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Characterizing protein conformation and dynamics using hydrogen-deuterium exchange electrospray ionization mass spectrometry
Monitoring hydrogen-deuterium exchange of proteins can yield a wealth of information about not only the native state, but also not native states of proteins. These non-native states have important biological roles including protein folding intermediates as well as ligand binding/delivery and protein-protein interactions. Structural and dynamic properties of a partially folded conformation (A-state) of ubiquitin are studied using amide hydrogen exchange in solution (HDX) and mass spectrometric detection. A clear distinction between the native state of the protein and the A-state can be made when HDX is carried out in a semicorrelated regime. Furthermore, combination of HDX and protein ion fragmentation in the gas phase by means of collision-induced dissociation (CAD)] is used to evaluate the conformational stability of various protein segments specifically in the molten globular state. Chain flexibility appears to be distributed very unevenly in this non-native conformation. This study also demonstrates the power of mass spectrometry as a tool in providing conformer-specific information about the structure and dynamics of both native and non-native protein states coexisting in solution under equilibrium. This dissertation has been broken down into several subsections. First, we evaluate electrospray ionization amide hydrogen exchange collision assisted dissociation mass spectrometry's (ESI HDX CAD MS) methodology to better understand the determinants of hydrogen scrambling in the gas phase, which can be used to probe non-native states of proteins. Secondly we examined the structure of a molten globule of ubiquitin using HDX CAD MS under mildly denaturing conditions and compare this with the proposed NMR and crystal structures of the A-state and native state, respectively. Lastly, we have conducted studies include ESI MS studies of non-covalent interactions between cellular retinoic acid binding protein II (CRABP II) and the ligand-binding domain (LBD) of retinoic acid receptor (RAR) as it pertains to delivery of retinoic acid (RA). The backbone dynamics of CRABP II will be investigated in order better understand the ligand binding and delivery properties of CRABP II and how they differ from CRABP I.^
Joshua K Hoerner,
"Characterizing protein conformation and dynamics using hydrogen-deuterium exchange electrospray ionization mass spectrometry"
(January 1, 2007).
Electronic Doctoral Dissertations for UMass Amherst.