Use of hydrogen/deuterium exchange-mass spectrometry in the study of cell wall degrading enzymes and their inhibitors
Bahrainwala, Tasneem M
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Mass Spectrometry has developed in the last decade into a mature tool for the study of biomolecules and proteins. It has proven to be an indispensable tool in determining the primary structure of proteins and more recently has been heavily utilized in the studies on secondary and tertiary protein structures. Mass spectrometry is also used to study protein complexes and their interactions with enzymes, ligands or substrates. The study of Aspergillus niger endopolygalacturonases, EPG-I and EPG-II, in the presence of substrate as well as in the presence of different polygalacturonase inhibiting proteins (PGIPs) are described. The cell wall degrading enzymes produced by the fungus Aspergillus niger have many commercial applications and are important in the study of plant pathogenesis. At the same time, PGIPs are part of a plant’s first lines of defense against the attack of fungi. PGIPs are extracellular proteins, ionically bound to the cell wall which limit fungal invasion by counter acting EPG activity and permitting for the induction of defense elicitors. The basis of the research in this dissertation is the utilization of mass spectrometry techniques coupled with hydrogen/deuterium exchange to study the interactions between a protein-carbohydrate binding system as well as protein-protein binding. Hydrogen/deuterium exchange-mass spectrometry offers several advantages, such as a high speed of analysis, sensitivity and reduced sample requirement as compared to other methods such as NMR and X-ray crystallography for studying protein-carbohydrate and protein-protein interactions.