Instantaneous fibrillation of egg white proteome with ionic liquid and macromolecular crowding

The study addresses the challenge of producing protein amyloid-like fibrils at scale, rapidly, and from low-cost sources. Fibrils are relevant both to amyloidogenic diseases and as functional biomaterials due to their mechanical strength and bio-based origin, yet conventional production requires purified proteins, prolonged incubation, and is not easily scalable. The authors hypothesize that mimicking biological macromolecular crowding and leveraging an ionic liquid (IL) capable of multiple interactions (electrostatic, hydrogen bonding, hydrophobic, and π–π) can induce instantaneous fibrillation directly from a complex proteome (egg white) at room temperature. The aims are to (i) screen ILs for fibrillation efficacy, (ii) monitor kinetics and structural transitions, (iii) identify participating proteins and clarify mechanisms via spectroscopy and molecular docking, (iv) assess thermal, mechanical, colloidal, and cytocompatibility properties, and (v) demonstrate utility as enzyme supports.

Protein fibrillation is driven by misfolding and aggregation mechanisms influenced by environmental stressors and leads to cross-β-rich amyloid structures. Two kinetic mechanisms are reported: monomer misfolding with a lag phase and oligomer seeding without a lag phase. Prior in vitro studies modulated pH, heat, and osmotic conditions, noting roles for hydrophobicity, secondary-structure changes, and specific interactions. ILs have been explored: some promote fibrillation (e.g., protic ILs for lysozyme) with long timescales, others inhibit (e.g., carboxylate-containing ILs). Common limitations include use of pure proteins, long processing times, and small batch scale. Macromolecular crowding (MC), ubiquitous in vivo, affects folding, binding, and aggregation, but prior MC-fibrillation studies focused on single proteins at high concentrations or polymer crowders. The present work integrates IL design (aromatic ions for π-stacking) with biological MC by using egg white proteome as a crowded, heterogeneous protein pool.

• Materials and source: Fresh chicken egg white (EW) diluted 1:10 (v/v) in water, stirred, and filtered to remove insoluble lipids; initial pH 8.82. EW proteome (EWP) was acid-denatured to pH 2.0 with 1 M HCl to promote unfolded intermediates.
• Ionic liquid screening: Cholinium methylsulfonate ([Cho][CH3SO3]), cholinium chloride ([Cho]Cl), benzylcholinium chloride ([BzCho]Cl), and cholinium tosylate ([Cho][Tos]) tested at 0.1 M and 1 M added to acidified EWP. Additional ion-pair controls: 1 M Na[Tos], 1 M [Cho]Cl, equimolar [Cho]Cl + Na[Tos], and [Cho][Tos].
• Fibrillation assessment and morphology: Visual turbidity; optical microscopy; AFM (semi-contact mode) on mica; TEM (carbon-coated grids). Time evolution imaged.
• Kinetics and structure: Thioflavin T (ThT, 30 µM) fluorescence monitored up to 7.1 h (λex 412 nm, λem 482 nm). Circular dichroism (CD) in far-UV (180–260 nm) to monitor secondary structure transitions over time (5, 10, 20 min; mature fibril). FT-IR (amide I, 1600–1700 cm−1) with deconvolution to quantify secondary-structure components before and after fibrillation.
• Protein identification: SDS-PAGE of supernatants and UV–vis spectral features to identify proteins participating in fibrillation.
• Molecular modeling: Molecular docking of IL ions ([Tos]−, [Cho]+, [BzCho]+, [CH3SO3]−, Cl−) with EWP proteins (ovalbumin, ovotransferrin, flavoprotein, ovomucoid, lysozyme) to compute interaction energies and interaction types; PASTA 2.0 to estimate aggregation propensity and secondary-structure changes (self- and co-aggregation free energies; β-strand content).
• Crowding and cross-seeding tests: Fibrillation of individual proteins (ovalbumin, lysozyme) and their mixture at pH 2.0 with [Cho][Tos]; CD and ThT kinetics compared to EWP to assess MC and cross-seeding contributions.
• Thermal/mechanical/colloidal characterization: TGA (25–800 °C under N2), DMA (E′, E″, tanδ, 20–200 °C, 1 and 10 Hz), zeta potential of fibril dispersions across pH 2–12.
• Cytocompatibility: L929 fibroblasts exposed to 5–25% (v/v) fibrils for 24 h; Live/Dead (Calcein-AM/PI), morphology (F-actin phalloidin/DAPI), and alamarBlue metabolic activity.
• Enzyme immobilization: Cytochrome c (Cyt c) immobilized on fibrils; AFM to visualize attachment; UV–vis to monitor redox state; peroxidase-like activity with ABTS/H2O2; report loading (µg Cyt c per mg fibrils) and activity units compared to aqueous control.
• Cost estimate: Yield and cost per gram from a single egg (Supplementary Note 3).

