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
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
