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Title: Combining single-molecule fluorescence and single-channel recording in droplet interface bilayers
Author: Weatherill, Eve Elizabeth
ISNI:       0000 0004 6352 9905
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2015
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Single-channel recordings (SCR) and single-molecule Fluorescence (SMF) are invaluable tools for studying membrane proteins. Combining these two techniques would allow for structural dynamics to be correlated with function at the single-molecule level. However, the different requirements of SCR and SMF measurements present a long-standing challenge. Droplet interface bilayers (DIBs) are a model membrane system in which parallelisation of such measurements is possible using optical single-channel recording to resolve the conductance of multiple proteins simultaneously. In this project, two approaches were evaluated for obtaining simultaneous measurements in DIBs: the first sought to report on structural changes associated with a gating bacterial mechanosensitive channel, MscS; the second sought to resolve kinetics of the spontaneous β-barrel folding mechanism. For both approaches, functional, labelled protiens were produced and characterised using ensemble measurements to confirm that the state-transition of interest was accompanied by a change in fluorescent signal. Characterisation of MscS in DIBs revealed that control of lateral tension (a requirement to stimulate mechanosensitive channel gating) posed a major experimental challenge, rendering further pursuit of this candidate impossible. However in the case of β-barrel folding, the single-molecule fluorescence alone revealed a rare but fast transition from a membrane-associated unfolded to a folded state. While further work is needed to fully exploit simultaneous measurements, significant progress towards this goal is presented in this work, in particular the development of reagents, experimental procedures and analytical tools.
Supervisor: Wallace, Mark Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available