Direct Observation of the Self‐Aggregation of R3R4 Bi‐repeat of Tau Protein

: Different cryo‐EM derived atomic models of in vivo tau filaments from patients with tauopathies consisted of R3 and R4 repeats of the microtubule‐binding domain. In comparison, only the R3 repeat forms the core of the heparin‐induced fibrils of the three repeat tau isoforms. For developing therapeutics, it is desirable to have an in vitro tau aggregation system producing fibrils corresponding to the disease morphology. Here we report the self‐aggregation of truncated tau segment R3R4 peptide without requiring heparin for aggregation induction. We used NMR spectroscopy and other biophysical methods to monitor the self‐aggregation of R3R4. We identified the hexapeptide region in R3 and β‐turn region in R4 as the aggregation initiating region of the protein. The solid‐state NMR of self‐aggregated R3R4 fibrils demonstrated that in addition to R3 residues, residues of R4 were also part of the fibril filaments. The presence of both R3 and R4 residues in the aggregation process and the core of fibril filaments suggest that the aggregation of R3R4 might resemble the in vivo aggregation process. Self‐aggregation of R3R4 bi‐repeat of tau is distinct from the heparin induced aggregation of the protein. Solution and solid‐state NMR spectroscopy confirm the significant role of R4 residues in the self‐aggregation.