Fibril-forming motifs are essential for the fibrillization of Tau_244–372.

<p>(A) Location of the two fibril-forming motifs ²⁷⁵VQIINK²⁸⁰ (PHF6*) and ³⁰⁶VQIVYK³¹¹ (PHF6) at Tau_244–372: VQIINK is in the R2 part of Tau_244–372, and VQIVYK is in the R3 part of Tau_244–372. (B) Negative-stain transmission electron micrographs of Tau_244–372, Tau_244–372/ΔPHF6, Tau_244–372/ΔPHF6*, and Tau_244–372/ΔPHF6/ΔPHF6* (scale bar 400 nm). (<b>C</b>) Kinetic curves for the aggregation of Tau_244–372, Tau_244–372/ΔPHF6, Tau_244–372/ΔPHF6*, and Tau_244–372/ΔPHF6/ΔPHF6*, monitored by the turbidity at 350 nm. The concentration of Tau protein was 50 µM, and 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM DTT and 12.5 µM heparin was used. The assays were carried out at 37°C. Tau_244–372, Tau_244–372/ΔPHF6, and Tau_244–372/ΔPHF6* finished their progress of aggregation in a short time of observation (15 h), but in a relatively long time of observation (100 h) Tau_244–372/ΔPHF6/ΔPHF6* did not show any clue of aggregation (the inset of C). (D) Kinetic curves for the aggregation of Tau_244–372, Tau_244–372/ΔPHF6, Tau_244–372/ΔPHF6*, and Tau_244–372/ΔPHF6/ΔPHF6*, monitored by ThT fluorescence. The long time incubation of Tau_244–372/ΔPHF6/ΔPHF6* is shown in the inset of D. The concentration of Tau protein was 8 µM, and 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM DTT and 2 µM heparin was used. The assays were carried out at 37°C. The observation time was 12 h for Tau_244–372, Tau_244–372ΔPHF6, and Tau_244–372ΔPHF6*, and 100 h for Tau_244–372/ΔPHF6/ΔPHF6*.