Most proteins and their complexes are flexible and dynamic in solution, occupying several conformations over time. Therefore, complex formation can be thought of as following a trajectory along... Show moreMost proteins and their complexes are flexible and dynamic in solution, occupying several conformations over time. Therefore, complex formation can be thought of as following a trajectory along which a loosely associated, weakly interacting encounter complex acts to pre-orient the binding partners before they proceed to the final stereo-specific state. Encounter complexes often play a large role for complexes that must balance a biological requirement for a high turnover rate with the necessity of forming a specific interaction. This is particularly the case for electron transfer complexes, such as the complex between yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP). The work described in this thesis focuses on the use of paramagnetic nuclear magnetic resonance (NMR) spectroscopy to study dynamic (transient) protein complexes, using the Cc-CcP complex as a model. Paramagnetic NMR has proven to be an extremely powerful technique for studying lowly populated states such as those of the encounter complex. It relies on the magnetic effects generated by an unpaired electron within a paramagnetic centre that disturb the local magnetic field experienced by nearby nuclei. This results in measureable changes in the NMR signals from which distance and orientation information for protein structure modelling can be extracted. 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