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Title: Exploring protein conformation with mass spectrometry
Author: Florane, H.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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The first part of this thesis describes the development and viability of a phase I screening system for obtaining a rank order of affinity of novel ligands against the immunophilin, Cyclophilin A (CypA). The naturally occurring inhibitor Cyclosporin A (CsA) was used as a positive control to validate a method for calculating the dissociation constant (Kd). An HPLC autosampler and pumping system was used as a high throughput on-line electrospray ionisation (ESI)-MS sampling system. Optimised ESI conditions were then used to screen novel ligands from 3 combinatorial libraries and approaches for data analysis is discussed. Hydrogen/deuterium exchange (HDX) can be used directly and indirectly as a means for studying protein conformations. Melittin, the major component of honey bee venom is taken here as a model system for studying secondary structure in solution and the gas phase. Comprising a 26 amino acid polypeptide, melittin occupies a random coil in aqueous conditions which can be transformed into an α-helix under increasingly hydrophobic conditions. A variety of HDX techniques were utilised: i) comparing rats of deuterium (d-) uptake by direct infusion – ESI at different pDs and methanol concentrations; ii) PLIMSTEX (protein-ligand interactions by mass spectrometry, titration and HDX) at high and low salt concentrations with varying pDs; iii) gas phase exchange in an LCQ ion trap using He/-methanol as the bath gas. Melittin was pre-incubated in a variety of methanol concentrations. Comparing results from these different approaches, α-helical retention has been shown to exist in the N-terminal half of the peptide. All the afore-mentioned techniques developed using melittin were adapted for CypA. Comparisons of d-uptake in the presence and absence of CsA shows the ligand to have a stabilising affect on the protein.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available