of classical biophysical methods to analyze protein oligomers is challenging. We here present a method to determine the number of membrane proteins inserted into lipid nanodiscs. It is based on the ability to selectively quantify the amount of a small and robust fusion protein that can be proteolytically cleaved off from a membrane protein after incorporation into lipid nanodiscs. A detailed knowledge of the number of membrane proteins per nanodisc at defined assembly conditions is essential to estimate the tendency for oligomerization, but also for guiding sample optimization for structural investigations that require the presence of a homogenous oligomeric state. We show that this method can efficiently be used to determine the number of VDAC1 channels in nanodiscs at various assembly conditions, as confirmed by negative stain EM. The presented method is suitable in particular for membrane proteins that cannot be probed easily by other methods such as single span transmembrane helices. This assay can be applied to any membrane protein that can be incorporated into a nanodisc without the requirement for special instrumentation and will thus be widely applicable and complementary to other methods that quantify membrane protein insertion in lipid nanodiscs.
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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
Keywords Bclxl ; Glycophorin ; Nanodisc ; Transmembrane Helices ; Vdac1; Dynamics
ISSN (print) / ISBN 0005-2736
e-ISSN 1879-2642
Journal Biochimica et Biophysica Acta - Biomembranes
Quellenangaben Volume: 1862, Issue: 4, Pages: , Article Number: 183190 Supplement: ,
Publisher Elsevier
Publishing Place Radarweg 29, 1043 Nx Amsterdam, Netherlands
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Institute of Structural Biology (STB)