| Περίληψη: | <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>
|
|---|