Edinburgh Research Archive logo

Edinburgh Research Archive

University of Edinburgh homecrest
View Item 
  •   ERA Home
  • Biological Sciences, School of
  • Biological Sciences thesis and dissertation collection
  • View Item
  •   ERA Home
  • Biological Sciences, School of
  • Biological Sciences thesis and dissertation collection
  • View Item
  • Login
JavaScript is disabled for your browser. Some features of this site may not work without it.

Probing protein-ligand interactions via solution phase hydrogen exchange mass spectrometry

View/Open
Esswein2010.pdf (5.473Mb)
Date
2010
Author
Esswein, Stefan Theo
Metadata
Show full item record
Abstract
Mass spectrometry is a versatile, sensitive and fast technique with which to probe biophysical properties in biological systems and one of the most important analytical tools in the multidisciplinary field of proteomics. The study of nativestate proteins and their complexes in the gas-phase is well established and direct infusion electrospray ionisation mass spectrometry (DI-ESI-MS) techniques are becoming increasingly popular as a tool for screening and determining quantitative information on protein-protein and protein-ligand interactions. However, complexes retained by ESI-MS are not always representative of those in solution and care must be taken in interpreting purely gas-phase results. This thesis details modification and advancement of solution phase techniques devised by Gross et al. utilising ESI-MS and Fitzgerald et al. applying matrix assisted laser desorption ionisation (MALDI)-MS termed PLIMSTEX (protein-ligand interactions by mass spectrometry, titration and hydrogen-deuterium-exchange)[1] and SUPREX (Stability of unpurified proteins from rates of H/D exchange)[2] to quantify these interactions with regards to high throughput analysis. The first part of this thesis describes the different developmental stages of the devised HPLC-front ends and their optimisation with myoglobin and insulin. The successfully developed HPLC-front end in conjunction with PLIMSTEX and SUPREX and ESI-MS then gets tested with self expressed and purified cyclophilin A(CypA)- cyclosporin A (CsA) system, followed by a test screen with potential CypA binding ligands. Dissociation constants (Kd’s) within one order of magnitude to reported values are determined. In the third part of this thesis the application of the devised ESI-SUPREX methodology has been applied to anterior gradient 2 (AGr2) and the factor H complement control proteins module 19-20 (fH19-20) exhibiting binding potential to a taggedhexapeptide and a synthetic pentasaccharide, respectively, resulting in thermodynamical data for these protein-ligand interactions. For the AGr2 system another dimension of investigation has been added by temperature controlling the devised ESI-SUPREX approach, revealing a phase transition in the protein at higher temperatures. The final part of this thesis describes the application of the ESI-SUPREX methodology to probe folding properties of CypA in the presence of the self expressed and purified E. coli chaperonin groEL. Thereby the denaturing properties of groEL have been emphasised along with the stabilisation of a denatured CypA species.
URI
http://hdl.handle.net/1842/4638
Collections
  • Biological Sciences thesis and dissertation collection

Library & University Collections HomeUniversity of Edinburgh Information Services Home
Privacy & Cookies | Takedown Policy | Accessibility | Contact
Privacy & Cookies
Takedown Policy
Accessibility
Contact
feed RSS Feeds

RSS Feed not available for this page

 

 

All of ERACommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsPublication TypeSponsorSupervisorsThis CollectionBy Issue DateAuthorsTitlesSubjectsPublication TypeSponsorSupervisors
LoginRegister

Library & University Collections HomeUniversity of Edinburgh Information Services Home
Privacy & Cookies | Takedown Policy | Accessibility | Contact
Privacy & Cookies
Takedown Policy
Accessibility
Contact
feed RSS Feeds

RSS Feed not available for this page