Unraveling\nthe Structure and Dynamics of Ac-PHF6-NH<sub>2</sub> Tau Segment Oligomers

The aggregation of the proteins tau and amyloid-β\nis a salient\nfeature of Alzheimer’s disease, the most common form of neurodegenerative\ndisorders. Upon aggregation, proteins transition from their soluble,\nmonomeric, and functional state into insoluble, fibrillar deposits\nthrough a complex process involving a variety of intermediate species\nof different morphologies, including monomers, toxic oligomers, and\ninsoluble fibrils. To control and direct peptide aggregation, a complete\ncharacterization of all species present and an understanding of the\nmolecular processes along the aggregation pathway are essential. However,\nthis is extremely challenging due to the transient nature of oligomers\nand the complexity of the reaction networks. Therefore, we have employed\na combined approach that allows us to probe the structure and kinetics\nof oligomeric species, following them over time as they form fibrillar\nstructures. Targeting the tau protein peptide segment Ac-PHF6-NH<sub>2</sub>, which is crucial for the aggregation of the full protein,\nsoft nano-electrospray ionization combined with ion mobility mass\nspectrometry has been employed to study the kinetics of heparin-induced\nintact oligomer formation. The oligomers are identified and characterized\nusing high-resolution ion mobility mass spectrometry, demonstrating\nthat the addition of heparin does not alter the structure of the oligomeric\nspecies. The kinetics of fibril formation is monitored through a Thioflavin\nT fluorescence assay. Global fitting of the kinetic data indicates\nthat secondary nucleation plays a key role in the aggregation of the\nAc-PHF6-NH<sub>2</sub> tau segment, while the primary nucleation rate\nis greatly accelerated by heparin.