Amyloid crystals, a form of ordered protein aggregates documented relatively recently, have not been studied as extensively as amyloid fibres. This study investigates the formation of amyloid crystals with low frequency ultrasound (20 kHz) using β-lactoglobulin, as a model protein for amyloid synthesis. Acoustic cavitation generates localised zones of intense shear, with extreme heat and pressure that could potentially drive the formation of amyloid structures at ambient bulk fluid temperatures (20 ± 1 °C). Thioflavin T fluorescence and electron microscopy showed that low-frequency ultrasound at 20 W/cm3 input power induced β-stacking to produce amyloid crystals in the mesoscopic size range, with a mean length of approximately 22 µm. FTIR spectroscopy indicated a shift towards increased intermolecular antiparallel β-sheet content. An increase in sonication time (0–60 min) and input power (4–24 W/cm3) increased the mean crystal length, but this increase was not linearly proportional to sonication time and input power due to the delayed onset of crystal growth. We propose that acoustic cavitation causes protein unfolding and aggregation and imparts energy to aggregates to cross the torsion barrier, to achieve their lowest energy state as amyloid crystals. The study contributes to a further understanding of protein chemistry relating to the energy landscape of folding and aggregation. Ultrasound presents opportunities for practical applications of amyloid structures, presenting a more adaptable and scalable approach for synthesis.
Graphical abstract
This study investigated the effect of low-frequency (20kHz) and high-frequency (414kHz) ultrasound treatment on the amino acid and secondary structural integrity of dairy proteins. Sonicated skim milk proteins were hydrolysed and analysed with reverse-phase high-performance liquid chromatography to investigate the amino acid content of the processed samples. It was successfully demonstrated that both low-frequency and high-frequency ultrasound did not adversely affect the amino acid content, even after prolonged extreme processing conditions (6h, 355kHz). This finding was supplemented with protein secondary structure data (Fourier-transform (FT)-IR secondary derivatives of the amide I band, 1700–1600cm−1) that showed that ultrasound was capable of causing structural modifications to the dairy proteins. This study shows that ultrasound can be used to influence protein–protein interactions in skim milk via alterations to the secondary structure without degrading the amino acids in the proteins.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.