St Andrews Research Repository

St Andrews University Home
View Item 
  •   St Andrews Research Repository
  • Chemistry (School of)
  • Chemistry
  • Chemistry Theses
  • View Item
  •   St Andrews Research Repository
  • Chemistry (School of)
  • Chemistry
  • Chemistry Theses
  • View Item
  •   St Andrews Research Repository
  • Chemistry (School of)
  • Chemistry
  • Chemistry Theses
  • View Item
  • Login
JavaScript is disabled for your browser. Some features of this site may not work without it.

Introducing spin labels into proteins to determine their solution conformation by pulsed EPR methods

Thumbnail
View/Open
EmmaBraniganPhDThesis.pdf (112.0Mb)
Date
2013
Author
Branigan, Emma
Supervisor
Naismith, James
Metadata
Show full item record
Abstract
In bacteria, mechanosensitive ion channels are essential for the cellular response to hypoosomitic shock, relieving the build up of membrane pressure. This thesis focuses on the Mechanosensitive Channel of Small Conductance (MscS) for which three conflicting gating models exist. These models were generated from structural studies of the closed and open conformations of MscS using three different experimental techniques. Pulsed Electron Double Resonance (PELDOR) spectroscopy was applied to MscS in the detergent solubilised state and the membrane-like bilayer. The distances between selectively introduced unpaired electrons in MscS were measured. PELDOR data in the detergent solubilised state were only consistent with the crystal structure depicting an open conformation of MscS, indicating that the transmembrane helices were unperturbed during crystallisation in detergent. MscS was reconstituted into membrane bilayer mimics, bicelles and nanodiscs, and PELDOR data in these environments suggested that both closed and open conformations determined by X-ray crystallography are stabilised in the membrane bilayer. The second part of this thesis involved stabilisation of an enzyme complex of the ubiquitin (Ub) pathway for structural analysis. This pathway is a eukaryotic signalling system involving post-translational modification of target protein amino groups with Ub. The variety of modification provided by Ub and its interplay with other Small Ubiquitin-like Modifier (SUMO) signalling proteins controls an array of cellular responses. The pathway functions to activate the Ub C-terminus, forming highly reactive thioester conjugates between Ub and the active site cysteine of a series of enzymes: E1, E2 and in some cases E3. This thesis explores the use of an isopeptide linkage to stabilise an E2~Ub conjugate in complex with an E3 enzyme. Sample conditions were optimised for the future use of PELDOR spectroscopy to structurally analyse the E2~Ub alone and in complex with E3.
Type
Thesis, PhD Doctor of Philosophy
Collections
  • Chemistry Theses
URI
http://hdl.handle.net/10023/4446

Items in the St Andrews Research Repository are protected by copyright, with all rights reserved, unless otherwise indicated.

Advanced Search

Browse

All of RepositoryCommunities & CollectionsBy Issue DateNamesTitlesSubjectsClassificationTypeFunderThis CollectionBy Issue DateNamesTitlesSubjectsClassificationTypeFunder

My Account

Login

Open Access

To find out how you can benefit from open access to research, see our library web pages and Open Access blog. For open access help contact: openaccess@st-andrews.ac.uk.

Accessibility

Read our Accessibility statement.

How to submit research papers

The full text of research papers can be submitted to the repository via Pure, the University's research information system. For help see our guide: How to deposit in Pure.

Electronic thesis deposit

Help with deposit.

Repository help

For repository help contact: Digital-Repository@st-andrews.ac.uk.

Give Feedback

Cookie policy

This site may use cookies. Please see Terms and Conditions.

Usage statistics

COUNTER-compliant statistics on downloads from the repository are available from the IRUS-UK Service. Contact us for information.

© University of St Andrews Library

University of St Andrews is a charity registered in Scotland, No SC013532.

  • Facebook
  • Twitter