Mazurenko, I orcid.org/0000-0003-2563-3130, Hatzakis, NS and Jeuken, LJC orcid.org/0000-0001-7810-3964 (2019) Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy. Journal of Visualized Experiments, 144. e58896. ISSN 1940-087X
Abstract
Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2019 Journal of Visualized Experiments. Reproduced in accordance with the publisher's self-archiving policy. |
Keywords: | Biochemistry; Issue 144; cytochrome bo₃; single enzyme; proteoliposomes; pH-sensitive dye; proton translocation; bioelectrochemistry |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) |
Funding Information: | Funder Grant number BBSRC BB/P005454/1 |
Depositing User: | Symplectic Publications |
Date Deposited: | 28 Sep 2018 11:53 |
Last Modified: | 21 Feb 2021 01:38 |
Status: | Published |
Publisher: | Journal of Visualized Experiments |
Identification Number: | 10.3791/58896 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:136317 |