The majority of the work presented in this thesis involves the design and synthesis of paramagnetic NMR probes, including lanthanoids caged probes and spin labels. An overview of the development of... Show moreThe majority of the work presented in this thesis involves the design and synthesis of paramagnetic NMR probes, including lanthanoids caged probes and spin labels. An overview of the development of different types of lanthanoids caged probes is given. Among all of the reported lanthanoid probes, the caged lanthanoid NMR probe version 5 (CLaNP-5) shows the largest paramagnetic effects due to the fact that it is tagged to proteins via two-point attachment and its free complex presents a single conformation. Although CLaNP-5 is successfully applied to study proteins and protein complexes, the net charge of Ln CLaNP-5 complex and the weak disulfide linker are the drawbacks. A new paramagnetic probe, CLaNP-7, was synthesized, for which the net charge of lanthanoids complexes was reduced to +1 by introducing p-nitrophenol. The __-tensor of CLaNP-7 is pH-dependent when a histidine residue is located close to the attachment site. It is proposed that the pH dependence is due to the fact that the histidine forms a hydrogen bond with a water that acts as the ninth ligand of the lanthanoid. In order to enhance the stability of the tag linkers, two approaches, thioether and bioorthogonal reactions, were investigated. The results showed that a new thio-reactive CLaNP-9 was successfully attached to protein and the reaction product was stable in the reductive conditions. Moreover, inhibitor-based paramagnetic probes were also synthesized and the co-crystal structures of protein with inhibitor-based probes were also presented. Show less
Recent studies have provided experimental evidence for the existence of an encounter complex, a transient intermediate in the formation of protein complexes. We have used paramagnetic relaxation... Show moreRecent studies have provided experimental evidence for the existence of an encounter complex, a transient intermediate in the formation of protein complexes. We have used paramagnetic relaxation enhancement NMR spectroscopy in combination with Monte Carlo simulations to characterize and visualize the ensemble of encounter orientations in the short-lived electron transfer complex of yeast Cc and CcP. The complete conformational space sampled by the protein molecules during the dynamic part of the interaction was mapped experimentally. Our results demonstrate that the encounter complex is populated for 30% of the time, where Cc samples only about 15% of the surface area of CcP. We have also shown that the occupancy of the encounter complex can be modulated across a broad range by single point mutations of interfacial residues. Thus, by adjusting the amount of the encounter complex through a judicious choice of point mutations, we can remodel the energy landscape of a protein complex and tune its binding specificity. It has not been well established whether binding hot spots, which are frequently found in strong static complexes, also govern transient protein interactions. To address this issue, we have investigated an electron transfer complex of physiological partners from yeast: yeast Cc and yeast CcP. Using NMR spectroscopy and double mutant cycle, we show that Cc R13 is a hot-spot residue, as R13A mutation has a strong destabilizing effect on binding. Based on our analysis, we propose that binding energy hot spots, which are prevalent in static protein complexes, could also govern transient protein interactions. We have also investigated the effect of interface mutations on the structure and dynamics of the horse Cc __ yeast CcP complex using NMR spectroscopy and X-ray crystallography. The horse Cc forms a more dynamic complex with yeast CcP as compared to the native yeast Cc-CcP complex, and the two Cc molecules acquire different orientations in complex with CcP. Interestingly, a single interface mutation makes the complex more specific, with the horse Cc in an orientation resembling that of the native yeast Cc. 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
For all organisms populating the Earth, motion is synonymous with life. In the living cell, protein molecules - its most important building blocks and work horses - are constantly on the move.... Show moreFor all organisms populating the Earth, motion is synonymous with life. In the living cell, protein molecules - its most important building blocks and work horses - are constantly on the move. Traditional static models are no longer sufficient for a thorough description of interactions among these biomolecules. Here we present a direct, experimental approach for assessing the dynamics of interactions between proteins, which for a long time has been an exclusive realm of theoretical studies. Show less
