Protein–protein complexes are formed via transient states called encounter complexes that greatly influence the formation of the stereospecific complex. Electrostatic charges on the protein... Show moreProtein–protein complexes are formed via transient states called encounter complexes that greatly influence the formation of the stereospecific complex. Electrostatic charges on the protein surfaces play a major role in encounter complexes of electron transfer proteins. The complex formed by cytochrome c (Cc) and cytochrome c peroxidase (CcP) has been studied intensively because it is an excellent model to explore the properties of transient protein-protein interactions. PRE experiments previously described the encounter complex formed by the two proteins in detail. In this thesis we tested to what degree the electrostatic patch on CcP is optimized to enhance the rate of the formation of the stereospecific complex. Using paramagnetic NMR in combination with Monte Carlo simulations and stopped flow spectrophotometry, we investigate several CcP mutants with reengineered charged patches to create new encounter complexes and measure their effects on electron transfer from Cc to CcP. The results indicate that the interactions with Cc are affected more by the total charge of CcP surface than the specific distribution of the charges, bringing into question the concept of electrostatic patches being highly optimized by evolution. 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
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
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
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 interactions between proteins are of central importance for virtually every process in a living cell. It has long been a mystery how two proteins associate to form a complex in a complicated... Show moreThe interactions between proteins are of central importance for virtually every process in a living cell. It has long been a mystery how two proteins associate to form a complex in a complicated cellular context. Recently, it was found that an intermediate state called encounter state, of a protein complex, exists briefly before a final protein complex is formed. In the encounter state, one protein is rolling over on the surface of its partner, searching for the optimal orientation. In my PhD thesis, a transient electron transfer complex formed between a heme protein and an iron-sulfur protein was found to be trapped in this intermediate state, existing as a pure encounter complex. Thus, characterization of this dynamic complex by nuclear magnetic resonance spectroscopy advances our understanding of the general mechanism of protein-protein interaction Show less
