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

Protein fibrillation, while associated with amyloidogenic diseases, also holds significant promise in nanobiotechnology due to the unique mechanical properties and biocompatibility of the resulting fibrils. However, current methods are limited by scalability issues, the requirement for expensive purified proteins, and slow fibrillation kinetics. These limitations hinder widespread application in large-scale production of functional biomaterials. Biological fibril formation is a complex process influenced by various factors such as stress, mutations, and aging. These factors lead to protein misfolding and the subsequent formation of aggregates, which eventually transform into stable amyloid fibrils through various intermolecular interactions. The rate and efficiency of fibrillation are dependent on the protein's properties (hydrophobic/hydrophilic nature, secondary structure) and can be influenced by fibrillation agents. Two primary mechanisms are proposed for fibril formation: nucleation of misfolded monomers (with a lag phase) and formation of misfolded oligomers as seeds (without a lag phase). Ionic liquids (ILs), owing to their tunable properties, have shown potential as fibrillation agents. However, previous studies have typically utilized pure proteins and resulted in lengthy fibrillation processes at the laboratory scale. Furthermore, the influence of molecular crowding, a critical factor in the in vivo environment where proteins exist in a crowded mixture of molecules, has been relatively understudied in relation to protein fibrillation. This study leverages the principles of biological cooperativity and molecular crowding by using egg white (EW) as a low-cost source of a complex protein mixture. Different ILs are screened for their ability to induce fibrillation, with cholinium tosylate ([Cho][Tos]) demonstrating exceptional efficiency in causing instantaneous fibrillation at room temperature. The research aims to establish a scalable and cost-effective method for producing protein fibrils with enhanced properties for various applications.

The literature extensively documents the dual nature of protein fibrillation, linking it to both pathological conditions such as Alzheimer's and Parkinson's diseases and promising applications in nanotechnology. Previous studies have investigated in vitro fibrillation using various techniques, focusing on modifying the environment (pH, temperature, osmolytes) and using purified proteins or synthesized peptides. The use of ionic liquids (ILs) as fibrillation agents has shown some promise, but these studies often involve pure proteins, leading to slow kinetics and low scalability. The effect of macromolecular crowding, a key factor in the cellular environment, is also not fully understood in the context of fibrillation. This research builds upon these studies by incorporating the effects of molecular crowding using a natural protein mixture (egg white) and a novel ionic liquid.

The researchers used chicken egg white (EW) as a low-cost source of proteins. After removing insoluble lipids, the aqueous solution containing the egg white proteome (EWP) was treated with HCl to denature the proteins, promoting fibrillation. Several ionic liquids (ILs), including cholinium methylsulfonate, cholinium chloride, benzylcholinium chloride, and cholinium tosylate ([Cho][Tos]), were tested. Only [Cho][Tos] induced immediate fibrillation at both low (0.1 M) and high (1 M) concentrations. The fibrillation process was monitored by several techniques: * **Microscopy:** Optical, atomic force (AFM), and transmission electron (TEM) microscopy were used to visualize the fibrils' morphology. * **Spectroscopy:** UV-Vis, Fourier transform infrared (FTIR), and circular dichroism (CD) spectroscopies were employed to characterize the fibrils' structure and to monitor secondary structural changes. Thioflavin T (ThT) fluorescence spectroscopy was used to monitor the kinetics of fibrillation. * **Molecular Docking:** Computational molecular docking simulations were performed to investigate the interactions between the IL ions and various proteins present in the EWP (ovalbumin, ovotransferrin, lysozyme, ovomucoid, flavoprotein) and protein-protein interactions. * **Mechanical Properties:** Thermogravimetric analysis (TGA) determined the thermal stability, while dynamic mechanical analysis (DMA) measured the mechanical stiffness and loss modulus of the fibrils. * **Cytocompatibility:** The cytocompatibility of the fibrils was assessed using the mouse fibroblast L929 cell line through Live/Dead assays, metabolic activity (alamarBlue) and morphological studies. * **Enzyme Immobilization:** Cytochrome c (Cyt c) was immobilized on the fibrils, and its activity was compared to Cyt c in solution. Detailed procedures are described in the Supplementary Information.

The study's key findings include: 1. **Instantaneous Fibrillation:** Cholinium tosylate ([Cho][Tos]) induced instantaneous (within seconds) fibrillation of the EWP at room temperature, unlike other tested ILs. The absence of a lag phase in the ThT fluorescence kinetics supports the formation of misfolded oligomers as seeds, leading to rapid fibril formation. 2. **Structural Characterization:** Microscopy revealed the formation of highly branched fibril networks, with AFM showing entangled fibrils and oligomers. TEM showed thick bundles of fibrils. 3. **Secondary Structural Changes:** CD and FTIR spectroscopy confirmed the transition from an all-α secondary structure to an antiparallel β-sheet or cross-β structure upon fibrillation. This transition occurred rapidly (within 20 min). 4. **Protein Involvement:** SDS-PAGE and UV-vis spectroscopy indicated the involvement of abundant egg white proteins (ovomucin, ovotransferrin, ovalbumin, avidin, flavoprotein) in fibrillation. 5. **Molecular Docking:** Molecular docking studies showed that hydrogen bonding, hydrophobic interactions, and π-stacking interactions between [Cho][Tos] and the amino acids of the proteins are the driving forces behind fibrillation. The [Tos]⁻ anion, with its aromatic ring, played a significant role in promoting these interactions. 6. **Role of Molecular Crowding:** Studies with individual proteins (ovalbumin and lysozyme) and their mixtures demonstrated the importance of molecular crowding in accelerating fibrillation. Cross-seeding of misfolded proteins likely contributes to the rapid fibrillation in the EWP. 7. **Enhanced Properties:** The obtained fibrils exhibited high thermal stability (glass transition temperature around 95 °C) and significantly enhanced mechanical stiffness (E' ≈ 42 GPa), exceeding values reported for fibrils from single proteins. 8. **Cytocompatibility:** Live/Dead assays, metabolic activity, and morphological analysis showed excellent cytocompatibility of the fibrils with L929 cells, even at concentrations up to 25%. 9. **Enzyme Immobilization:** The fibrils successfully immobilized cytochrome c (Cyt c), leading to a 2.5-fold increase in its activity compared to Cyt c in solution, demonstrating their potential as enzyme supports.

This study successfully demonstrates a novel and highly efficient method for producing protein fibrils. The use of the ionic liquid [Cho][Tos] and the inherent molecular crowding of egg white overcomes the limitations of existing methods, resulting in instantaneous fibrillation at room temperature. The findings highlight the synergistic effect of the IL and molecular crowding, where the [Tos]⁻ anion facilitates crucial interactions (H-bonding, hydrophobicity, π-stacking) while crowding enhances the efficiency of fibril formation, likely through cross-seeding. The resulting fibrils' enhanced mechanical properties and cytocompatibility, combined with their ability to enhance enzyme activity, suggest their potential for diverse biotechnological applications. The low cost and scalability of this approach open doors for large-scale production of functional biomaterials.

This research presents a highly efficient and cost-effective method for producing protein fibrils using egg white as a starting material and cholinium tosylate as a fibrillation agent. The process results in fibrils with superior mechanical properties and cytocompatibility, showing great potential as enzyme supports and other applications. Future research could focus on optimizing the process for even larger-scale production and exploring a wider range of applications in biomedicine and nanotechnology.

The study's limitations include the focus on a single type of egg white and a limited exploration of other ILs. While the cytocompatibility is demonstrated, more extensive in vivo studies are needed. The mechanistic understanding of cross-seeding in the complex EWP mixture could be further refined through more sophisticated analysis.