• Instantaneous fibrillation: Only [Cho][Tos] induced immediate (seconds) fibrillation of the acidified EWP at both 0.1 M and 1 M; other ILs did not. Na[Tos] alone also induced instant fibrillation; [Cho]Cl did not, and [Cho]Cl + Na[Tos] required higher salt amounts, indicating tosylate’s dominant role with a balancing contribution of cholinium.
• Morphology: Optical and AFM showed highly branched, entangled fibril networks that thicken with time and IL concentration. TEM revealed 1D nanorod bundles with aspect ratio ~7–10.
• Kinetics (ThT): No lag phase observed, consistent with rapid oligomer formation and growth; ThT emission at 482 nm increased over time. EWP reached stationary phase faster with lower ThT intensity than single-protein systems, consistent with heterogeneous packing.
• Secondary-structure transition: CD showed fast transition from all-α to antiparallel β-sheet/cross-β within 20 min. FT-IR amide I deconvolution indicated growth of cross-β band shifting from 1626 to 1620 cm−1 with increased area; α-helix band shifted 1650→1647 cm−1, confirming cross-β formation.
• Participating proteins: SDS-PAGE and UV–vis indicated involvement of major EW proteins—ovomucin, ovotransferrin, ovalbumin, avidin, and flavoprotein. UV–vis showed disappearance of flavoprotein (365 nm) and ovotransferrin (450 nm) bands and pentafurcation/blue-shift of the 280 nm aromatic band, evidencing π-stacking/hydrophobic interactions with aromatic residues (Trp, Tyr, Phe).
• Molecular interactions: Docking interaction energy trend across proteins: [Tos]− > [BzCho]+ > [Cho]+ > [CH3SO3]− > Cl−. Strongest anion-protein interaction was [Tos]− with ovotransferrin (−4.8 kcal/mol). Interaction types involved hydrogen bonding and hydrophobic, plus π–π interactions (e.g., Phe645 of ovotransferrin). PASTA predicted increased β-strands upon fibrillation; ovalbumin showed highest self-aggregation free energy (−11.07 kcal/mol), and ovotransferrin–flavoprotein highest co-aggregation (−7.64 kcal/mol).
• Crowding and cross-seeding: Ovalbumin and ovalbumin/lysozyme mixtures formed cross-β structures upon redispersion; pure lysozyme formed a reversible charged complex at pH 2 and did not yield stable fibrils alone. Data support crowding-assisted cross-seeding in the proteome.
• Thermal/mechanical: Fibrils exhibited glass transition near 95 °C and thermal degradation above 307 °C. High stiffness with storage modulus E′ ≈ 42 GPa at 25 °C, exceeding many single-protein fibrils and comparable to Aβ40 ranges.
• Colloidal charge tunability: Zeta potential ranged from +31 mV (pH 2) to −21 mV (pH 10), indicating good colloidal stability and tunable charge.
• Cytocompatibility: L929 cells showed preserved membrane integrity and morphology up to 25% fibrils; ~100% viability at 5–25%, with only ~6% reduction at 25%.
• Enzyme immobilization: Cyt c loading ~25.7 µg per 14 mg fibrils; immobilization reduced Cyt c (Fe3+→Fe2+) and enhanced peroxidase activity to 2.4537 U µmol E−1 vs 0.976 in water (≈2.5-fold increase).
• Cost: Approx. 2 g fibrils produced per egg at <5 €, i.e., ~2.5 € per g at lab scale.

The findings demonstrate that an IL with an aromatic anion (tosylate) can harness hydrogen-bonding, hydrophobic, and π–π interactions to bypass the nucleation lag phase and trigger instantaneous fibrillation in a crowded proteome at room temperature and low cost. Macromolecular crowding in egg white promotes protein–protein interactions and cross-seeding, enabling proteins that do not fibrillate alone (e.g., lysozyme) to integrate into mixed fibrils. Spectroscopy and docking corroborate the central role of [Tos]−, with ovotransferrin and other β-rich proteins acting as strong interaction partners that seed propagation. The resulting fibrils show superior mechanical stiffness and thermal stability, are cytocompatible, and function as effective enzyme supports that can enhance catalytic activity (Cyt c case). Control experiments with Na[Tos] and [Cho]Cl clarify ion-specific effects, with cholinium moderating interaction strength to yield thinner fibrils than Na[Tos] alone. Overall, the approach addresses the scalability, speed, and cost barriers in fibril production and expands opportunities for biomaterial applications.

This work introduces a rapid, room-temperature, and cost-effective method to produce protein fibrils directly from a complex, inexpensive matrix (egg white) by exploiting macromolecular crowding and an ionic liquid (cholinium tosylate) that provides hydrogen-bonding, hydrophobic, and π–π interactions. Mechanistic insights from spectroscopy and molecular docking reveal tosylate-dominated interactions and crowding-enabled cross-seeding. The fibrils exhibit high stiffness, thermal stability, colloidal tunability, cytocompatibility, and serve as robust supports that enhance cytochrome c activity. The process yields ~2 g fibrils per egg at ~2.5 € per g (lab scale), suggesting strong potential for scale-up. Future work should optimize process scale-up, explore other proteomes and IL chemistries, validate broader biocatalytic and biomedical uses, and further elucidate cross-seeding pathways and fibril composition.

• The study demonstrates applications with a single enzyme (cytochrome c); broader enzyme and process generality remains to be shown.
• Cross-seeding in the full proteome is suggested but not unequivocally proven for all components; ThT/CD provide indirect evidence.
• The process relies on acidic conditions (pH 2.0), which may limit compatibility with certain biomolecules prior to neutralization.
• Results are at lab scale; although cost and yield estimates are provided, practical scale-up, continuous processing, and downstream purification were not demonstrated.
• IL specificity is high; [Cho][Tos] (or Na[Tos]) is effective, whereas other tested ILs are not, potentially limiting flexibility without further IL design.