Amyloid fibrils, defined by highly ordered β-sheet architectures, arise from protein self-assembly and are central to both pathological aggregation and functional biomaterial applications. Among proteins confirmed to form amyloids is stem bromelain (SB), a proteolytic enzyme widely used in food, pharmaceutical, and biomedical industries. While heating is a recognized trigger for SB fibrillation, the persistence of fibril growth during post-heating incubation at ambient conditions and its nanoscale morphological dynamics remain unclear. In this study, we investigated the time-dependent fibrillation of SB using atomic force microscopy (AFM). SB was first heated in borate buffer (pH 10.8) at 65 °C for 10 h, followed by incubation at room temperature for 1, 3, and 7 days. High-resolution AFM imaging directly visualized the morphological progression: from dispersed protofibrils on Day 1, to elongated and partially aligned fibrils on Day 3, culminating in dense, interconnected amyloid networks by Day 7. Quantitative analysis of AFM images revealed a progressive increase in alignment in the orientation distribution which shows directional growth of fibril on mica substrate. Moreover, there is a clear upward trend in fibril coverage-area over time, with Day 7 showing significantly higher coverage which implies structural organization. We also introduce a technique that provides an accessible, high-resolution approach for real time morphological studies of SB protofibril elongation and provides new insights into the kinetics and organizational dynamics of amyloid fibril formation.
Lugo et al. (2026) studied this question.