Individual Alzheimer’s disease (AD) patients have been shown to have structurally distinct amyloid-β (Aβ) aggregates, including fibrils, in their brain. These findings suggest the possibility of a relationship between AD progression and Aβ fibril structures. Thus, the characterization of the structural dynamics of Aβ could aid the development of novel therapeutic strategies and diagnosis. Protein structure and dynamics have typically been studied separately. Most of the commonly used biophysical approaches are limited in providing substantial details regarding the combination of both structure and dynamics. On the other hand, high-speed atomic force microscopy (HS-AFM), which simultaneously visualizes an individual protein structure and its dynamics in liquid in real time, can uniquely link the structure and the kinetic details, and it can also unveil novel insights. Although amyloidogenic proteins generate heterogeneously aggregated species, including transient unstable states during the aggregation process, HS-AFM elucidated the structural dynamics of individual aggregates in real time in liquid without purification and isolation. Here, we review and discuss the HS-AFM imaging of amyloid aggregation and strategies to optimize the experiments showing findings from Aβ and amylin, which is associated with type II diabetes, shares some common biological features with Aβ, and is reported to be involved in AD.

Bacterial collagenases involved in donor infection are widely applied in many fields due to their high activity and specificity; however, little is known regarding the mechanisms by which bacterial collagenases degrade insoluble collagen in host tissues. Using high-speed atomic force microscopy, we simultaneously visualized the hierarchical structure of collagen fibrils and the movement of a representative bacterial collagenase, Clostridium histolyticum type I collagenase (ColG), to determine the relationship between collagen structure and collagenase movement. Notably, ColG moved ~14.5 nm toward the collagen N terminus in ~3.8 s in a manner dependent on a catalytic zinc ion. While ColG was engaged, collagen molecules were not only degraded but also occasionally rearranged to thicken neighboring collagen fibrils. Importantly, we found a similarity of relationship between the enzyme-substrate interface structure and enzyme migration in collagen-collagenase and DNA-nuclease systems, which share a helical substrate structure, suggesting a common strategy in enzyme evolution.

Alzheimer's disease (AD) is the most prevalent age‐related neurodegenerative disorder, mainly characterized by amyloid β (Aβ) accumulation in the brain. Numerous new agents are currently undergoing clinical trials as disease‐modifying therapies (DMTs) targeting Aβ. ALZ‐801 is a promising candidate DMT for AD, with a phase 3 trial of ALZ‐801 ongoing specifically for apolipoprotein E (APOE) ε4 homozygous patients with early‐stage AD. This study aimed to examine the effects of ALZ‐801 on Aβ assembly and explore its toxicological profile. Thioflavin T (ThT) assays and two imaging modalities—transmission electron microscopy (TEM) and high‐speed atomic force microscopy (HS‐AFM)—were used to evaluate ALZ‐801's effects on Aβ assembly. To assess the effect of ALZ‐801 on Aβ ₄₂ ‐induced cytotoxicity, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assays and lactate dehydrogenase (LDH) assays were performed. ThT assays revealed increased lag time and decreased fluorescence in the presence of ALZ‐801, confirming inhibition of Aβ ₄₂ fibril formation, as confirmed by TEM. Real‐time observation using HS‐AFM revealed that ALZ‐801 inhibited the formation of Aβ ₄₂ fibril from low‐molecular‐weight (LMW)‐Aβ ₄₂ in the presence of Aβ ₄₂ seeds. HS‐AFM also revealed that globular aggregates from LMW‐Aβ ₄₂ were significantly larger with ALZ‐801, with few fibrils noted. MTT and LDH assays indicated that ALZ‐801 prevented LMW‐Aβ ₄₂ ‐induced cytotoxicity but did not reduce cytotoxicity induced by high‐molecular‐weight‐Aβ ₄₂ . ALZ‐801 can inhibit Aβ ₄₂ aggregation by preventing both nucleus formation and fibril elongation, while promoting large globular oligomer formation, and can significantly reduce LMW‐Aβ ₄₂ ‐induced cytotoxicity. These findings underscore the potential of ALZ‐801 as an effective DMT for APOE ε4 homozygous patients with AD.