Universiteit Leiden

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Making the invisible visible: paramagnetic NMR and the transient protein complex

Promotor: Prof.dr. M. Ubbink

J.T. Schilder
24 november 2015
Thesis in Leiden Repository

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 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.