Multicopper oxidase, laccase, can efficiently reduce oxygen to water and are mostly used in the enzymatic biofuel cells. However, they suffer from low stability when functionalized over an... Show moreMulticopper oxidase, laccase, can efficiently reduce oxygen to water and are mostly used in the enzymatic biofuel cells. However, they suffer from low stability when functionalized over an electrode. This can be overcome by designing artificial catalysts for the oxygen reduction reaction based on the active site of laccase which requires a detailed understanding of the active site. The current research is aimed at characterizing the active site of small laccase from Streptomyces coelicolor using a combination of paramagnetic NMR spectroscopy, EPR spectroscopy, mutagenesis and quantum mechanical (QM) calculations. The presence of chemical exchange at the active site of laccase attributed to the coordinating histidines is reported. QM calculations showed the importance of the orientation of the coordinating water derived ligand. Mutagenesis study showed the importance of second shell residue in stabilizing intermediates during the oxygen reduction reaction. It is also reported that by changing the pH, a new intermediate could be experimentally observed however, further research is needed to characterize this. The resonance assignment shown in the current research can be used as spies to characterize the active site of laccase. This might in future provide insight into the catalytic mechanism of oxygen reduction reaction by laccase. Show less
The aim of the research presented in this thesis was to develop new methods forchallenging systems in liquid-state NMR using paramagnetic effects generated by thetwo-armed probe CLaNP-5. Chapter 1... Show moreThe aim of the research presented in this thesis was to develop new methods forchallenging systems in liquid-state NMR using paramagnetic effects generated by thetwo-armed probe CLaNP-5. Chapter 1 is an introduction about NMR assignmentdevelopment and challenges and a brief opening to the theory of paramagnetic effectsand their applications. Chapter 2 describes the upgrade of PARAssign software and itsability to assign and assess methyl groups of a 25 kDa protein using experimentalpseudocontact shifts from 1 to 3 paramagnetic centers. Chapter 3 describes theimplementation of residual dipolar coupling into PARAssign software as extra datasetinput in addition to the pseudocontact shifts. Applications are given for residueselective isotope labeled protein. Chapter 4 depicts the use of pseudocontact shiftsused to probe small methyl group movements and re-orientation under ligand binding.Chapter 5 gives some clues about how paramagnetic effect such as RDC but also PCScan provide information for challenging highly dynamic system. Chapter 6 givesinsights for PCS application to very large multimeric protein, the challenges andlimitations are discussed. Chapter 7 provides a discussion and few perspectives aboutthe work carried out and presented in this thesis. Show less
This PhD thesis focuses on fundamental aspects of protein-protein interactions. A multidisciplinary methodology for the detection and visualization of transient, lowly-populated encounter protein... Show moreThis PhD thesis focuses on fundamental aspects of protein-protein interactions. A multidisciplinary methodology for the detection and visualization of transient, lowly-populated encounter protein complexes is described. The new methodology combined paramagnetic NMR spectroscopy with computational methods (ensemble docking approach and Monte Carlo simulations) to provide a new model to describe the formation of a protein complex on the basis of the physical forces involved in the process, namely electrostatic and hydrophobic interactions. The formation of a productive protein complex is a stepwise process, in which the free components evolve to the final complex passing through a transient, lowly-populated encounter state. For a long time the first step of association was thought to be exclusively driven by long-range electrostatic interactions. Experimental evidences and theoretical studies questioned this assumption and suggested also a role of hydrophobic interactions in protein association. To study the contribution of the different forces we study the highly dynamic complex formed by plastocyanin and cytochrome f, two redox partners in oxygenic photosynthesis, for which both electrostatic and hydrophobic interactions were shown to contribute to the stabilization of the final complex. Through the combination of paramagnetic relaxation enhancement NMR techniques and computational methods we were able to visualize the presence of hydrophobic interactions in the encounter state and to elucidate the contribution of either electrostatic or hydrophobic forces to the formation of the encounter complex. Show less
Protein-protein interactions play an important role in all cellular processes such as signal transduction, electron transfer, gene regulation, transcription, and translation. Understanding these... Show moreProtein-protein interactions play an important role in all cellular processes such as signal transduction, electron transfer, gene regulation, transcription, and translation. Understanding these protein-protein interactions at the molecular level, is an important aim in structural biology. The protein interactions studied in this work are principally involved in the signal transduction and in electron transfer. These protein interactions belong to the family of transient dynamic interactions, meaning they associate and dissociate rapidly. The work presented in chapters two and three gives a detailed description of a study to determine the binding orientation of focal adhesion kinase derived peptide on the Src SH3 domain using paramagnetic NMR spectroscopy. The work presented in chapter four gives detailed information on the cloning, expression and purification of the focal adhesion kinase domain with the SH3 and S H2 binding sequences (32k) using a baculovirus expression system. The results of NMR characterization were presented for the complexes of different Src domains and the 32k. The work presented in the chapters five and six describes the NMR characterization of the interactions between several redox proteins. The aim of the work was to convert the transiently bound weak protein complexes into specific complexes Show less
