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Applying complementary structural techniques to elucidate structure-function relationships of the bacterial Na+-hydantoin transporter Mhp1

Polyakova, Anna (2015) Applying complementary structural techniques to elucidate structure-function relationships of the bacterial Na+-hydantoin transporter Mhp1. PhD thesis, University of Leeds.

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Abstract

Secondary-active trans-membrane transporters couple the ‘downhill’ movement of ions with the ‘uphill’ movement of essential substrates, such as neurotransmitters or metabolites, across the cell membrane. Disruption of these vital processes is linked to some severe diseases in humans, for example Parkinson’s disease. The aim of this project is to investigate the structural basis of the transport mechanism of secondary-active transporters, using the bacterial sodium-hydantoin transporter Mhp1 as a model system, with a combination of X-ray crystallography and small-angle X-ray scattering (SAXS). A wide variety of strategies have been applied and optimised in the crystallisation, harvesting and cryo-protection steps to improve the data obtained from Mhp1 crystals. These include modifications to the crystal drop morphology to improve crystal size and quality, as well as on-line crystal dehydration and contact-less harvesting by photoablation to minimise damage during cryo-protection and harvesting. This has enabled the determination of several novel crystal structures of Mhp1 mutants in complex with various ligands. SAXS experiments have been used to obtain information about the overall shape and detergent organisation around the purified protein, in order to gain insight into crystallisation propensity and detergent packing in membrane protein crystals. The challenges imposed by the presence of the detergent molecules have been overcome by combining SAXS with sizeexclusion chromatography. The results obtained in this Thesis suggest that the altered ligand binding in the Mhp1 mutants studied in this work is a result of an altered protein conformational landscape, rather than loss of specific protein-ligand interactions. They have also shown that conformational selection during crystallisation can stabilise ligand binding states that are not strongly favoured in solution. Whilst this provides useful detail on ligand binding, it also highlights that a combination of biochemical, biophysical and structural data are required to understand how mutations perturb membrane protein function.

Item Type: Thesis (PhD)
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds)
The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute for Molecular and Cellular Biology (Leeds)
Identification Number/EthosID: uk.bl.ethos.677303
Depositing User: Anna Polyakova
Date Deposited: 11 Jan 2016 10:49
Last Modified: 25 Jul 2018 09:51
URI: http://etheses.whiterose.ac.uk/id/eprint/11607

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