title: An electrospray ms-coupled microfluidic device for sub-second hydrogen/deuterium exchange pulse-labelling reveals allosteric effects in enzyme inhibition.
abstract: In this work, we introduce an integrated, electrospray mass spectrometry-coupled microfluidic chip that supports the complete workflow for 'bottom up' hydrogen/deuterium exchange (HDX) pulse labelling experiments. HDX pulse labelling is used to measure structural changes in proteins that occur after the initiation of a reaction, most commonly folding. In the present case, we demonstrate the device on the β-lactamase enzyme TEM-1, identifying active site changes that occur upon acylation by a covalent inhibitor and subtle changes in conformational dynamics that occur away from the active site over a period of several second after the inhibitor is bound. Our results demonstrate the power of microfluidics-enabled sub-second HDX pulse labelling as a tool for studying allostery and show some intriguing correlations with mutagenesis studies.
authors: Chemistry Department, York University, Toronto, Canada M3J 1P3.
authors: Tamanna
authors: T
authors: Rob
authors: Preet Kamal
authors: PK
authors: Gill
authors: Dasantila
authors: D
authors: Golemi-Kotra
authors: Derek J
authors: DJ
authors: Wilson
mesh_terms: D000215:Acylation; D000494:Allosteric Regulation; D020134:Catalytic Domain; D003903:Deuterium / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D006859:Hydrogen / Q000737:chemistry; D006860:Hydrogen Bonding; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D065093:beta-Lactamase Inhibitors; D006859:Hydrogen; D003903:Deuterium; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1