Droplet-on-hydrogel bilayer based assay for functional study of membrane proteins
<p>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 pro...
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| Format: | Thesis |
| Language: | English |
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2022
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| _version_ | 1797110924488736768 |
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| author | Cocul’ová, Z |
| author2 | Berry, R |
| author_facet | Berry, R Cocul’ová, Z |
| author_sort | Cocul’ová, Z |
| collection | OXFORD |
| description | <p>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.</p>
<p>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.</p>
<p>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.</p> |
| first_indexed | 2024-03-07T08:03:08Z |
| format | Thesis |
| id | oxford-uuid:19367800-3e89-473d-97db-cf8731b4ac76 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T08:03:08Z |
| publishDate | 2022 |
| record_format | dspace |
| spelling | oxford-uuid:19367800-3e89-473d-97db-cf8731b4ac762023-10-12T12:36:17ZDroplet-on-hydrogel bilayer based assay for functional study of membrane proteinsThesishttp://purl.org/coar/resource_type/c_db06uuid:19367800-3e89-473d-97db-cf8731b4ac76BiophysicsEnglishHyrax Deposit2022Cocul’ová, ZBerry, R<p>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.</p> <p>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.</p> <p>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.</p> |
| spellingShingle | Biophysics Cocul’ová, Z Droplet-on-hydrogel bilayer based assay for functional study of membrane proteins |
| title | Droplet-on-hydrogel bilayer based assay for functional study of membrane proteins |
| title_full | Droplet-on-hydrogel bilayer based assay for functional study of membrane proteins |
| title_fullStr | Droplet-on-hydrogel bilayer based assay for functional study of membrane proteins |
| title_full_unstemmed | Droplet-on-hydrogel bilayer based assay for functional study of membrane proteins |
| title_short | Droplet-on-hydrogel bilayer based assay for functional study of membrane proteins |
| title_sort | droplet on hydrogel bilayer based assay for functional study of membrane proteins |
| topic | Biophysics |
| work_keys_str_mv | AT coculovaz dropletonhydrogelbilayerbasedassayforfunctionalstudyofmembraneproteins |