Thesis
Droplet-on-hydrogel bilayer based assay for functional study of membrane proteins
- Abstract:
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Functional study of membrane proteins, especially when a transmembrane potential is required for protein function, is notoriously difficult. In this thesis, I introduce an experimental assay that adapts the droplet-on-hydrogel technique for a high-resolution functional study of membrane proteins with independent control of the electrical and chemical transmembrane potential.
This experimental assay is the next step toward studying a membrane protein of interest embedded into an energised lipid bilayer. The bilayer is formed between a 200 nl water droplet and a supporting layer of hydrogel, granting a stable, functional, and accessible lipid bilayer. Delivery of proteins, labels, substrates, or ions to the droplet above the bilayer is achieved through a low-cost custom-built electronically controlled perfusion system. Experiments presented in this work show a stable perfusion with 26.68 ± 2.5 nl steps.
The main motivation for creating this experimental assay is a desire to observe the ATP synthase F1Fo in conditions mimicking the protein’s natural environment. This complex of two molecular motors - membrane-embedded Fo and water-soluble F1 - catalyses the synthesis of ATP molecules, the basic energy units of life. Using site-directed mutagenesis, I modified TA2.A1 F1Fo in two places, to be able to label the protein with a gold nanorod and anchor it to the agarose-streptavidin gel for a high-resolution functional study experiment. The assay may allow us to study the activation potential of the protein at different conditions, as well as to observe high-resolution single-molecule rotation steps of F1Fo at the time scale of a few microseconds.
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(Preview, Dissemination version, pdf, 60.0MB, Terms of use)
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Authors
Contributors
- Institution:
- University of Oxford
- Division:
- MPLS
- Department:
- Physics
- Sub department:
- Condensed Matter Physics
- Research group:
- Molecular motors group, Kavli institute
- Oxford college:
- St Catherine's College
- Role:
- Supervisor
- ORCID:
- 0000-0002-0331-473X
- Funding agency for:
- Cocul’ová, Z
- Grant:
- EP/L016044/1
- Funding agency for:
- Cocul’ová, Z
- Grant:
- EP/L016044/1
- Funder identifier:
- http://dx.doi.org/10.13039/100007013
- Funding agency for:
- Cocul’ová, Z
- DOI:
- Type of award:
- DPhil
- Level of award:
- Doctoral
- Awarding institution:
- University of Oxford
- Language:
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English
- Keywords:
- Subjects:
- Pubs id:
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2042888
- Local pid:
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pubs:2042888
- Deposit date:
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2023-10-09
- ARK identifier:
Terms of use
- Copyright holder:
- Cocul’ová, Z
- Copyright date:
- 2022
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