Protein Structural Studies by Oxidative Labeling and Mass Spectrometry
Despite siginificant number of previous studies, the area of protein structure, folding and conformational dynamics remains a critical subject in biological chemistry. Oxidative labeling combined with electrospray ionization (ESI) mass spectrometry (MS) offers enormous opportunities for studying protein structures at the level of individual residues. Such high selectivity combined with excellent sensitivity makes ESI-MS a primary tool for the study of protein folding reactions. This approach is verified here by exploring the behavior of a model protein, myoglobin. Hydroxyl radicals (∙OH) are generated by laser photolysis of H2O2 and react with the solvent exposed amino acids. In this way, information is obtained for differently folded states. These comprehensive investigations reveal useful insights into the structural differences of different protein conformers. The second part of this project focuses on kinetic techniques. It is clear that fastest events during protein folding occur in the sub-milliseconds range. However, not much is known regarding these fast events due to the lack of suitable instrumentation capable of monitoring rapid events with structural resolution. By using an ultra-rapid mixer with hydrodynamic focusing this thesis provides the groundwork for future covalent labeling studies that will employ ∙OH labeling with unsurpassed time resolution. Together with ESI-MS detection this approach has the potential to provide detailed structural information on early protein folding intermediates.
protein folding, holo-myoglobin, apo-myoglobin, electrospray ionization mass spectrometry, equilibrium, kinetics, oxidative labeling, ultra-rapid mixing.
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