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Amyloid fibrils have immense potential to become the basis of modern biomaterials. The formation of amyloid fibrils in vitro strongly depends on the solvent properties. Ionic liquids (ILs), alternative solvents with tunable properties, have been shown to modulate amyloid fibrillization. In this work, we studied the impact of five ILs with 1-ethyl-3-methylimidazolium cation [EMIM+] and anions of Hofmeisterseries hydrogen sulfate [HSO4−], acetate [AC−], chloride [Cl−], nitrate [NO3−], and tetrafluoroborate [BF4−] on the kinetics of insulin fibrillization and morphology, and the structure of insulin fibrils when applying fluorescence spectroscopy, AFM and ATR-FTIR spectroscopy. We found that the studied ILs were able to speed up the fibrillization process in an anion- and IL-concentration-dependent manner. At an IL concentration of 100 mM, the efficiency of the anions at promoting insulin amyloid fibrillization followed the reverse Hofmeister series, indicating the direct binding of ions with the protein surface. At a concentration of 25 mM, fibrils with different morphologies were formed, yet with similar secondary structure content. Moreover, no correlation with the Hofmeister ranking was detected for kinetics parameters. IL with the kosmotropic strongly hydrated [HSO4−] anion induced the formation of large amyloid fibril clusters, while the other kosmotropic anion [AC−] along with [Cl−] led to the formation of fibrils with similar needle-like morphologies to those formed in the IL-free solvent. The presence of the ILs with the chaotropic anions [NO3−] and [BF4−] resulted in longer laterally associated fibrils. The effect of the selected ILs was driven by a sensitive balance and interplay between specific protein–ion and ion–water interactions and non-specific long-range electrostatic shielding. [ABSTRACT FROM AUTHOR]

Amyloid fibrils have immense potential to become the basis of modern biomaterials. The formation of amyloid fibrils in vitro strongly depends on the solvent properties. Ionic liquids (ILs), alternative solvents with tunable properties, have been shown to modulate amyloid fibrillization. In this work, we studied the impact of five ILs with 1-ethyl-3-methylimidazolium cation [EMIM+] and anions of Hofmeisterseries hydrogen sulfate [HSO4−], acetate [AC−], chloride [Cl−], nitrate [NO3−], and tetrafluoroborate [BF4−] on the kinetics of insulin fibrillization and morphology, and the structure of insulin fibrils when applying fluorescence spectroscopy, AFM and ATR-FTIR spectroscopy. We found that the studied ILs were able to speed up the fibrillization process in an anion- and IL-concentration-dependent manner. At an IL concentration of 100 mM, the efficiency of the anions at promoting insulin amyloid fibrillization followed the reverse Hofmeister series, indicating the direct binding of ions with the protein surface. At a concentration of 25 mM, fibrils with different morphologies were formed, yet with similar secondary structure content. Moreover, no correlation with the Hofmeister ranking was detected for kinetics parameters. IL with the kosmotropic strongly hydrated [HSO4−] anion induced the formation of large amyloid fibril clusters, while the other kosmotropic anion [AC−] along with [Cl−] led to the formation of fibrils with similar needle-like morphologies to those formed in the IL-free solvent. The presence of the ILs with the chaotropic anions [NO3−] and [BF4−] resulted in longer laterally associated fibrils. The effect of the selected ILs was driven by a sensitive balance and interplay between specific protein–ion and ion–water interactions and non-specific long-range electrostatic shielding.

Plant proteins self-assembly into amyloid fibrils is a promising modification to be introduced in emerging food and materials applications. Although the valorization of protein-rich side streams in amaranth oil production is completely missing, we show that double salt extraction provides the recovery of globulin fraction of amaranth seed from food waste with high purity and complete protein solubility at acidic pH. We demonstrate in silico the self-assembly propensity with a high density of amyloidogenic regions of the 11S seed storage globulin of Amaranthus hypochondriacus. We further show that the recovered amaranth proteins can self-assemble into amyloid fibrils under heat-induced acidic hydrolysis. The amyloid nature of the fibrils was studied by thiazole orange (TO) fluorescence, SDS-PAGE, FTIR, CD, TEM, and AFM. We systematically investigated how the temperature of fibrillization affected protein unfolding, peptide release, as well as its effect on tuning the formation, growth, and polymorphism of amaranth amyloid fibrils. The β-sheet-rich structured proteins produced from the amaranth food waste exhibited mesoscopic polymorphism of the rigid amyloid fibrils with the manifestation of left-handed ribbons of varying polymorphism. We show that favourable conditions for extensive hydrolysis of the amaranth proteins promote the growth of well-ordered thick twisted ribbons with small-pitch and extraordinary rigidity and we interpret these finding based on structural mesoscopic models of amyloid fibrils. These results provide additional insight into the formation of plant amyloid fibrils as functional food and biocompatible materials, and introduce a pathway to convert amaranth food protein waste into amyloid nanofibrils. [Display omitted] • Amarantin-rich protein isolate (92%) is recovered from waste by double salt extraction. • Acidic conditions (pH ≤ 4) induce total solubility of the recovered amaranth proteins. • Amyloid-forming capacity of the 11S seed storage globulin is based on the high density of amylogenic regions. • Temperature affects the kinetics and polymorphism of amaranth fibril self-assembly. • Amaranth twisted ribbons combine high rigidity and a small pitch size. [ABSTRACT FROM AUTHOR]