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Title: A study of protein regulation by mass spectrometry-based methods
Author: Beilsten-Edmands, Victoria
ISNI:       0000 0004 7430 5229
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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Understanding the function and regulation of proteins is critical to medical and physiological research. To achieve this, the modification status, as well as the interactions of proteins need to be defined. Mass spectrometry is often used to catalogue modifications but their effect on interactions is less often reported. In this thesis these two strands are combined to address a series of challenging complexes that underpin cellular processes from stress responses to energy production. Following an introduction to mass spectrometry and its applications to the study of protein interactions (Chapters 1 and 2), three protein assemblies are considered; Hsp70 chaperones, eukaryotic translation initiation factors, and F- and V-type ATP synthases. First, the oligomerisation of Hsp70 is studied (Chapter 3). Dimerisation is shown to be dependent on a highly-conserved phosphorylation site, suggesting a role for this modification in the regulation of protein complex assembly and targeting to antagonistic pathways. A study of eukaryotic translation initiation factor 2B (eIF2B) is presented (Chapter 4), with proteomics revealing clusters of modifications at protein interfaces. Chemical cross-linking is applied, eliminating proposed structural models and supporting a decameric assembly. A cognition-enhancing drug (known as ISRIB) is an inhibitor of the integrated stress response, and was found to stabilise the active decameric eIF2B assembly, rationalising the observed therapeutic effects of ISRIB. Finally, ATP synthases, membrane protein complexes which exhibit multiple forms of regulation, are investigated (Chapter 5). Positional information of small membrane subunits and the effect of post-translational modifications is deduced. The role of an endogenous inhibitor protein is investigated providing insights into its location, oligomeric state, and influence on nucleotide binding. Overall this thesis applies mass spectrometry to three critical protein assemblies and contributes a more complete understanding of their activity by uncovering aspects of their carefully-tuned regulatory mechanisms.
Supervisor: Robinson, Carol V. Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available