A specific form of prefibrillar aggregates that functions as a precursor of amyloid nucleation

Non-fibrillar protein aggregates that appear in the earlier stages of amyloid fibril formation are sometimes considered to play a key role in amyloid nucleation; however, the structural features of these aggregates currently remain unclear. We herein identified a characteristic pathway of fibril formation by human insulin B chain, in which two major species of prefibrillar aggregates were identified. Based on the time-resolved tracking of this pathway with far-UV circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and (1)H-NMR spectroscopy, the first prefibrillar aggregate with a hydrodynamic diameter of approximately 70 nm accumulated concomitantly with the formation of a β-sheet structure, and the size further evolved to 130 nm with an additional structural development. These prefibrillar aggregates were metastable and survived at least 24 hours as long as they were maintained under quiescent conditions. The energy barrier for nucleation was overcome by shaking or even by applying a single short ultrasonic pulse. Furthermore, an investigation where nucleation efficiency was monitored by fibrillation rates with varying the timing of the ultrasonic-pulse treatment revealed that the second prefibrillar aggregate specifically produced amyloid nuclei. These results suggest that the second form of the prefibrillar aggregates acts as a direct precursor for the amyloid nucleation.