This study investigates the structure and biological activity of amyloid beta-protein (Aβ) protofibrils, which are intermediate structures in the fibrillogenesis process of Aβ. The research team, led by Dominic M. Walsh and colleagues, discovered that protofibrils are in equilibrium with low molecular weight Aβ (LMW Aβ), have a secondary structure characteristic of amyloid fibrils, and can form mature amyloid-like fibrils. They also found that protofibrils affect the normal metabolism of cultured neurons. The study used size exclusion chromatography (SEC) to prepare homogeneous populations of Aβ, and electron microscopy to examine the morphology of protofibrils. The results showed that protofibrils are short, flexible fibrils with a diameter of 4–10 nm and length up to 200 nm. They are transient during Aβ fibrillogenesis and are precursors of longer, more rigid amyloid-type fibrils. The study also found that protofibrils bind dyes such as Congo red and thioflavin T, indicating the presence of β-sheet structure. Circular dichroism spectroscopy revealed that protofibrils contain significant β-sheet structure, along with some random coil and α-helix. The biological activity of protofibrils was assessed using the MTT assay, which showed that protofibrils significantly reduced MTT reduction in cultured neurons, indicating their toxicity. The study also found that protofibrils are precursors of fibrils and that fibrils do not readily dissociate into protofibrils or LMW Aβ. The findings suggest that protofibrils are important intermediates in the fibrillogenesis process and that targeting protofibrils may be a promising therapeutic strategy for Alzheimer's disease. The study highlights the importance of understanding the structure and function of protofibrils in the development of therapies for Alzheimer's disease.This study investigates the structure and biological activity of amyloid beta-protein (Aβ) protofibrils, which are intermediate structures in the fibrillogenesis process of Aβ. The research team, led by Dominic M. Walsh and colleagues, discovered that protofibrils are in equilibrium with low molecular weight Aβ (LMW Aβ), have a secondary structure characteristic of amyloid fibrils, and can form mature amyloid-like fibrils. They also found that protofibrils affect the normal metabolism of cultured neurons. The study used size exclusion chromatography (SEC) to prepare homogeneous populations of Aβ, and electron microscopy to examine the morphology of protofibrils. The results showed that protofibrils are short, flexible fibrils with a diameter of 4–10 nm and length up to 200 nm. They are transient during Aβ fibrillogenesis and are precursors of longer, more rigid amyloid-type fibrils. The study also found that protofibrils bind dyes such as Congo red and thioflavin T, indicating the presence of β-sheet structure. Circular dichroism spectroscopy revealed that protofibrils contain significant β-sheet structure, along with some random coil and α-helix. The biological activity of protofibrils was assessed using the MTT assay, which showed that protofibrils significantly reduced MTT reduction in cultured neurons, indicating their toxicity. The study also found that protofibrils are precursors of fibrils and that fibrils do not readily dissociate into protofibrils or LMW Aβ. The findings suggest that protofibrils are important intermediates in the fibrillogenesis process and that targeting protofibrils may be a promising therapeutic strategy for Alzheimer's disease. The study highlights the importance of understanding the structure and function of protofibrils in the development of therapies for Alzheimer's disease.