Hen Egg White Lysozyme Fibrillation Research Articles

The volatile constituents of rose, saffron and cardamom suppress the fibrillation of tau and not HEWL through the formation of non-toxic tau oligomers

The formation of fibrillar structures of tau is correlated with taupathies including Alzheimer's disease (AD). This study has aimed to find ways to prevent tau fibril formation. Here, we used dietary compounds including cinnamon (CN), damask rose (Rose), saffron (Saf) and green cardamom (Car), to evaluate the effects of their volatile constituents, on hen egg white lysozyme (HEWL), as a model protein (commonly used for fibrillation studies), as well as the brain-related tau protein. The study was done using different spectroscopic techniques as well as SDS-PAGE, AFM and MTT assay. While the results suggested that the volatile constituents were unable to prevent HEWL fibril formation, most of the dietary compounds, in particular Saf, Rose and Car, were able to interfere with the mature fibril formation, by either maintaining the native form of tau or resulting in the entrapment of non-toxic oligomeric forms of tau. Moreover, the neurotoxicity analysis of tau samples on neuroblastoma SH-SY5Y cells indicated that tau treated with Saf, Rose and Car were the least toxic. Overall, the findings indicate that the potential therapeutic impacts of the volatile constituents of Rose, Car and in particular Saf, may demonstrate significant efficacy in addressing neurodegenerative diseases such as AD.

Isoflavones and lysozyme interplay: Molecular insights into binding mechanisms and inhibitory efficacies of isoflavones against protein modification

In this study, we investigated the complexation of bioactive isoflavones, specifically biochanin A (BCA) and genistein (GEN), with hen egg white lysozyme (HEWL) and explored their inhibitory effects on HEWL modification using a combination of multi-spectroscopic and computational methods. The observed binding affinity was of a moderate nature (on the order of 104 M−1), and a static quenching mechanism was identified in the fluorescence quenching process. Notably, the binding constant (Kb) for GEN (4.449 ± 0.262 × 104 M−1) was found to be higher than that for BCA (3.707 ± 0.108 × 104 M−1) towards HEWL. Our spectroscopic measurements, complemented by molecular docking calculations, suggested the involvement of Trp62 in the binding site of the isoflavones within the geometry of HEWL. The micro-environment surrounding the Trp-residues exhibited an increase in hydrophilicity, as indicated by Synchronous fluorescence (SFS) and three-dimensional fluorescence (3D) studies. Interestingly, circular dichroism (CD) studies revealed no marked alteration in the secondary structure of HEWL upon binding with the isoflavones. Furthermore, our investigation into the interaction patterns, employing FTIR and molecular docking studies, revealed a predominance of hydrogen bonding and hydrophobic interactions. Beyond the binding study, the isoflavones demonstrated a promising inhibitory effect on the d-ribose-mediated glycation of HEWL, as well as on HEWL fibrillation, as evidenced by fluorescence emission studies. Our findings not only exhibited an excellent correlation with experimental observations but also provided precise insights into the location and dynamics of isoflavones within the binding site through detailed analyses of molecular docking and molecular dynamics simulation data.

Graphene acid quantum dots: A highly active multifunctional carbon nano material that intervene in the trajectory towards neurodegeneration

Carbon dots (CDs) are carbon nano materials (CNMs) that find use across several biological applications because of their water solubility, biocompatible nature, eco-friendliness, and ease of synthesis. Additionally, their physiochemical properties can be chemically tuned for further optimization towards specific applications. Here, we investigate the efficacy of C70-derived Graphene Acid Quantum Dots (GAQDs) in mitigating the transformation of soluble, monomeric Hen Egg-White Lysozyme (HEWL) to mature fibrils during its amyloidogenic trajectory. Our findings reveal that GAQDs exhibit dose-dependent inhibition of HEWL fibril formation (up to 70 % at 5 mg/mL) without affecting mitochondrial membrane potential or inducing apoptosis at the same density. Furthermore, GAQDs scavenged reactive oxygen species (ROS); achieving a 50 % reduction in ROS levels at a mere 100 µg/mL when exposed to a standard free radical generator. GAQDs were not only found to be biocompatible with a human neuroblastoma-derived SHSY-5Y cell line but also rescued the cells from rotenone-induced apoptosis. The GAQD-tolerance of SHSY-5Y cells coupled with their ability to restitute cells from rotenone-dependent apoptosis, when taken in conjunction with the biocompatibility data, indicate that GAQDs possess neuroprotective potential. The data position this class of CNMs as promising candidates for resolving aberrant cellular outputs that associate with the advent and progress of multifactorial neurodegenerative disorders including Parkinson’s (PD) and Alzheimer’s diseases (AD) wherein environmental causes are implicated (95 % etiology). The data suggest that GAQDs are a multifunctional carbon-based sustainable nano-platform at the intersection of nanotechnology and neuroprotection for advancing green chemistry-derived, sustainable healthcare solutions.

