ArticleThe Journal of membrane biology2022
A Limitation of Using Dithionite Quenching to Determine the Topology of Membrane-inserted Proteins.
Article in The Journal of membrane biology, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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4 citing papers in PubMed.
- DDHD2 possesses both lipase and transacylase capacities that remodel triglyceride acyl chains.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- DDHD2 possesses both lipase and transacylase capacities that remodel triglyceride acyl chains.bioRxiv : the preprint server for biology · 2025Article
- Allosteric coupling of substrate binding and proton translocation in MmpL3 transporter frommBio · 2024Article
- Spectroscopic evidence of tetanus toxin translocation domain bilayer-induced refolding and insertion.Biophysical journal · 2021Article
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1 author.
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Abstract
Determining the topology of membrane-inserted proteins and peptides often relies upon indirect fluorescent measurements. One such technique uses NBD, an environmentally sensitive fluorophore that can be covalently linked to proteins. Relative to a hydrophilic environment, NBD in a hydrophobic environment shows an increase in emission intensity and a shift to shorter wavelengths. To gain further insight, NBD fluorescence can be chemically quenched using dithionite. As dithionite is an anion, it is only expected to penetrate the outer leaflet interfacial region and should be excluded from the hydrocarbon core, the inner leaflet, and the lumen of LUV. This assumption holds at neutral pH, where a large number of NBD/dithionite experiments are carried out. Here, we report control experiments in which LUV were directly labeled with NBD-PE to assess dithionite quenching in acidic conditions. Results showed that at acidic pH, dithionite moved more freely across the bilayer to quench the inner leaflet. For the buffer conditions used, dithionite exhibited a sharp change in behavior between pH 5.5 and 6.0. Therefore, in acidic conditions, dithionite could not differentiate in which leaflet the NBD resided.
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Registered trials
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