Universiteit Leiden

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Proefschrift

Understanding functional dynamics and conformational stability of beta-glycosidases

Due to their central physiological roles in living organisms, retaining beta-glycosidases have been the subject of tremendous research efforts to examine their structure/function relation using numerous biophysical and biochemical approaches.

Auteur
Ben, Bdira F.
Datum
20 februari 2018
Links
Thesis in Leiden Repository

Due to their central physiological roles in living organisms, retaining beta-glycosidases have been the subject of tremendous research efforts to examine their structure/function relation using numerous biophysical and biochemical approaches. Since the proposition of the hydrolysis mechanism in the late fifties by Koshland1, the fundamental research on retaining b-glycosidases has been revolutionized by the discovery of multiple reversible and irreversible inhibitors. One of the most successful class of inhibitors are mechanism based inactivators, which were extensively used to identify the nucleophilic catalytic residues and to comprehend the catalytic mechanism and substrate itinerary. Subsequently, covalent inhibitors were used as warheads to synthesize chromogenic activity based probes (ABPs), which were widely used to selectively label and discover new retaining beta-glycosidases in complex biological samples. The organic synthesis and biological applications of these ABPs has become routine. Nevertheless, their binding mechanism and influences on protein conformation and dynamics remained unexplored. Therefore, this work is aimed to establish a bridge between the two research disciplines, using ABP technology to understand functional dynamics and conformational stability of retaining bglycosidases in solution and in vivo. The research relied on standard biochemistry and advanced NMR spectroscopy research approaches.