Detection and disaggregation of amyloid fibrils by luminescent amphiphilic platinum(II) complexes.

Cyclometallated Pt(II) complexes possessing hydrophobic 2-phenylpyridine (ppy) ligands and hydrophilic acetonylacetone (acac) ligands have been investigated for their ability to detect amyloid fibrils via luminescence response. Using hen egg-white lysozyme (HEWL) as a model amyloid protein, Pt(II) complexes featuring benzanilide-substituted ppy ligands and ethylene glycol-functionalized acac ligands demonstrated enhanced luminescence in the presence of HEWL fibrils, whereas Pt(II) complexes lacking complementary hydrophobic/hydrophilic ligand sets displayed little to no emission enhancement. An amphiphilic Pt(II) complex incorporating a bis(ethylene glycol)-derivatized acac ligand was additionally found to trigger restructuring of HEWL fibrils into smaller spherical aggregates. Amphiphilic Pt(II) complexes were generally non-toxic to SH-SY5Y neuroblastoma cells, and several complexes also exhibited enhanced luminescence in the presence of Aβ42 fibrils associated with Alzheimer's disease. This study demonstrates that easily prepared and robust (ppy)PtII(acac) complexes show promising reactivity toward amyloid fibrils and represent attractive molecular scaffolds for design of small-molecule probes targeting amyloid assemblies.

Interaction of gallium, indium, and vanadyl curcumin complexes with hen egg-white lysozyme (HEWL): Mechanistic aspects and evaluation of antiamyloidogenic activity

Many proteins and peptides can aggregate into amyloid fibrils with high-ordered and cross-β rich structure characteristics. Amyloid deposition is a common feature of neurodegenerative diseases called amyloidosis. Various natural polyphenolic compounds such as curcumin exhibited antiamyloidogenic activities, but less researches were focused on the metal complexes of these compounds. In this study, the inhibitory effects of gallium curcumin (Ga(cur)3), indium curcumin (In(cur)3), and vanadyl curcumin (VO(cur)2) on the amyloid fibrillation of hen egg white lysozyme (HEWL) have been investigated. Moreover, the details of binding interactions of these metal complexes with HEWL have been explored. The results of fluorescence quenching analyses revealed that In(cur)3 and VO(cur)2 have much higher binding affinities than Ga(cur)3 toward HEWL. The interactions of these metal complexes were accompanied by partial conformational changes in the tertiary structure of HEWL. The kinetic curves of the fibrillation process demonstrated that In(cur)3 and VO(cur)2 have higher inhibitory effects than Ga(cur)3 on the amyloid fibrillation of HEWL. The strength of binding to HEWL is completely in accordance with inhibitory activities of these metal complexes of curcumin.

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.

Interaction of gallium, indium and vanadyl diacetylcurcumin complexes with lysozyme: mechanistic aspects and evaluation of antiamyloidogenic activity.

Diacetylcurcumin as a derivative of curcumin is a strong nitric oxide (NO) and O2-.anion scavenger. One strategy to improve stability of curcumin and its derivatives is complexation with metal. In this study, the binding interactions of gallium diacetylcurcumin (Ga(DAC)3), indium diacetylcurcumin (In(DAC)3), and vanadyl diacetylcurcumin (VO(DAC)2) with hen egg white lysozyme (HEWL) have been investigated. The results of fluorescence quenching analyses revealed that In(DAC)3 and VO(DAC)2 have higher binding affinities than Ga(DAC)3 towards HEWL. The interactions of these metal complexes were not accompanied by considerable conformational changes in the tertiary structure of HEWL. Furthermore, the inhibitory effects of these complexes on the amyloid fibrillation of HEWL were confirmed by the thioflavin T fluorescence assays. The kinetic curves of the fibrillation process illustrated that VO(DAC)2 has the highest inhibitory activity and In(DAC)3 has a significant delaying effect on the formation of amyloid fibrils of HEWL.

Inhibitory effect of plain and functionalized graphene nanoplateles on hen egg white lysozyme fibrillation.

Protein fibrillation is a phenomenon associated with misfolding and the production of highly ordered nanofibrils, which may cause serious degenerative diseases such as Parkinson's disease, Alzheimer's disease, and type 2 diabetes. Upon contact with biological fluids, the nanomaterials are immediately covered by proteins and interact with them. In this study, the effects of Graphene NanoPlateles (Plain-GNPs) and their modified forms with a carboxyl group (GNPs -COOH) and an amine group (GNPs -NH2) are evaluated on the fibrillation process of Hen Egg White Lysozyme (HEWL). The fibrillation process of HEWL was studied using thioflavin-T, Circular Dichroism spectrometry, and Atomic Force Microscopy. Plain-GNPs significantly decreased the fibrillation process at different stages, including nucleation, exponential fibrillation phases, and end-mature fibril products. However, GNPs-COOH and GNPs-NH2 affected the final fluorescence of ThT. The species formed in the presence of Plain-GNPs showed less toxicity in SH-SY5Y cells, which could be applicable for therapeutic purposes.

The impact of modified polystyrene on lysozyme fibrillation studied by surface-enhanced Raman spectroscopy (SERS)

Nanoplastics could modulate the fibrillation of amyloid proteins. However, many chemical functional groups are adsorbed to change the interfacial chemistry of nanoplastics in the real world. Herein, this study aimed to investigate the effects of polystyrene (PS), carboxyl modified PS (PS-COOH), and amino modified PS (PS-NH2) on the fibrillation of hen egg-white lysozyme (HEWL). Due to the differences in the interfacial chemistry, concentration was considered an essential factor. PS-NH2 (10 μg/mL) could promote the fibrillation of HEWL similar to PS (50 μg/mL) and PS-COOH (50 μg/mL). Moreover, promoting the primary nucleation step of amyloid fibril formation was the primary reason. The differences in spatial conformation of HEWL were characterized by Fourier transform-infrared spectroscopy and surface enhanced Raman spectroscopy (SERS). Strikingly, a particular signal of SERS of HEWL incubated with PS-NH2 at 1610 cm−1 was found due to the interaction between amino group of PS-NH2 and tryptophan (or tyrosine) of HEWL. Therefore, a new perspective was provided to understand the regulation of interfacial chemistry of nanoplastics on the fibrillation of amyloid proteins. Additionally, this study suggested that SERS could be a powerful method to investigate the interactions between proteins and nanoparticles.

Differential effects of DTT on HEWL amyloid fibrillation and fibril morphology at different pH

Proteins can transform from their native state to a state having fibrillar aggregates characterized by cross β sheet structure. The fibrillar aggregates are known as amyloid and have been linked to several disorders. Disulfide bonds in proteins are one of the important factors that determine the propensity of aggregation. Hen Egg White Lysozyme (HEWL) was used by us as a model protein to decipher the role disulfide bonds play in the amyloid fibril formation and fibril morphology by using Dithiothreitol (DTT) as reducing agent at pH 2.7 and pH 7.4. We found that DTT can have different effects on HEWL amyloid depending on pH and the buffer used for preparing the amyloid fibrils. Our studies highlight the critical role of non-native disulfide bonds in amyloidogenesis and how disruption of these bonds can greatly affect the fibrillation process. Overall, these studies throw light on the fibrillation mechanism and can be explored further in designing effective inhibitors against amyloidosis.

Elucidating the inhibitory effects of rationally designed novel hexapeptide against hen egg white lysozyme fibrillation at acidic and physiological pH

Inhibition of highly ordered cross-β-sheet-rich aggregates of misfolded amyloid proteins using rationally designed sequence-based short peptides is a promising therapeutic strategy for the treatment of neurodegenerative diseases. Here, we have explored the anti-amyloidogenic potency of a rationally designed hexapeptide (Tyr-Pro-Gln-Ile-Pro-Asn) on in vitro hen egg white lysozyme (HEWL) amyloid fibril formation at acidic pH and physiological pH using computational docking as well as various biophysical techniques such as fluorescence spectroscopy, UV–vis spectroscopy, FTIR spectroscopy, confocal microscopy and TEM. The peptide was designed based on the aggregation-prone region (APR) of HEWL and thus referred to as SqP1 (Sequence-based Peptide 1). SqP1 showed over 70% inhibition of HEWL amyloid formation at pH 2.2 and approximately 50% inhibition at pH 7.5. We propose that SqP1 binds to the APR of HEWL and interacts strongly with the Trp62/Trp63, ultimately stabilizing monomeric HEWL at both the pH conditions and preventing conformation changes in the structure of HEWL, leading to the formation of amyloidogenic fibrillar structures. A sequence-based peptide inhibitor of HEWL amyloid formation was not reported previously, making this a critical study that will further emphasize the importance of short synthetic peptides as amyloid inhibitors.

Inhibition of hen egg white lysozyme fibrillation by a self-assembled nanostructured lysozyme and graphene oxide conjugate

Self-assembled nanostructured lysozyme and graphene oxide conjugate (GO-snLYZ) shows efficient inhibitory effects towards fibrillation of HEWL.

Inhibition of lysozyme amyloid fibrillation by curcumin-conjugated silver nanoparticles: A multispectroscopic molecular level study

Inhibition of amyloid fibrillation of hen egg white lysozyme (HEWL) was targeted by using curcumin-conjugated silver nanoparticles (c-AgNPs). Fluorescence assays (thioflavin T, ANS binding, tryptophan (Trp) fluorescence), circular dichroism and microscopic imaging techniques (HRTEM and fluorescence) were adopted to study fibrillation inhibition. Results suggested that c-AgNPs having hydrodynamic radius ∼ 22 nm can inhibit fibrillation of HEWL and restricted the protein fibrillation to smaller fibrils. Moreover, in the form of c-AgNPs, curcumin remained water-soluble and was more effective than curcumin. Further, interactions between HEWL and c-AgNPs were studied using fluorescence and circular dichroism spectroscopic techniques. The nanoparticles had caused static quenching of Trp fluorescence of native HEWL. This was also confirmed through determination of excited state lifetime of Trp of HEWL in absence and the presence of c-AgNPs. The association and quenching constant for the conjugation of HEWL with c-AgNPs were determined ∼ 107 M−1. Hydrophobic interactions were found to be involved in HEWL-c-AgNPs complex. Polarity of Trp microenvironment in HEWL was not perturbed by c-AgNPs as supported by synchronous and three-dimensional fluorescence spectroscopy. Far-UV CD spectra suggested that the secondary structure of native HEWL was not affected by c-AgNPs.

One- and Two-Photon Excited Autofluorescence of Lysozyme Amyloids.

Autofluorescence properties of amyloid fibrils are of much interest but, to date, the attention has been given mostly to one-photon excited fluorescence (1PEF), while the two-photon excited fluorescence (2PEF) properties of amyloids are much less explored. We investigate 1PEF and 2PEF of hen egg-white lysozyme (HEWL) in the form of monomers and fibrils. HEWL monomers feature some autofluorescence, which is enhanced in the case of fibrils. Moreover, by varying NaCl content, we introduce changes to fibrils morphology and show how the increase of the salt concentration is linked with an increase of 1PEF and 2PEF intensities. Interestingly, we observe 2PEF emission red-shifted in comparison to 1PEF. We confirm the presence of different relaxation pathways upon one- or two-photon excitation by different lifetimes of the fluorescence decays. Finally, we correlate the changes in optical properties of HEWL fibrils and monomers with salt-mediated changes in their morphology and the secondary structure.

Repression Effects of Hydrolysates from Hen-Egg Proteins on Amyloid Fibril Formation.

Amyloid fibrils, which are formed from aggregates of aberrant proteins, can cause various forms of amyloidosis (including Alzheimer's disease). Such disorders often occur in elderly populations and are suspected to be lifestyle related. Thus, it has been speculated that some foodstuffs could be beneficial for preventing amyloidosis. In this study, we determine whether fibril formation by the hen egg white lysozyme (HEWL) could be inhibited by conducting a thioflavin T assay followed by fluorescence and electron microscopy observations. The results demonstrated that four peptide specimens prepared by the hydrolysis of crude proteins from the egg white, egg yolk, chalazae, and eggshell membrane of hen eggs effectively inhibited HEWL fibril formation. Among the four specimens, peptides from chalazae exhibited the highest preventive ability. The superiority of chalaza peptides was also observed when fibril formation was assayed using a full-length human lysozyme and human amyloid β peptide 1-42, which is the key factor for the development of Alzheimer's disease. Our study of the fibrillization of the human lysozyme also showed that metal ions (Zn2+, Ca2+, Co2+, Mn2+ and Al3+) promoted fibrillization, and their effects were abolished by the peptide specimens (especially by chalaza peptides). Thus, we conclude that chicken-egg proteins could be a convenient source of therapeutic materials for amyloidosis.

Mechanistic In Vitro Dissection of the Inhibition of Amyloid Fibrillation by n-Acetylneuraminic Acid: Plausible Implication in Therapeutics for Neurodegenerative Disorders.

A variety of neurodegenerative disorders including Parkinson's disease are due to fibrillation in amyloidogenic proteins. The development of therapeutics for these disorders is a topic of extensive research as effective treatments are still unavailable. The present study establishes that n-acetylneuraminic acid (Neu5ac) inhibits the amyloid fibrillation of hen egg-white lysozyme (HEWL) and α-synuclein (SYN), as observed using various biophysical techniques and cellular assays. Neu5ac inhibits the amyloid formation in both proteins, as suggested from the reduction in the ThT fluorescence and remnant structures in transmission electron microscopy micrographs observed in its presence. In HEWL fibrillation, Neu5ac decreases the hydrophobicity and resists the transition of the α-helix to a β-sheet, as observed by an ANS binding assay, circular dichroism (CD) spectra, and Fourier transform infrared measurements, respectively. Neu5ac stabilizes the states that facilitate the amyloid formation in HEWL and SYN, as demonstrated by an enhanced intrinsic fluorescence in its presence, which is further confirmed by an increase in Tm obtained from differential scanning calorimetry thermograms and an increase in the near-UV CD signal for HEWL with Neu5ac. However, the increase in stability is not a manifestation of Neu5ac binding to amyloid facilitating (partially folded or native) states of both proteins, as verified by isothermal titration calorimetry and fluorescence binding measurements. Besides, Neu5ac also attenuates the cytotoxicity of amyloid fibrils, as evaluated by a cell toxicity assay. These findings provide mechanistic insights into the Neu5ac action against amyloid fibrillation and may establish it as a plausible inhibitor molecule against neurodegenerative disorders.

UV Resonance Raman explores protein structural modification upon fibrillation and ligand interaction

Amyloids are proteinaceous deposits considered an underlying pathological hallmark of several degenerative diseases. The mechanism of amyloid formation and its inhibition still represent challenging issues, especially when protein structure cannot be investigated by classical biophysical techniques as for the intrinsically disordered proteins (IDPs). In this view, the need to find an alternative way for providing molecular and structural information regarding IDPs prompted us to set a novel, to our knowledge, approach focused on UV Resonance Raman (UVRR) spectroscopy. To test its applicability, we study the fibrillation of hen-egg white lysozyme (HEWL) and insulin as well as their interaction with resveratrol, employing also intrinsic fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and atomic force microscopy (AFM). The increasing of the β-sheet structure content at the end of protein fibrillation probed by FTIR occurs simultaneously with a major solvent exposure of tryptophan (Trp) and tyrosine (Tyr) residues of HEWL and insulin, respectively, as revealed by UVRR and intrinsic fluorescence spectroscopy. However, because the latter technique is successfully used when proteins naturally contain Trp residues, it shows poor performances in the case of insulin, and the information regarding its tertiary structure is exclusively provided by UVRR spectroscopy. The presence of an increased concentration of resveratrol induces mild changes in the secondary structure of both protein fibrils while remodeling HEWL fibril length and promoting the formation of amorphous aggregates in the case of insulin. Although the intrinsic fluorescence spectra of proteins are hidden by resveratrol signal, UVRR Trp and Tyr bands are resonantly enhanced, showing a good sensitivity to the presence of resveratrol and marking a modification in the noncovalent interactions in which they are involved. Our findings demonstrate that UVRR is successfully employed in the study of aggregation-prone proteins and of their interaction with ligands, especially in the case of Trp-lacking proteins.

Surface-modified magnetite nanoparticles affect lysozyme amyloid fibrillization

BackgroundThe surface of nanoparticles (NPs) is an important factor affecting the process of poly/peptides' amyloid aggregation. We have investigated the in vitro effect of trisodium citrate (TC), gum arabic (GA) and citric acid (CA) surface-modified magnetite nanoparticles (COAT-MNPs) on hen egg-white lysozyme (HEWL) amyloid fibrillization and mature HEWL fibrils. MethodsDynamic light scattering (DLS) was used to characterize the physico-chemical properties of studied COAT-MNPs and determine the adsorption potential of their surface towards HEWL. The anti-amyloid properties were studied using thioflavin T (ThT) and tryptophan (Trp) intrinsic fluorescence assays, and atomic force microscopy (AFM). The morphology of amyloid aggregates was analyzed using Gwyddion software. The cytotoxicity of COAT-MNPs was determined utilizing Trypan blue (TB) assay. ResultsAgents used for surface modification affect the COAT-MNPs physico-chemical properties and modulate their anti-amyloid potential. The results from ThT and intrinsic fluorescence showed that the inhibitory activities result from the more favorable interactions of COAT-MNPs with early pre-amyloid species, presumably reducing nuclei and oligomers formation necessary for amyloid fibrillization. COAT-MNPs also possess destroying potential, which is presumably caused by the interaction with hydrophobic residues of the fibrils, resulting in the interruption of an interface between β-sheets stabilizing the amyloid fibrils. ConclusionCOAT-MNPs were able to inhibit HEWL fibrillization and destroy mature fibrils with different efficacy depending on their properties, TC-MNPs being the most potent nanoparticles. General significanceThe study reports findings regarding the general impact of nanoparticles' surface modifications on the amyloid aggregation of proteins.

Elevated temperatures accelerate the formation of toxic amyloid fibrils of hen egg‐white lysozyme

The formation of amyloid fibrils is critical for neurodegenerative diseases. Some physiochemical conditions can promote the conversion of proteins from soluble globular shapes into insoluble well‐organized amyloid fibrils. The aim of this study was to investigate the effect of temperatures on amyloid fibrils formation in vitro using the protein model of hen egg‐white lysozyme (HEWL). The HEWL fibrils were prepared at temperatures of 37, 45, 50 and 57°C in glycine solution of pH 2.2. Under transmission electron microscopy, we found the well‐organized HEWL amyloid fibrils at temperatures of 45, 50 and 57°C after 10 days of incubation. Thioflavin T and Congo red florescence assays confirmed that the formation and growth of HEWL fibrils displayed a temperature‐dependent increase, and 57°C produced the most amounts. Meanwhile, the surface hydrophobicity of aggregates was greatly increased by ANS binding assay, and β‐sheet contents by circular dichroism analysis were increased by 17.8%, 22.0% and 34.9%, respectively. Furthermore, the HEWL fibrils formed at 57°C caused significant cytotoxicity in SH‐SY5Y cells after 48 hr exposure, and the cell viability determined by MTT assay was decreased, with 81.35 ± 0.29% for 1 μM, 61.45 ± 2.62% for 2 μM, and 11.58 ± 0.39% (p < .01) for 3 μM. Nuclear staining results also confirmed the apoptosis features. These results suggest that the elevated temperatures could accelerate protein unfolding of the native structure and formation of toxic amyloid fibrils, which can improve understanding the mechanisms of the unfolding and misfolding process of prion protein.

Hydrophilic AIE-Active Tetraarylethenes for Fluorescence Sensing and Super-Resolution Imaging of Amyloid Fibrils from Hen Egg White Lysozyme

Hen egg white lysozyme (HEWL) is frequently applied as a model protein for research on protein folding, unfolding, and fibrillization identified by featured fluorescent probes. Here, a series of hydrophilic, pH-sensitive tetraarylethene (TAE)-type AIEgens are synthesized via a geminal cross-coupling (GCC) reaction and evaluated for their capabilities of fluorescence sensing and super-resolution localization imaging of HEWL fibrils. With superior optical and sensing properties, the selected TAE-type AIEgen probe is weakly emissive in aqueous media, without dependence on the pH value and buffer concentration, but exhibits "turn-on" fluorescence upon interaction with HEWL amyloid fibrils in a spontaneous and reversible way that just meets the requirement of fluorescence random switching for super-resolution imaging. The selected probe has the strongest fluorescence response to HEWL amyloid fibrils exhibiting a limit of detection of 0.59 nmol/L and enables super-resolution fluorescence imaging of amyloid aggregates with a high resolution of 40 nm.