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Title: $Unraveling the structure of monomeric and fibrilized 441-residue tau with NMR spectroscopy
Abstract: The microtubule-associated, intrinsically disordered protein tau is a key- protein in maintaining the axonal transport in neurons. The association to and the dissociation from microtubules is regulated via a complex pattern of phosphorylation that is still largely unknown. Moreover, a clear relationship between Alzheimer’s disease (AD) and tau was established. Tau-mediated neurodegenerative diseases are characterized by insoluble tau-aggregates, term- ed paired helical filaments that eventually assemble into neurofibrilary tan- gles. However, it is unclear, which extracellular or intracellular factors might trigger the transformation from a physiological important protein into a pathological agent. In this thesis, several aspects of tau were investigated using NMR spec- troscopy. In the first part of the results section, paramagnetic relaxation experiments were conducted that allowed the characterization of the global folding of monomeric tau in solution with great detail. The 2288 distance re- straints derived from these experiments has allowed for an ensemble structure calculation, that resulted in a residue-specific model. The second part of the results section investigates the performance of disorder predictors. We found a good correlation of the residue-contacts predicted with IUPred when compared to the contact map of tau. Protein- binding sites prediction with ANCHOR is in very good agreement with the hot-spots of microtubule-binding. For the secondary structure prediction, a combined approach of predictors allowed for the correct secondary structure assignment to the strongest residual secondary structure propensities. The occurrence of paired helical filaments is accompanied with a hy- perphosphorylated state of tau. To avoid incomplete and unspecific phos- phorylation, we decided to mimic phosphorylation by replacing serines and threonines with glutamic acid in epitopes of AD-diagnostic antibodies (AT8, AT100 and PHF1). The global and local structural characteristics were investigated and compared to the wildtype of tau. We demonstrate, that pseudophosphorylation results in an opening of the paperclip conformation. The higher aggregation rate found for this pseudophosphorylated tau-mutant underpins the importance of the transient network of intramolecular inter- actions. We found, that a weakening of the transient interactions precedes local structural changes. In the fourth part, the NMR-investigations were conducted on the fuzzy coat of tau paired helical filaments. Due to the size of the aggregates of more than 1 MDa, the use of high resolution magic angle spinning was imperative. The presence and assignment of residues 1M-T212 and 399V-L441 indicate a highly dynamic N-terminal and C-terminal fuzzy coat, that transiently con- tact the PHF-core. In particular the very N-terminus shows a tightened electrostatic interaction with the PHF-core. These findings rationalize the affinity of conformation-specific PHF-tau antibodies that exhibit two dis- continuous epitopes, one at the very N-terminus and the second within the PHF-core.