Identifying and characterizing the early oligomeric species of tau proteins is the key to understanding the mechanism of protein aggregation and, eventually, guiding the possible therapeutic intervention in their pathologic effects. The experimental studies of these transient and dynamic oligomeric intermediates have been difficult with existing experimental tools, and the mechanistic details about the aggregation process and tau self-assembly are currently not well understood. We utilize the combination of the Overhauser Dynamic Nuclear Polarization (ODNP), NMR and Electron Spin Resonance (EPR) spectroscopy, in combination with the protein site directed mutagenesis and spin-labeling approach to identify and characterize the different aggregation species of tau187 peptide (residues 255--441 of human tau) and to study the aggregation mechanism in solution, at ambient conditions.

Our experimental approach employs the Overhauser Dynamic Nuclear Polarization (ODNP) to investigate the translational dynamics of hydration water, at relevant time-scales (∼ 10-1000 ps.), near (∼ 5A) the spin-labeled protein surface. This is possible by measuring the ODNP Enhancement of the 1H of liquid water, diffusing near the paramagnetic spin-label, and extracting the dynamic information from the measured ODNP factors by considering the relevant spectral density function. We present our experimental findings, where tau187 monomers, upon incubation with heparin, suddenly self-assemble and populate the heterogeneous oligomeric species, where the overall increasing structural order and conformational rearrangements of tau187 continue throughout extended period of time, prior to the elongation of the fibrils.

We also carried out EPR experimental analysis, where the inter-mixtures of the paramagnetic with diamagnetic labeled tau187 species allowed us to study the organization of peptide regions into parallel beta-sheets during fibril formation. This methodology has also enabled us to study the oligomer dynamics and sub-unit exchange in different aggregation species of tau187 and to provide experimental insight into elongation process of tau fibrils via annealing of highly ordered tau187 oligomers. Overall we successfully showcased our novel experimental approach for characterization of the early protein aggregation events.