Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.701731
Title: Characterising membrane protein-surfactant complexes using mass spectrometry methods
Author: Watkinson, Thomas Geoffrey
ISNI:       0000 0004 5993 1619
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2016
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Thesis embargoed until 01 Feb 2022
Access from Institution:
Abstract:
Membrane proteins (MPs) facilitate a large number of essential physiological functions and are the targets for greater than 50 % of licensed pharmaceuticals. MPs, however, are difficult to study in vitro as a result of their inherent properties, such as their insolubility in aqueous solution without the presence of a suitable surfactant to maintain the native-like state. For this reason, high resolution structural data of MPs are lacking relative to their water soluble counterparts. This has inspired a need for improvement in methods of solubilisation and analysis of MPs. Amphipols (APols), are a class of novel surfactants for solubilisation of MPs in a native-like state. APols provide a means for solubilisation of MPs with very high kinetic stability using materials and an approach that can be applied, possibly indiscriminately, to any MP. Mass spectrometry (MS) is a multi-faceted technique used in structural biology. Native-MS, which emerged following the development of “soft” ionisation methods such as electrospray ionisation (ESI), has been used extensively to deliver proteins in to the gas phase for analysis of protein ion m/z, whilst maintaining their native state and non-covalent interactions. The combination of native-MS and ion mobility spectrometry (IMS) allows simultaneous measurement of the collision cross section (CCS) and m/z of protein ions. This can be used to study the conformational state of proteins and protein complexes. Native MS is being used more frequently for the study of MPs. Collisional activation of MP:surfactants allows for liberation of MPs and MP complexes, and determination of CCS values using IMS-MS to evaluate the conformational state and investigate the topology and stability of MP complexes. In addition to native-MS, structural proteomics is a growing field for MS-based study of MP structural biology. In work shown here, Fast Photochemical Oxidation of Proteins (FPOP) and Hydrogen-Deuterium Exchange (HDX) coupled to liquid chromatography (LC)-MS allow for observation of solvent accessible regions of the bacterial outer membrane protein (OMP) OmpT in detergent micelles and APols. Work presented here in three experimental chapters shows APols to improve on detergent micelles in the ability to observe the most native-like forms of MPs in the gas phase, finding that the MPs OmpT, Mhp1 and GalP are only observed in the most lowly charged and, hence, more native-like states by nESI-IMS-MS when released from A8-35 and not from DDM micelles. Certain properties of APols (such as molecular weight and charge density) further benefit or hinder delivery of OMPs in to the gas phase. OmpT, tOmpA and PagP are less readily observed by nESI-IMS-MS when solubilised in the largest and most-negatively charged APols. Following from this, FPOP-LC-MS data suggest that this protective effect of APols is mediated through extra contacts with the non-transmembrane regions of MPs, preventing unfolding and protecting MPs from excess charging during ionisation (as evidenced by a decreased degree of oxidation of reactive residue side chains near the boundaries of the transmembrane domain of OmpT). This same degree of protection is not observed using HDX-MS. In fact, data suggests that NAPol (a non-ionic APol with significantly different chemical composition than the previously described A8-35-like APols) promotes greater flexibility of OmpT in the extramembrane regions relative to solubilisation in DDM micelles, despite it being thought to provide the most native-like environment for MPs. Altogether, this thesis shows the power of A8-35-like APols as a solubilisation tool for studying MP structure and function using MS-based techniques. Data shown here also informs on the nature of APol interactions with MPs and how this may manifest in the improved stability of MPs in solution.
Supervisor: Ashcroft, Alison ; Radford, Sheena Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.701731  DOI: Not available
Share: