Fibrillation Kinetics Research Articles

Different charged biopolymers induce α-synuclein to form fibrils with distinct structures

The aggregation of α-synuclein (α-syn) into amyloid fibrils, a key process in the development of Parkinson's disease (PD) and other synucleinopathies, is influenced by a range of factors such as charged biopolymers, chaperones, and metabolites. However, the specific impacts of different biopolymers on α-syn fibril structure are not well understood. In our work, we found that different polyanions and polycations, such as polyphosphate (polyP), polyuridine (polyU), and polyamines (including putrescine, spermidine, and spermine), markedly altered the fibrillation kinetics of α-syn in vitro. Furthermore, seeding assay revealed distinct cross-seeding capacities across different biopolymer-induced α-syn fibrils, suggesting the formation of structurally distinct strains under different conditions. Utilizing cryo-electron microscopy (cryo-EM), we further examined the detailed structural configuration of α-syn fibrils formed in the presence of these biopolymers. Notably, we found that while polyamines do not change the atomic structure of α-syn fibrils, polyU and polyP induce the formation of distinct amyloid fibrils, exhibiting a range of structural polymorphs. Our work offers valuable insights into how various charged biopolymers affect the aggregation process and the resultant structures of α-syn fibrils, thereby enhancing our understanding of the structural variations in α-syn fibrils across different pathological conditions.

E22G Aβ40 fibril structure and kinetics illuminate how Aβ40 rather than Aβ42 triggers familial Alzheimer’s

Arctic (E22G) mutation in amyloid-β (Aβ enhances Aβ40 fibril accumulation in Alzheimer’s disease (AD). Unlike sporadic AD, familial AD (FAD) patients with the mutation exhibit more Aβ40 in the plaque core. However, structural details of E22G Aβ40 fibrils remain elusive, hindering therapeutic progress. Here, we determine a distinctive W-shaped parallel β-sheet structure through co-analysis by cryo-electron microscopy (cryoEM) and solid-state nuclear magnetic resonance (SSNMR) of in-vitro-prepared E22G Aβ40 fibrils. The E22G Aβ40 fibrils displays typical amyloid features in cotton-wool plaques in the FAD, such as low thioflavin-T fluorescence and a less compact unbundled morphology. Furthermore, kinetic and MD studies reveal previously unidentified in-vitro evidence that E22G Aβ40, rather than Aβ42, may trigger Aβ misfolding in the FAD, and prompt subsequent misfolding of wild-type (WT) Aβ40/Aβ42 via cross-seeding. The results provide insight into how the Arctic mutation promotes AD via Aβ40 accumulation and cross-propagation.

Monitoring insulin fibrillation kinetics using chromatographic analysis

Insulin is a small protein widely used to treat patients with diabetes and is a commonly used model for protein fibrillation studies. Under specific conditions, such as low pH and high temperature, insulin monomers aggregate to form fibrils. This aggregation is problematic for manufacturing and storage of insulin. The thioflavin T (ThT) assay is commonly used to study amyloid fibrillation but suffers from several limitations, such as the effect of protein concentration, the size of the amyloid fibrillar bundles, competitive binding, and fibril aggregation, all of which hinder precise quantitative analysis. Here, we present a method for studying the kinetics of insulin fibrillation utilizing ultra-performance liquid chromatography (UPLC). This method enables the quantitative detection of soluble insulin components, including chemically modified components. The formation of a deamidated species could be monitored at the early stage of fibrillation, and this species was likely included in the fibrils. In addition, in the presence of inhibitors known to compete with ThT for binding to fibrils, UPLC analysis showed the disappearance of soluble components even though the ThT assay did not indicate the presence of fibrils. These results suggest that the UPLC-based analysis presented here can complement the ThT assay for investigating the kinetics of protein fibrillation.

Lutein, a carotenoid found in numerous plants and the human eye, demonstrates the capacity to bundle collagen fibrils

Collagen fibrils serve as the building blocks of the extracellular matrix, providing a resilient and structural framework for tissues. However, the bundling of collagen fibrils is of paramount importance in maintaining the structural integrity and functionality of various tissues in the human body. In this scenario, there is limited exploration of molecules that promote the bundling of collagen fibrils. Investigating the interactions of well-known carotenoids, commonly associated with ocular health, particularly in the retina, with collagen presents a novel and significant area of study. Here, we studied the influence of lutein, a well-known carotenoid present in many plant tissues and has several biological properties, on the structure, thermal stability, self-assembly, and fibrillation of collagen. Fibrillation kinetics and electron microscopic analyses indicated that lutein did not interfere with fibrillation process of collagen, whereas it enhances the lateral fusion of collagen fibrils leading to the formation of compact bundles of thick fibrils under physiological conditions. The hydrophobic and hydrogen bonding interactions between lutein and collagen fibrils are most likely the cause of the bundling of the fibrils. This study establishes the first investigation of collagen-carotenoid interactions, showcasing the unique property of lutein in bundling collagen fibrils, which may find potential application in tissue engineering.

Inhibition of α-Synuclein fibrillation by curcumin and difluoro boron derivatized curcumin complexes in aqueous environment

Fibrillation of α-Synuclein (α-Syn) is a key pathogenic event in Parkinson’s disease (PD). In the present work, we have investigated the effects of Curcumin (Cur) and its derivatives; curcumin-BF2 (Cur-BF2) and iodinated-curcumin-BF2 (I-Cur-BF2) on the inhibition of α-Syn fibrillation. The ITC results showed that all three curcumins bind via a combination of both H-bonding and hydrophobic interactions. The fibrillation kinetics studies demonstrate that Cur and its derivatives inhibit its fibrillation and increase α-Syn solubility. I-Cur-BF2 is the most efficient in fibrillation inhibition followed by Cur-BF2 and Cur, attributed to the ability to increase the hydrophobic interactions. Transmission electron microscopy (TEM) confirmed the presence of thinner, lesser, and smaller α-Syn fibrils in the presence of the Cur and its derivatives. The fibrillar aggregates formed in the presence of Cur and its derivatives are unable to induce fibrillation in healthy monomeric α-Syn. Cur and its derivatives also have the ability to disaggregate elongating and preformed fibrils and sequester them in globular condensates which prevent further fibrillation in α-Syn. Together, our findings provide unique information about the mechanism of binding of Cur and its derivatives with α-Syn and demonstrate that curcumin compounds regulate α-Syn amyloid formation in the aqueous environment.

Effects of molecular weight of hydrolysate on the formation of soy protein isolate hydrolysate nanofibrils: Kinetics, structures, and interactions

Enzymatic hydrolysis prior to protein fibrillation was an effective way to facilitate the formation of nanofibrils. This study aimed to investigate the effects of molecular weights of hydrolysate on the kinetics, structures, and interactions of soy protein isolate (SPI) hydrolysate nanofibrils. The results showed that hydrolysate with molecular weight > 10 kDa showed a distinct fibrillation kinetics curve and a higher apparent rate constant (27.72) during fibrillation, indicating their vital role in determining the fibrillation. Hydrolysate with molecular weight > 10 kDa could form nanofibrils with higher radius gyration (17.11 ± 0.77 Å) due to stronger hydrophobic interaction, showing a stronger fibrillation ability. Hydrolysate with molecular weight within 5–10 kDa exhibited enhanced π-π stacking interactions during fibrillation, thereby promoting the extension of nanofibrils, and contributing to the formation of more nanofibrils. Hydrolysate with molecular weight < 5 kDa tended to randomly aggregate during fibrillation, resulting in a significant loss of cross-β structures in nanofibrils. Therefore, hydrolysate with different molecular weights exhibited synergistic effects during fibrillation.

Mechanistic Insights Behind the Self-Assembly of Human Insulin under the Influence of Surface-Engineered Gold Nanoparticles.

Elucidating the underlying principles of amyloid protein self-assembly at nanobio interfaces is extremely challenging due to the diversity in physicochemical properties of nanomaterials and their physical interactions with biological systems. It is, therefore, important to develop nanoscale materials with dynamic features and heterogeneities. In this work, through engineering of hierarchical polyethylene glycol (PEG) structures on gold nanoparticle (GNP) surfaces, tailored nanomaterials with different surface properties and conformations (GNPs-PEG) are created for modulating the self-assembly of a widely studied protein, insulin, under amyloidogenic conditions. Important biophysical studies including thioflavin T (ThT) binding, circular dichroism (CD), surface plasmon resonance (SPR), and atomic force microscopy (AFM) showed that higher-molecular weight GNPs-PEG triggered the formation of amyloid fibrils by promoting adsorption of proteins at nanoparticle surfaces and favoring primary nucleation rate. Moreover, the modulation of fibrillation kinetics reduces the overall toxicity of insulin oligomers and fibrils. In addition, the interaction between the PEG polymer and amyloidogenic insulin examined using MD simulations revealed major changes in the secondary structural elements of the B chain of insulin. The experimental findings provide molecular-level descriptions of how the PEGylated nanoparticle surface modulates protein adsorption and drives the self-assembly of insulin. This facile approach provides a new avenue for systematically altering the binding affinities on nanoscale surfaces by tailoring their topologies for examining adsorption-induced fibrillogenesis phenomena of amyloid proteins. Together, this study suggests the role of nanobio interfaces during surface-induced heterogeneous nucleation as a primary target for designing therapeutic interventions for amyloid-related neurodegenerative disorders.

The pH effects on thermal amyloid fibrillation kinetics of hen egg‐white lysozyme using new normalization factor for Raman spectroscopy

AbstractAmyloid fibrillation kinetics of proteins associated with neurodegenerative diseases has been extensively studied using Raman spectroscopy. The normalization factor for the spectra is crucial for obtaining correct kinetics of Raman indicators, especially vibrational band intensities. Here, we compared the concentration dependences between the absorption at 280 nm in UV–vis spectroscopy and the phenylalanine (Phe) Raman band intensity at 1003 cm−1 in amyloid fibrillation kinetics of lysozyme. The former exhibits better performance as normalization factor. Using this new normalization factor, the effect of pH value on the transformation of hen egg‐white lysozyme (HEWL) tertiary and secondary structures was studied subsequently. With increasing acidity, the unfolding of tertiary structures and the transformation of secondary structures are significantly accelerated. Notably, the populations of various secondary structures in the final state remain in the pH &lt; 2.0 solutions, indicating that the branching ratios of “on‐pathway” to amyloid fibrils and “off‐pathway” to gel‐like aggregates are independent on the pH value in the range of 1.1–1.9.

Cyclic-NDGA Effectively Inhibits Human γ-Synuclein Fibrillation, Forms Nontoxic Off-Pathway Species, and Disintegrates Preformed Mature Fibrils.

Parkinson's disease arises from protein misfolding, aggregation, and fibrillation and is characterized by LB (Lewy body) deposits, which contain the protein α-synuclein (α-syn) as their major component. Another synuclein, γ-synuclein (γ-syn), coexists with α-syn in Lewy bodies and is also implicated in various types of cancers, especially breast cancer. It is known to seed α-syn fibrillation after its oxidation at methionine residue, thereby contributing in synucleinopathy. Despite its involvement in synucleinopathy, the search for small molecule inhibitors and modulators of γ-syn fibrillation remains largely unexplored. This work reveals the modulatory properties of cyclic-nordihydroguaiaretic acid (cNDGA), a natural polyphenol, on the structural and aggregational properties of human γ-syn employing various biophysical and structural tools, namely, thioflavin T (ThT) fluorescence, Rayleigh light scattering, 8-anilinonaphthalene-1-sulfonic acid binding, far-UV circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR) spectroscopy, atomic force microscopy, ITC, molecular docking, and MTT-toxicity assay. cNDGA was observed to modulate the fibrillation of γ-syn to form off-pathway amorphous species that are nontoxic in nature at as low as 75 μM concentration. The modulation is dependent on oxidizing conditions, with cNDGA weakly interacting (Kd ∼10-5 M) with the residues at the N-terminal of γ-syn protein as investigated by isothermal titration calorimetry and molecular docking, respectively. Increasing cNDGA concentration results in an increased recovery of monomeric γ-syn as shown by sodium dodecyl sulfate and native-polyacrylamide gel electrophoresis. The retention of native structural properties of γ-syn in the presence of cNDGA was further confirmed by far-UV CD and FTIR. In addition, cNDGA is most effective in suppression of fibrillation when added at the beginning of the fibrillation kinetics and is also capable of disintegrating the preformed mature fibrils. These findings could, therefore, pave the ways for further exploring cNDGA as a potential therapeutic against γ-synucleinopathies.

A Model Study to Assess Fibrillation and Product Stability to Support Peptide Drug Design.

The fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and can accelerate product development by guiding the selection of solution-stable candidates and formulations. The studies reported here investigated the effects of structural modifications on the fibrillation of a 29-residue peptide (PepA) and two sequence modified variants (PepB, PepC). The C-terminus of PepA was amidated, whereas both PepB and PepC retained the carboxylate, and Ser16 in PepA and PepB was substituted with a helix-stabilizing residue, α-aminoisobutyric acid (Aib), in PepC. In thermal denaturation studies by far-UV CD spectroscopy and fibrillation kinetic studies by fluorescence and turbidity measurements, PepA and PepB showed heat-induced conformational changes and were found to form fibrils, whereas PepC did not fibrillate and showed only minor changes in the CD signal. Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed a high degree of protection from HD exchange in mature PepA fibrils and its proteolytic fragments, indicating that most of the sequence had been incorporated into the fibril structure and occurred nearly simultaneously throughout the sequence. The effects of the net peptide charge and formulation pH on fibrillation kinetics were investigated. In real-time stability studies of two formulations of PepA at pH's 7.4 and 8.0, analytical methods detected significant changes in the stability of the formulations at different time points during the study, which were not observed during accelerated studies. Additionally, PepA samples were withdrawn from real-time stability and subjected to additional stress (40 °C, continuous shaking) to induce fibrillation; an approach that successfully amplified oligomers or prefibrillar species previously undetected in a thioflavin T assay. Taken together, these studies present an approach to differentiate and characterize fibrillation risk in structurally related peptides under accelerated and real-time conditions, providing a model for rapid, iterative structural design to optimize the stability of therapeutic peptides.

Enhancing anti-amyloidogenic properties and antioxidant effects of Scutellaria baicalensis polyphenols through novel nanoparticle formation

Protein aggregation and oxidative stress have gained significant research attention due to their association with a group of diseases known as amyloidosis. Among the strategies developed to prevent amyloidosis, utilization of polyphenols stands out as one of the most commonly employed approaches. Scutellaria baicalensis is renowned as one of the foremost herbal sources of polyphenols. In this study, we employed a direct oxidative pyrolysis method for polymerizing S. baicalensis's polyphenols (SBPPs) after their extraction, resulting in the formation of novel SBPPs nanoparticles. Upon polymerization, SBPPs nanoparticles showed remarkable properties including heightened water solubility, increased surface area, modified surface functional groups, and enhanced stability. As a result of these diverse factors, there was a considerable enhancement in the anti-amyloidogenic properties and antioxidant effects of SBPPs nanoparticles compared to its bulk form. The fibrillation kinetics, AFM images, and cytotoxicity assays strongly indicate that SBPPs nanoparticles are more effective than SBPPs at preventing amyloid fibril formation and associated cell toxicity. Additionally, SBPPs nanoparticles demonstrated more effective prevention of reactive oxygen species (ROS) production. In conclusion, the use of SBPPs in nanoparticle form presents a promising strategy to enhance anti-amyloidogenic properties, mitigate oxidative stress, and offer potential therapeutic benefits for amyloidosis-related diseases.

Charge manipulation of the human insulin B chain C-terminal to shed light on the complex mechanism of insulin fibrillation

Insulin fibrillation poses a significant challenge in the development and treatment of diabetes. Current efforts to unravel its mechanisms have thus far remained incomplete. To shed light on the intricate processes behind insulin fibrillation, we employed mutagenesis techniques to introduce additional positive charge residues into the C-terminal region of the insulin B chain which plays an important role in insulin dimerization. We employed our investigation with various spectroscopic methods, electron microscopy, and molecular dynamics simulations. These methods allowed us to explore the structure and fibrillation behavior of the engineered B chains following their expression in a bacterial host and successful purification. This manipulation had a pronounced impact on the oligomerization behavior of the insulin B chain. It appears that these mutations delay the formation of the dimeric state in the process of transitioning to larger oligomers, consequently, leading to an alteration in the kinetics of fibrillation. Our findings also indicated that the mutant insulin B chains (Di-R, Di-K, and Di-H) displayed resistance to the initiation of fibrillation. This resistance can be attributed to the repulsive forces generated by the introduced positive charges, which disrupt the attractive interactions favoring nucleation. Notably, the mutant B chains formed shorter and less abundant oligomers and fibrils, which can be ascribed to the alterations induced by repulsion. Our engineered mutant B chains exhibited enhanced stability against stress-induced fibrillation, hinting at their potential utility in the development of new insulin analogs. This study underscores the significance of the C-terminal region in the initial stages of insulin B chain fibrillation, providing valuable insights into the intricate mechanisms involved and their potential pharmaceutical applications.

In situ Raman spectral observation of succinimide intermediates in amyloid fibrillation kinetics

Succinimide intermediates play the crucial role in the nucleation process for protein amyloid fibril formation, as they can usually induce a non-native conformation in a fraction of soluble proteins to render amyloidogenicity and neurotoxicity. Thus, in situ detection of succinimide intermediates during amyloid fibrillation kinetics is of considerable importance, albeit challenging, because these succinimides are generally unstable in physiological conditions. Here, we found an in situ Raman spectral fingerprint to trace the succinimide intermediates in amyloid fibril formation, wherein the carbonyl symmetric stretching of cyclic imide in the succinimide derivative is located at ca. 1790 cm−1. Using its intensity as an indicator of succinimide intermediates, we have in situ detected and unravelled the role of succinimide intermediates during the oligomer formation from the Bz-Asp-Gly-NH2 dipeptide or the amyloid fibrillation kinetics of lysozyme with thermal/acid treatment.

N-Formylation modifies membrane damage associated with PSMα3 interfacial fibrillation.

The virulence of Staphylococcus aureus, a multi-drug resistant pathogen, notably depends on the expression of the phenol soluble modulins α3 (PSMα3) peptides, able to self-assemble into amyloid-like cross-α fibrils. Despite remarkable advances evidencing the crucial, yet insufficient, role of fibrils in PSMα3 cytotoxic activities towards host cells, the relationship between its molecular structures, assembly propensities, and modes of action remains an open intriguing problem. In this study, combining atomic force microscopy (AFM) imaging and infrared spectroscopy, we first demonstrated in vitro that the charge provided by the N-terminal capping of PSMα3 alters its interactions with model membranes of controlled lipid composition without compromising its fibrillation kinetics or morphology. N-formylation eventually dictates PSMα3-membrane binding via electrostatic interactions with the lipid head groups. Furthermore, PSMα3 insertion within the lipid bilayer is favoured by hydrophobic interactions with the lipid acyl chains only in the fluid phase of membranes and not in the gel-like ordered domains. Strikingly, our real-time AFM imaging emphasizes how intermediate protofibrillar entities, formed along PSMα3 self-assembly and promoted at the membrane interface, likely disrupt membrane integrity via peptide accumulation and subsequent membrane thinning in a peptide concentration and lipid-dependent manner. Overall, our multiscale and multimodal approach sheds new light on the key roles of N-formylation and intermediate self-assembling entities, rather than mature fibrils, in dictating deleterious interactions of PSMα3 with membrane lipids, likely underscoring its ultimate cellular toxicity in vivo, and in turn S. aureus pathogenesis.

Highly Potent Peptide Therapeutics To Prevent Protein Aggregation in Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disorder resulting from a significant amplification of CAG repeats in exon 1 of the Huntingtin (Htt) gene. More than 36 CAG repeats result in the formation of a mutant Htt (mHtt) protein. These amino-terminal mHtt fragments lead to the formation of misfolded proteins, which then form aggregates in the relevant brain regions. Therapies that can delay the progression of the disease are imperative to halting the course of the disease. Peptide-based drug therapies provide such a platform. Inhibitory peptides were screened against monomeric units of both wild type (Htt(Q25)) and mHtt fragments, Htt(Q46) and Htt(Q103). Fibril kinetics was studied by utilizing the Thioflavin T (ThT) assay. Atomic force microscopy was also used to study the influence of the peptides on fibril formation. These experiments demonstrate that the chosen peptides suppress the formation of fibrils in mHtt proteins and can provide a therapeutic lead for further optimization and development.

Frequency dependence of ultrasonic effects on the kinetics of hen egg white lysozyme fibrillation

Our study aimed to investigate the effects of ultrasound on the fibrillation kinetics of HEWL (hen egg white lysozyme) and its physicochemical properties. Ultrasound, a mechanical wave, can induce conformational changes in proteins. To achieve this, we developed an ultrasound exposure system and used various biophysical techniques, including ThT fluorescence spectroscopy, ATR-FTIR, Far-UV CD spectrophotometry, Fluorescence microscopy, UV-spectroscopy, and seeding experiments. Our results revealed that higher frequencies significantly accelerated the fibrillation of lysozyme by unfolding the native protein and promoting the fibrillation process, thereby reducing the lag time. We observed a change in the secondary structure of the sonicated protein change to the β-structure, but there was no difference in the Tm of native and sonicated proteins. Furthermore, we found that higher ultrasound frequencies had a greater seeding effect. We propose that the effect of frequency can be explained by the impact of the Reynolds number, and for the Megahertz frequency range, we are almost at the transition regime of turbulence. Our results suggest that laminar flows may not induce any significant change in the fibrillation kinetics, while turbulent flows may affect the process.

Effects of ultrasonic pretreatment on fibrillation kinetics, morphologies, and functional properties of bovine serum albumin fibrils

Amyloid fibrils (AFs) are generally believed to contribute to the functional properties of food matrix. In order to improve the application accessibility of protein fibrils, a better understanding of the mechanisms regulating their functional properties and fibrillation is needed. In this work, the fibrillation kinetics of bovine serum albumin (BSA) under static acid-heating and how ultrasonic pretreatment (USP) affects their ability to form stable emulsions and foams were investigated. BSA fibrils were generated under diverse acidic pHs (pH 2.0 to 4.0) by heating at 65 °C for 12 h. The formation kinetics, physicochemical properties, and morphologies of BSA fibrils were studied using thioflavin T fluorescence, electrophoretic gel, spectroscopic methods, viscosity profiling, and transmission electron microscope. Emulsions and foams were made and evaluated for functional capacity and stability. Results showed that the fibrillated BSA at pH 3.0 possesses the best performance in emulsifying and foaming properties. USP can lead to finer, shorter, and more flexible fibrils, compared to the untreated control. The mature BSA fibrils mediated by 20-min USP may be efficient in covering around oil droplets/bubbles, resulting in stronger emulsifying and foaming properties. The attempts were made to provide referable meaning to the functional application of BSA in the food industry.

Self-Aggregating Tau Fragments Recapitulate Pathologic Phenotypes and Neurotoxicity of Alzheimer's Disease in Mice.

In tauopathy conditions, such as Alzheimer's disease (AD), highly soluble and natively unfolded tau polymerizes into an insoluble filament; however, the mechanistic details of this process remain unclear. In the brains of AD patients, only a minor segment of tau forms β-helix-stacked protofilaments, while its flanking regions form disordered fuzzy coats. Here, it is demonstrated that the tau AD nucleation core (tau-AC) sufficiently induced self-aggregation and recruited full-length tau to filaments. Unexpectedly, phospho-mimetic forms of tau-AC (at Ser324 or Ser356) show markedly reduced oligomerization and seeding propensities. Biophysical analysis reveal that the N-terminus of tau-AC facilitates the fibrillization kinetics as a nucleation motif, which becomes sterically shielded through phosphorylation-induced conformational changes in tau-AC. Tau-AC oligomers are efficiently internalized into cells via endocytosis and induced endogenous tau aggregation. In primary hippocampal neurons, tau-AC impaired axon initial segment plasticity upon chronic depolarization and is mislocalized to the somatodendritic compartments. Furthermore, it is observed significantly impaired memory retrieval in mice intrahippocampally injected with tau-AC fibrils, which corresponds to the neuropathological staining and neuronal loss in the brain. These findings identify tau-AC species as a key neuropathological driver in AD, suggesting novel strategies for therapeutic intervention.

Modulation of the Fibrillation Kinetics and Morphology of a Therapeutic Peptide by Cucurbit[7]uril.

Fibrillation is a challenge commonly encountered in the formulation and development of therapeutic peptides. Cucurbit[7]urils (CB[7]), a group of water soluble macrocycles, have been reported to suppress fibrillation in insulin and human calcitonin through association with Phe and Tyr residues which drive fibril formation. Here, we report the effect of CB[7] on the fibrillation behavior of the HIV fusion inhibitor enfuvirtide (ENF) that contains N-terminal Tyr and C-terminal Phe residues. Thioflavin T fluorescence, CD spectroscopy, and transmission electron microscopy were used to monitor fibrillation behavior. Fibrillation onset showed a strong pH dependency, with pH 6.5 identified as the condition most suitable to monitor the effects of CB[7]. Binding of CB[7] to wild-type ENF was measured by isothermal titration calorimetry and was consistent with a single site (Ka = 2.4 × 105 M-1). A weaker interaction (Ka = 2.8 × 103 M-1) was observed for an ENF mutant with the C-terminal Phe substituted for Ala (ENFm), suggesting that Phe was the specific site for CB[7] recognition. The onset of ENF fibrillation onset was delayed, rather than fully suppressed, in the presence of CB[7]. The ENFm mutant showed a greater delay in fibrillation onset but with no observable effect on fibrillation kinetics in the presence of CB[7]. Interestingly, ENF/CB[7] and ENFm fibrils exhibited comparable morphologies, differing from those observed for ENF alone. The results indicate that CB[7] is capable of modulating fibrillation onset and the resulting ENF fibrils by specifically binding to the C-terminal Phe residue. The work reinforces the potential of CB[7] as an inhibitor of fibrillation and highlights its role in determining fibril morphologies.

Modulation of Insulin Amyloid Fibrillization in Imidazolium-Based Ionic Liquids with Hofmeister Series Anions.

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.