Exploring protein conformation with mass spectrometry

• Main Author: Florane, H.
• Published: University of Edinburgh 2008
• Subjects: 571.4
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.650980

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 (K_d). 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 (<i>d</i>-) 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/<i>d­</i>-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 <i>d</i>-uptake in the presence and absence of CsA shows the ligand to have a stabilising affect on the protein.