Amyloidogenic Growth Observation of Stem Bromelain via Atomic Force Microscopy

TL;DR

This study uses atomic force microscopy to observe how stem bromelain forms amyloid fibrils over time at room temperature.

Contribution

The paper introduces a high-resolution method for real-time observation of stem bromelain fibril growth and organization.

Findings

SB forms protofibrils on day 1, which develop into dense amyloid networks by day 7.

Fibril coverage and alignment increase progressively over time, indicating structural organization.

The study provides insights into the kinetics and dynamics of amyloid formation in SB.

Abstract

In this paper, we report on the amyloidogenic fibril
formation
of stem bromelain (SB) by using atomic force microscopy (AFM). Stem
bromelain (SB), a proteolytic enzyme, is widely used in industries
and medicine, making it essential to understand the factors affecting
aggregation. Amyloid formation entails the assembly of proteins into
highly ordered, β-sheet-rich fibrillar structures; yet while
heating is a recognized trigger for SB fibrillation, the extent of
continued fibril growth at room temperature incubation and its nanoscopic
morphological observation remain unexplored. Here, SB was heated in
pH 10.8 borate buffer at 65 °C for 10 h,
then incubated at room temperature for 1, 3, and 7 days, respectively.
A time-course imaging directly visualized the morphological progression
from small, dispersed protofibrils on day 1 to increasingly
pronounced fibrillar bundles on day 3 and 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.

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