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... Show moreDue 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. Show less
Most proteins and their complexes are flexible and dynamic in solution, occupying several conformations over time. Therefore, complex formation can be thought of as following a trajectory along... Show moreMost proteins and their complexes are flexible and dynamic in solution, occupying several conformations over time. Therefore, complex formation can be thought of as following a trajectory along which a loosely associated, weakly interacting encounter complex acts to pre-orient the binding partners before they proceed to the final stereo-specific state. Encounter complexes often play a large role for complexes that must balance a biological requirement for a high turnover rate with the necessity of forming a specific interaction. This is particularly the case for electron transfer complexes, such as the complex between yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP). The work described in this thesis focuses on the use of paramagnetic nuclear magnetic resonance (NMR) spectroscopy to study dynamic (transient) protein complexes, using the Cc-CcP complex as a model. Paramagnetic NMR has proven to be an extremely powerful technique for studying lowly populated states such as those of the encounter complex. It relies on the magnetic effects generated by an unpaired electron within a paramagnetic centre that disturb the local magnetic field experienced by nearby nuclei. This results in measureable changes in the NMR signals from which distance and orientation information for protein structure modelling can be extracted. Show less
By utilizing paramagnetic NMR techniques, the structure and dynamics of the P450cam system were investigated. The analysis of PCS and RDC illuminated the stereo-specific final complex of Pdx and... Show moreBy utilizing paramagnetic NMR techniques, the structure and dynamics of the P450cam system were investigated. The analysis of PCS and RDC illuminated the stereo-specific final complex of Pdx and P450cam, while the results of PRE demonstrated the presence of a transient encounter complex. Furthermore, the significant insights of the interaction in the interface were uncovered by X-ray crystallography. Currently, the nature of Pdx effector activity is under debate. Since paramagnetic NMR experiments are applicable to solution studies at ambient temperature, PCS, RDC and PRE methods can further resolve the molecular mechanism of P450cam in the future. Show less
The work described in this thesis focuses on the application of various NMR techniques to the study of interactions between proteind and small molecules and proteins and peptides, incluiding the... Show moreThe work described in this thesis focuses on the application of various NMR techniques to the study of interactions between proteind and small molecules and proteins and peptides, incluiding the well-established classical NMR approaches, and the precently developed paramagnetic NMR methods. computational tools have been used to complement and visualized the experimental data. Show less
