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
Spin labeling and electron paramagnetic resonance (EPR) have been employed to study structure and dynamics of proteins. The surface polarity of four single cysteine mutants of the Zn-azurin in... Show moreSpin labeling and electron paramagnetic resonance (EPR) have been employed to study structure and dynamics of proteins. The surface polarity of four single cysteine mutants of the Zn-azurin in frozen solution were studied using 275 GHz EPR (J-band), with the advantage compared to 9 GHz (X-band) and 95 GHz (W-band) to allow differentiating between sites having small differences in polarity. The polarity/proticity profiles of the four mutants have been obtained and compared with data already reported in literature of spin labels in different solvents. In order to detect distances at the nm scale, azurin was used as a known model system for double electron-electron spin resonance. The conformation of the spin label linker was modelled. The model was found to be in good agreement with experimental results and can be extended to other proteins. The dynamics of the transient complex of Nostoc sp. PCC 7119 cytochrome f __ plastocyanin was investigated by NMR using the paramagnetic relaxation enhancements. The experimental distance restraints, used in docking calculations, are best interpreted by the presence of a dynamic ensemble of protein-protein orientations within the complex, rather than by a single, well-defined structure, and can be described with an encounter complex model. Show less
The biological processes that are the basis of all life forms are mediated largely by protein-protein interactions. The protein complexes involved in these interactions can be categorised by their... Show moreThe biological processes that are the basis of all life forms are mediated largely by protein-protein interactions. The protein complexes involved in these interactions can be categorised by their affinity, which results in a range from static to transient complexes. Electron transfer complexes, which have to combine high turn-over with specificity are typically transient complexes. The transient complexes under investigation in this work are all part of the photosynthetic redox chain, in which electrons are transferred from membrane bound cytochrome f (cytf) to either plastocyanin (Pc) or cytochrome c6 (cytc6). This work describes the investigation of several aspects of transient protein-protein interactions that are applicable to many other transient complexes. The use of NMR as a tool of choice for these studies has proven it an invaluable technique that provides insight in rules that govern the world of transient complex formation. New approaches such as PRE and the creation of ensembles of structures are investigated. They will hopefully lead to a full understanding of dynamics and the variety of ways that transient complexes use dynamics to perform their function in the cell. Show less
