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

Häusler, Elisabeth; Fredriksson, Kai; Goba, I.; Peters, Carsten; Raltchev, Kolio; Sperl, Laura E.; Steiner, Andrea; Weinkauf, Sevil; Hagn, Franz

doi:10.1016/j.bbamem.2020.183190

Cited by 12 publications

(13 citation statements)

References 33 publications

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“…Therefore, we investigated whether the TMH alone has a tendency for oligomerization, which would suggest a potential active role in the oligomerization process of full‐length Bak in a lipid environment—a key step in activation (Bleicken et al , 2017; Cosentino & García‐Sáez, 2017; Moldoveanu & Czabotar, 2020). Oligomerization of the TMH in lipid nanodiscs was probed by a recently reported fusion protein assay (Häusler et al , 2020). Briefly, we assembled nanodiscs using different ratios of GB1‐fused Bak‐TMH and membrane scaffold protein (MSP) of different sizes.…”

Section: Resultsmentioning

confidence: 99%

“…However, a high-resolution structure was not yet available. For high-level production in E. coli, the Bak-TMH was produced as an N-terminal fusion protein with the expression helper and solubility tag GB1 (Zhou & Wagner, 2010), similar to what has previously been reported for the BclxL-TMH and other systems (Raltchev et al, 2018;Steiner et al, 2020). Using multidimensional NMR experiments, we obtained backbone resonance assignments of 89% of all non-proline residues in the Bak-TMH protein construct both in dodecylphosphocholine (DPC) micelles (Appendix Fig S1A) and in DMPC/DMPG (3:1) MSP1D1DH5 nanodiscs (Hagn et al, 2013(Hagn et al, , 2018Klo ¨pfer & Hagn, 2019) (Fig 2B).…”

Section: Structural Analysis Of Full-length Bak In Lipid Nanodiscs Using a Segmental Approachmentioning

confidence: 99%

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High‐resolution analysis of the conformational transition of pro‐apoptotic Bak at the lipid membrane

et al. 2021

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Permeabilization of the outer mitochondrial membrane by pore‐forming Bcl2 proteins is a crucial step for the induction of apoptosis. Despite a large set of data suggesting global conformational changes within pro‐apoptotic Bak during pore formation, high‐resolution structural details in a membrane environment remain sparse. Here, we used NMR and HDX‐MS (Hydrogen deuterium exchange mass spectrometry) in lipid nanodiscs to gain important high‐resolution structural insights into the conformational changes of Bak at the membrane that are dependent on a direct activation by BH3‐only proteins. Furthermore, we determined the first high‐resolution structure of the Bak transmembrane helix. Upon activation, α‐helix 1 in the soluble domain of Bak dissociates from the protein and adopts an unfolded and dynamic potentially membrane‐bound state. In line with this finding, comparative protein folding experiments with Bak and anti‐apoptotic BclxL suggest that α‐helix 1 in Bak is a metastable structural element contributing to its pro‐apoptotic features. Consequently, mutagenesis experiments aimed at stabilizing α‐helix 1 yielded Bak variants with delayed pore‐forming activity. These insights will contribute to a better mechanistic understanding of Bak‐mediated membrane permeabilization.

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Section: Resultsmentioning

confidence: 99%

Section: Structural Analysis Of Full-length Bak In Lipid Nanodiscs Using a Segmental Approachmentioning

confidence: 99%

High‐resolution analysis of the conformational transition of pro‐apoptotic Bak at the lipid membrane

et al. 2021

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“…Concentration determination of protein and fluorophores was performed using the Lambert–Beer law and published extinction coefficients (see Methods section and Table S1). Unfortunately, the contribution of MSP 1 D 1 to the 280 nm absorbance could not be deconvoluted, because methods to perform such a deconvolution only became available quite recently [58]. The labelled fraction (~ 11–12 mL) expected to contain the two OpuA components in equimolar ratio was selected for subsequent testing in biochemical and biophysical assays (Fig.…”

Section: Resultsmentioning

confidence: 99%

Single‐molecule studies of conformational states and dynamics in the ABC importer OpuA

et al. 2021

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The current model of active transport via ABC importers is mostly based on structural, biochemical and genetic data. We here establish single-molecule F€ orster resonance energy transfer (smFRET) assays to monitor the conformational states and heterogeneity of the osmoregulatory type I ABC importer OpuA from Lactococcus lactis. We present data probing both intradomain distances that elucidate conformational changes within the substrate-binding domain (SBD) OpuAC, and interdomain distances between SBDs or transmembrane domains. Using this methodology, we studied ligand-binding mechanisms, as well as ATP and glycine betaine dependences of conformational changes. Our work expands the scope of smFRET investigations towards a class of so far unstudied ABC importers, and paves the way for a full understanding of their transport cycle in the future.

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“…Concentration determination of protein and fluorophores was performed using the Lambert-Beer law and published extinction coefficients (see methods section and Table S1). Unfortunately, the contribution of MSP 1 D 1 to the 280 nm absorbance could not be deconvoluted, because methods to perform such a deconvolution only became available quite recently [53]. The labelled fraction (~11-12 ml) expected to contain the two OpuA components in equimolar ratio, was selected for subsequent testing in biochemical and biophysical assays (Figure 6c, d).…”

Section: Resultsmentioning

confidence: 99%

Single-molecule studies of conformational states and dynamics in the ABC importer OpuA

Tassis

Vietrov

Koning

et al. 2020

Preprint

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The current model of active transport via ABC importers is mostly based on structural, biochemical and genetic data. We here establish single-molecule Förster-resonance energy transfer (smFRET) assays to monitor the conformational states and heterogeneity of the type-I ABC importer OpuA from Lactococcus lactis. Our studies include intradomain assays that elucidate conformational changes within the substrate-binding domain (SBD) OpuAC and interdomain assays between SBDs or transmembrane domains. Using the methodology, we studied ligand-binding mechanisms as well as ATP and glycine betaine dependences of conformational changes. Our study expands the scope of smFRET investigations towards a class of so far unstudied ABC importers, and paves the way for a full understanding of their transport cycle in the future.

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

Cited by 12 publications

References 33 publications

High‐resolution analysis of the conformational transition of pro‐apoptotic Bak at the lipid membrane

High‐resolution analysis of the conformational transition of pro‐apoptotic Bak at the lipid membrane

Single‐molecule studies of conformational states and dynamics in the ABC importer OpuA

Single-molecule studies of conformational states and dynamics in the ABC importer OpuA

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