Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.595051
Title: Mass spectrometric analysis of pilus assembly, amyloid fibril formation, and membrane proteins in their native state
Author: Leney, Aneika Corrine
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2012
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Abstract:
Structural analysis of proteins and their complexes is crucial to understanding protein function. This thesis demonstrates the application of mass spectrometry (MS) to the study of pilus assembly on Gram-negative bacteria and amyloid formation in dialysisrelated amyloidosis. In addition, it also tackles the challenging area of membrane protein analysis by MS. Pili are hair-like appendages located on the outer membrane of bacteria that are involved in the transmission of infection. A periplasmic chaperone and an outer membrane usher protein coordinate pilin subunit assembly. Electrospray ionisation (ESI)-MS was used to elucidate the mechanism of subunit assembly. Experiments revealed the specific amino acids on the N-terminal extension of the pilus subunits that are important in catalysing the subunit assembly process. Further MS/MS analysis indicated differences in the stability of the chaperone-subunit-usher ternary complexes formed, providing new insights into the role of the usher in orchestrating pilus biogenesis. Ion mobility spectrometry (IMS)-MS was next used to characterise oligomeric intermediates formed during beta-2 microglobulin (β2m) assembly into amyloid fibrils. Analysis of the oligomers formed by a range of β2m point mutants that affect the kinetics of amyloid fibril formation highlighted the complexity of this fibril-forming process. Further detailed characterisation of the β2m mutant H51A revealed subtle differences in the subunit exchange dynamics of the oligomers involved in fibril formation. Finally, this thesis shows a novel method for solubilising membrane proteins for MS analysis. Amphipathic polymers, termed amphipols, were used to fold and enhance the stability of two bacterial outer membrane proteins, OmpT and PagP. The utility of amphipols to study the structural and functional properties of membrane proteins by ESI-IMS-MS was then developed. Together these data show the power of ESI-IMS-MS in separating conformationally-distinct populations of amphipathic polymers from the amphipol-membrane complex whilst maintaining a ‘native-like’ membrane protein structure in the gas phase.
Supervisor: Ashcroft, A. ; Radford, S. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.595051  DOI: Not available
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