01whole.pdf (15.82 MB)
Download file

MdfA - a model multidrug efflux pump for single-molecule investigations of transport

Download (15.82 MB)
thesis
posted on 29.03.2022, 00:55 by Stephanie Sabina Nagy
MdfA is an Escherichia coli multidrug efflux pump that has a broad - substrate profile that encompasses cationic, neutral and zwitterionic antimicrobials. MdfA is proposed to operate by an alternating access mechanism, a major substrate translocation mechanism thought to occur throughout a diverse range of transport proteins. However, direct visual evidence for this transport mechanism has not been obtained for any protein. The aim of this research is to explore MdfA as a model for conducting single - molecule Förster resonance energy transfer (sm - FRET) experiments to allow for direct visualisation of the alternating access mechanism, by direct observation of fluorescent substrate transport in parallel with protein domain movement. A prerequisite for this work is to identify a highly fluorescent substrate. This thesis aimed to determine whether MdfA and a Cys - variant of MdfA designed for FRET studies can recognise and transport the highly fluorescent monovalent cation rhodamine 6G (R6G) using fluorescence binding and transport assays. The results demonstrate that R6G is a substrate of MdfA, and that the Cys - variant of MdfA retains sufficient activity as a model for future sm - FRET studies. Moreover, this thesis reports on the first direct visualisation of fluorescence transport mediated by a multidrug efflu x pump at the single - cell level in real - time.

History

Table of Contents

Chapter 1. Introduction -- Chapter 2. Materials and methods -- Chapter 3. Characterisation of MdfA mediated efflux of R6G -- Chapter 4. Single - cell imaging of direct R6G efflux by MdfA_CL_V44C/V307C in E. coli -- Chapter 5. Conclusions and future directions.

Notes

Bibliography: pages i-vii Theoretical thesis.

Awarding Institution

Macquarie University

Degree Type

Thesis MRes

Degree

MRes, Macquarie University, Faculty of Science and Engineering, Department of Molecular Sciences

Department, Centre or School

Department of Molecular Sciences

Year of Award

2017

Principal Supervisor

Ian Paulsen

Rights

Copyright Stephanie Sabina Nagy 2017. Copyright disclaimer: http://mq.edu.au/library/copyright

Language

English

Extent

1 online resource (VI, 50, vii pages)

Former Identifiers

mq:71204 http://hdl.handle.net/1959.14/1271926