Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.788747
Title: Investigating the metamorphic properties of annexin A5 during membrane integration
Author: Stewart, David John Ossian
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2019
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
Abstract:
The annexin superfamily is comprised of a range of Ca2+ dependent phospholipid binding proteins. Each of these proteins are metamorphic, which means that they possesses the capacity to dynamically and reversibly refold themselves into two or more stable conformations. Annexin A5 (ANXA5) has been linked with a variety of cellular functions including: endocytosis, Ca2+ transfer, anticoagulation, membrane repair and exocytosis. Although this protein has been characterised as having pH-dependent and Ca2+-dependent phosphatidylserine (PS) binding the full extent of these mechanisms are not understood. In this study the changes in ANXA5 conformation as it transitions through its binding states was investigated. Recombinant human ANXA5 was produced in E. coli and subjected to analysis by circular dichroism (CD) and Förster resonance energy transfer (FRET). This revealed that the Ca2+ driven metamorphic refolding of ANXA5 involves minor adjustments of the protein to facilitate trimerisation upon PS binding. However, these experiments also revealed that ANXA5 at acidic conditions undergoes a significant change in conformation upon exposure to liposomes. When under these conditions ANXA5 was demonstrated to bind both PS-containing and PS-free liposomes independent of Ca2+. This indicates that ANXA5 membrane association at acidic conditions relies upon hydrophobic interactions. This variance between the Ca2+- and pH-dependent binding mechanisms is suggestive of alternate cellular localisation and function. Furthermore, this study also examined the mechanisms behind the Ca2+ channel activity of ANXA5. In particular, the necessity for ANXA5 trimerisation. This investigation demonstrated that Ca2+ flux could be readily induced, with a delayed onset, across PS-containing liposomes in the presence of nanomolar concentrations of ANXA5, but was lost when trimer formation was disrupted. Furthermore, this investigation also observed the formation of a transmembrane ANXA5 complex by changes in the rate of FRET across a membrane. Similar to the ANXA5-mediated Ca2+ flux this structure was lost upon the prevention of ANXA5 trimerisation, which suggests that the ANXA5 trimer is able to sufficiently alter the structure of the membrane to allow for the formation of a Ca2+ channel.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.788747  DOI:
Keywords: Q Science (General) ; QR Microbiology
Share: