Sulfated Polysaccharides as a Fighter with Protein Non-Physiological Aggregation: The Role of Polysaccharide Flexibility and Charge Density.

Proteins can lose native functionality due to non-physiological aggregation. In this work, we have shown the power of sulfated polysaccharides as a natural assistant to restore damaged protein structures. Protein aggregates enriched by cross-ß structures are a characteristic of amyloid fibrils related to different health disorders. Our recent studies demonstrated that model fibrils of hen egg white lysozyme (HEWL) can be disaggregated and renatured by some negatively charged polysaccharides. In the current work, using the same model protein system and FTIR spectroscopy, we studied the role of conformation and charge distribution along the polysaccharide chain in the protein secondary structure conversion. The effects of three carrageenans (κ, ι, and λ) possessing from one to three sulfate groups per disaccharide unit were shown to be different. κ-Carrageenan was able to fully eliminate cross-ß structures and complete the renaturation process. ι-Carrageenan only initiated the formation of native-like ß-structures in HEWL, retaining most of the cross-ß structures. In contrast, λ-carrageenan even increased the content of amyloid cross-ß structures. Furthermore, κ-carrageenan in rigid helical conformation loses its capability to restore protein native structures, largely increasing the amount of amyloid cross-ß structures. Our findings create a platform for the design of novel natural chaperons to counteract protein unfolding.

Hen Egg White Lysozyme (HEWL) Confers Resistance to Verticillium Wilt in Cotton by Inhibiting the Spread of Fungus and Generating ROS Burst.

Verticillium wilt is a soil-borne vascular disease caused by the fungal pathogen Verticillium dahliae. It causes great harm to upland cotton (Gossypium hirsutum) yield and quality. A previous study has shown that Hen egg white lysozyme (HEWL) exerts strong inhibitory activity against V. dahliae in vitro. In the current study, we introduced the HEWL gene into cotton through the Agrobacterium-mediated transformation, and the exogenous HEWL protein was successfully expressed in cotton. Our study revealed that HEWL was able to significantly inhibit the proliferation of V. dahlia in cotton. Consequently, the overexpression of HEWL effectively improved the resistance to Verticillium wilt in transgenic cotton. In addition, ROS accumulation and NO content increased rapidly after the V. dahliae inoculation of plant leaves overexpressing HEWL. In addition, the expression of the PR genes was significantly up-regulated. Taken together, our results suggest that HEWL significantly improves resistance to Verticillium wilt by inhibiting the growth of pathogenic fungus, triggering ROS burst, and activating PR genes expression in cotton.

The Effect of Arginine on the Phase Stability of Aqueous Hen Egg-White Lysozyme Solutions.

The effect of arginine on the phase stability of the hen egg-white lysozyme (HEWL) has been studied via molecular dynamics computer simulations, as well as experimentally via cloud-point temperature determination. The experiments show that the addition of arginine increases the stability of the HEWL solutions. The computer simulation results indicate that arginine molecules tend to self-associate. If arginine residues are located on the protein surface, the free arginine molecules stay in their vicinity and prevent the way protein molecules "connect" through them to form clusters. The results are not sensitive to a particular force field and suggest a possible microscopic mechanism of the stabilizing role of arginine as an excipient.

pH-Dependent HEWL-AuNPs Interactions: Optical Study.

Optical methods (spectroscopy, spectrofluorometry, dynamic light scattering, and refractometry) were used to study the change in the state of hen egg-white lysozyme (HEWL), protein molecules, and gold nanoparticles (AuNPs) in aqueous colloids with changes in pH, and the interaction of protein molecules with nanoparticles was also studied. It was shown that changing pH may be the easiest way to control the protein corona on gold nanoparticles. In a colloid of nanoparticles, both in the presence and absence of protein, aggregation-deaggregation, and in a protein colloid, monomerization-dimerization-aggregation are the main processes when pH is changed. A specific point at pH 7.5, where a transition of the colloidal system from one state to another is observed, has been found using all the optical methods mentioned. It has been shown that gold nanoparticles can stabilize HEWL protein molecules at alkaline pH while maintaining enzymatic activity, which can be used in practice. The data obtained in this manuscript allow for the state of HEWL colloids and gold nanoparticles to be monitored using one or two simple and accessible optical methods.

Influence of Urea and Dimethyl Sulfoxide on K-Peptide Fibrillation.

Protein fibrillation leads to formation of amyloids-linear aggregates that are hallmarks of many serious diseases, including Alzheimer's and Parkinson's diseases. In this work, we investigate the fibrillation of a short peptide (K-peptide) from the amyloidogenic core of hen egg white lysozyme in the presence of dimethyl sulfoxide or urea. During the studies, a variety of spectroscopic methods were used: fluorescence spectroscopy and the Thioflavin T assay, circular dichroism, Fourier-transform infrared spectroscopy, optical density measurements, dynamic light scattering and intrinsic fluorescence. Additionally, the presence of amyloids was confirmed by atomic force microscopy. The obtained results show that the K-peptide is highly prone to form fibrillar aggregates. The measurements also confirm the weak impact of dimethyl sulfoxide on peptide fibrillation and distinct influence of urea. We believe that the K-peptide has higher amyloidogenic propensity than the whole protein, i.e., hen egg white lysozyme, most likely due to the lack of the first step of amyloidogenesis-partial unfolding of the native structure. Urea influences the second step of K-peptide amyloidogenesis, i.e., folding into amyloids.

Study on Antibacterial Activity and Structure of Chemically Modified Lysozyme.

Lysozyme is a natural protein with a good bacteriostatic effect, but its poor inhibition of Gram-negative bacteria limits its development potential as a natural preservative. Therefore, the modification of natural lysozyme to expand the antimicrobial spectrum become the focus of lysozyme study. Egg white lysozyme has low cost, rich content in nature, is easy to obtain, strong stability, and high enzyme activity, so it can be applied in the modification of lysozyme. Egg white lysozyme was modified by chemical methods using organic acids. Caffeic acid and p-coumaric acid in organic acids were used as modifiers, and 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxy succinimide were used as dehydration condensation agents during modification. A certain degree of modified lysozyme was obtained through appropriate modification conditions. The antibacterial properties and structure of the obtained two organic acid-modified lysozymes were compared with natural enzymes. The results showed that compared with the native enzyme, the activity of modified lysozyme decreased, but the inhibitory effect on Gram-negative bacteria was enhanced. The minimum inhibitory concentrations of caffeic acid-modified enzyme and p-coumaric acid-modified enzyme on Escherichia coli and Pseudomonas aeruginosa were 0.5 mg/mL and 0.75 mg/mL, respectively. However, the antibacterial ability of modified lysozyme to Gram-positive bacteria was lower than that of the natural enzyme. The minimum inhibitory concentration of caffeic acid-modified enzyme and p-coumaric acid-modified enzyme to Staphylococcus aureus and Bacillus subtilis was 1.25 mg/mL. The peak fitting results of the amide-I band absorption peak in the infrared spectroscopy showed that the content of the secondary structure of the two modified enzymes obtained after modification was different from that of natural enzymes. In the study, two organic acids were used to modify egg white lysozyme, which enhanced the enzyme's inhibition of Gram-negative bacteria, and analyzed the mechanisms for the change in the enzyme's antibacterial ability from the perspective of the structural change of the modified enzyme, providing a new idea for lysozyme modification.

Investigation on the effect of three isoflavones on the fibrillation of hen egg-white lysozyme.

In this paper, the effects of three isoflavones including daidzein, genistein, and puerarin on fibrillation of hen egg-white lysozyme were investigated by various analytical methods. The results demonstrated that all isoflavones could effectively inhibit the fibrillogenesis of hen egg-white lysozyme and destabilized the preformed fibrils of hen egg-white lysozyme in a dose-dependent manner. To further understand the inhibition mechanism, molecular modeling was carried out. The docking results demonstrated that the isoflavones could bind to two key fibrogenic sites in hen egg-white lysozyme through van der Waals force, electrostatic forces, and hydrogen bonding, as well as σ-π stacking. By these means, isoflavones could not only obviously enhance the hydrophobicity of the binding sites, but also greatly stabilize the native state of HEWL, which was able to postpone the fibrosis process of hen egg-white lysozyme.

Effect of polyphenols from Syringa vulgaris on blood stasis syndrome.

In this study, we employed a previously developed in vivo assay system to determine whether the flowers and leaves of Syringa vulgaris (S. vulgaris; commonly known as "lilac") can prevent blood stasis syndrome, known as oketsu in Japanese. This syndrome is considered an important pathology in traditional Chinese and Japanese medicine, and is related to diseases such as peripheral vascular disorders, blood vessel inflammation, and platelet aggregation, whose severities are augmented owing to lipid peroxidation, free radicals, and oxidative stress. The assay system employed in this study monitored the blood flow decrease in the tail vein of mice subjected to sensitization with hen egg white lysozyme. Through bioassay-guided fractionation of different S. vulgaris extracts, five polyphenols were isolated and identified. Among them, quercetine 3-glucoside, quercetin 3-rutinoside, and acteoside were identified as active compounds, as they significantly mitigated blood flow reduction. These findings indicate that the polyphenols obtained from S. vulgaris could be useful for preventing oketsu and improve the quality of life of individuals with disorders and diseases such as gynecopathy, cold sensitivity, poor circulation, allergy, and lifestyle-related diseases.

Multi-spectroscopic and molecular dynamics simulations investigation of the binding mechanism of polybrominated diphenyl ethers to hen egg white lysozyme.

Three PBDEs (BDE25, BDE47, and BDE154) were selected to investigate the interactions between PBDEs and hen egg white lysozyme (HEWL) by molecular modeling, fluorescence spectroscopy, and FT-IR spectra. The docking results showed that hydrogen bonds were formed between BDE25 and residue TRP63 and between BDE47 and TRP63 with bond lengths of 2.178 Å and 2.146 Å, respectively. The molecular dynamics simulations indicated that van der Waals forces played a predominant role in the binding of three PBDEs to HEWL. The observed fluorescence quenching can be attributed to the formation of complexes between HEWL and PBDEs, and the quenching mechanism is a static quenching. According to Förster's non-radiative energy transfer theory, the binding distances r were < 7 nm, indicating a high probability of energy transfer from HEWL to the three PBDEs. The synchronous fluorescence showed that the emission maximum wavelength of tryptophan (TRP) residues emerged a red-shift. FT-IR spectra indicated that BDE25, BDE47 and BDE154 induced the α-helix percentage of HEWL decreased from 32.70% ± 1.64% to 28.27% ± 1.41%, 27.50% ± 1.38% and 29.78% ± 1.49%, respectively, whereas the percentage of random coil increased from 26.67% ± 1.33% to 27.60% ± 1.38%, 29.18% ± 1.46% and 30.59% ± 1.53%, respectively.

Effect of Technically Relevant X-Ray Doses on the Structure and Function of Alcohol Dehydrogenase and Hen Egg-White Lysozyme.

PURPOSE: The effect of different irradiation doses on the structure and activity of lyophilized powders of Hen Egg-White Lysozyme (HEWL) and alcohol dehydrogenase (ADH) was investigated using these substances as models for robust and sensitive proteins, respectively. Three doses were selected to cover the ranges of radio-sterilization (25kGy), treatment of blood products (25Gy) and annual background radiation dose (approximately 2mGy). The results offer an initial screening of different irradiation doses and support the development of X-ray imaging methods as non-destructive process analytical technology (PAT) tools for detecting the visible particulate matters in such products. METHODS: HEWL and ADH were exposed to X-rays in the solid state. The effect of irradiation was determined directly after irradiation and after storage. Structural changes and degradation were investigated using SAXS, SDS-PAGE and HPLC-MS. Protein functionality was assessed via activity assays. RESULTS: Lower irradiation doses of 25Gy and 2mGy had no significant impact on the structure and enzyme activity. The dose of 25kGy caused a significant decrease in the enzyme activity and structural changes immediately after irradiation of ADH and after storage of irradiated HEWL at -20°C. CONCLUSION: The results emphasize the importance of careful selection of radiation doses for development of X-ray imaging methods as PAT tools inspection of solid biopharmaceutical products.

Effect of silybin on the fibrillation of hen egg-white lysozyme.

In this study, the fibrillation of hen egg-white lysozyme (HEWL) in the absence and presence of different concentrations of silybin was studied by thioflavin T spectroscopy, Congo red binding assays, 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence assay, circular dichroism, and transmission electron microscopy. The experimental results indicated that not only the fibrillation of HEWL at high temperature (65°C) and low pH (pH = 2.0) could be inhibited effectively by silybin but also the inhibition of HEWL by silybin followed a dose-dependent manner. Molecular docking studies indicated that 2 possible binding modes could be found in the interaction between silybin and HEWL via van der Waals forces and electrostatic forces as well as hydrogen bonding. One of these 2 conformations was directly entered into the cavity of HEWL (binding site I); the other was bound to the surface of HEWL (binding site II). In this way, silybin could not only increase the hydrophobicity of the cavity or the surface of HEWL but also influence the microenvironment of the binding site, which was able to stabilize the structure of HEWL and delay the process of HEWL fibrosis.

Effect of temperature on the interaction of cisplatin with the model protein hen egg white lysozyme.

The products of the reaction between cisplatin (CDDP) and the model protein hen egg white lysozyme (HEWL) at 20, 37 and 55 °C in pure water were studied by UV-Vis absorption spectroscopy, intrinsic fluorescence and circular dichroism, dynamic and electrophoretic light scattering and inductively coupled plasma mass spectrometry. X-ray structures were also solved for the adducts formed at 20 and 55 °C. Data demonstrate that high temperature facilitates the formation of CDDP-HEWL adducts, where Pt atoms bind ND1 atom of His15 or NE2 atom of His15 and NH1 atom of Arg14. Our study suggests that high human body temperature (fever) could increase the rate of drug binding to proteins thus enhancing possible toxic side effects related to CDDP administration.

Low Abundant N-linked Glycosylation in Hen Egg White Lysozyme Is Localized at Nonconsensus Sites.

Although wild-type hen egg white lysozyme (HEL) is lacking the consensus sequence motif NX(S/T), in 1995 Trudel et al. (Biochem. Cell Biol. 1995, 73, 307-309) proposed the existence of a low abundant N-glycosylated form of HEL; however, the identity of active glycosylation sites in HEL remained a matter of speculation. For the first time since Trudel's initial work, we report here a comprehensive characterization by means of mass spectrometry of N-glycosylation in wild-type HEL. Our analytical approach comprised ZIC-HILIC enrichment of N-glycopeptides from HEL trypsin digest, deglycosylation by (18)O/PNGase F as well as by various endoglycosidases, and LC-MS/MS analysis of both intact and deglycosylated N-glycopeptides engaging multiple techniques of ionization and fragmentation. A novel data interpretation workflow based on MS/MS spectra classification and glycan database searching enabled the straightforward identification of the asparagine-rich N-glycopeptide [34-45] FESNFNTQATNR and allowed for compositional profiling of its modifying N-glycans. The overall heterogeneity profile of N-glycans in HEL comprised at least 26 different compositions. Results obtained from deglycosylation experiments provided clear evidence of asparagine residues N44 and N39 representing active glycosylation sites in HEL. Both of these sites do not fall into any known N-glycosylation-specific sequence motif but are localized in rarely observed nonconsensus sequons (NXN, NXQ).

Hydrolytic enzymes conjugated to quantum dots mostly retain whole catalytic activity.

BACKGROUND: Tagging a luminescent quantum dot (QD) with a biological like enzyme (Enz) creates value-added entities like quantum dot-enzyme bioconjugates (QDEnzBio) that find utility as sensors to detect glucose or beacons to track enzymes in vivo. For such applications, it is imperative that the enzyme remains catalytically active while the quantum dot is luminescent in the bioconjugate. A critical feature that dictates this is the quantum dot-enzyme linkage chemistry. Previously such linkages have put constraints on polypeptide chain dynamics or hindered substrate diffusion to active site, seriously undermining enzyme catalytic activity. In this work we address this issue using avidin-biotin linkage chemistry together with a flexible spacer to conjugate enzyme to quantum dot. METHODS: The catalytic activity of three biotinylated hydrolytic enzymes, namely, hen egg white lysozyme (HEWL), alkaline phosphatase (ALP) and acetylcholinesterase (AChE) was investigated post-conjugation to streptavidin linked quantum dot for multiple substrate concentrations and varying degrees of biotinylation. RESULTS: We demonstrate that all enzymes retain full catalytic activity in the quantum dot-enzyme bioconjugates in comparison to biotinylated enzyme alone. However, unlike alkaline phosphatase and acetylcholinesterase, the catalytic activity of hen egg white lysozyme was observed to be increasingly susceptible to ionic strength of medium with rising level of biotinylation. This susceptibility was attributed to arise from depletion of positive charge from lysine amino groups after biotinylation. CONCLUSIONS: We reasoned that avidin-biotin linkage in the presence of a flexible seven atom spacer between biotin and enzyme poses no constraints to enzyme structure/dynamics enabling retention of full enzyme activity. GENERAL SIGNIFICANCE: Overall our results demonstrate for the first time that streptavidin-biotin chemistry can yield quantum dot enzyme bioconjugates that retain full catalytic activity as native enzyme.

High-pressure protein crystallography of hen egg-white lysozyme.

Crystal structures of hen egg-white lysozyme (HEWL) determined under pressures ranging from ambient pressure to 950 MPa are presented. From 0.1 to 710 MPa, the molecular and internal cavity volumes are monotonically compressed. However, from 710 to 890 MPa the internal cavity volume remains almost constant. Moreover, as the pressure increases to 950 MPa, the tetragonal crystal of HEWL undergoes a phase transition from P43212 to P43. Under high pressure, the crystal structure of the enzyme undergoes several local and global changes accompanied by changes in hydration structure. For example, water molecules penetrate into an internal cavity neighbouring the active site and induce an alternate conformation of one of the catalytic residues, Glu35. These phenomena have not been detected by conventional X-ray crystal structure analysis and might play an important role in the catalytic activity of HEWL.

Hen egg-white lysozyme crystallisation: protein stacking and structure stability enhanced by a Tellurium(VI)-centred polyoxotungstate.

As synchrotron radiation becomes more intense, detectors become faster and structure-solving software becomes more elaborate, obtaining single crystals suitable for data collection is now the bottleneck in macromolecular crystallography. Hence, there is a need for novel and advanced crystallisation agents with the ability to crystallise proteins that are otherwise challenging. Here, an Anderson-Evans-type polyoxometalate (POM), specifically Na6 [TeW6 O24 ]⋅22 H2 O (TEW), is employed as a crystallisation additive. Its effects on protein crystallisation are demonstrated with hen egg-white lysozyme (HEWL), which co-crystallises with TEW in the vicinity (or within) the liquid-liquid phase separation (LLPS) region. The X-ray structure (PDB ID: 4PHI) determination revealed that TEW molecules are part of the crystal lattice, thus demonstrating specific binding to HEWL with electrostatic interactions and hydrogen bonds. The negatively charged TEW polyoxotungstate binds to sites with a positive electrostatic potential located between two (or more) symmetry-related protein chains. Thus, TEW facilitates the formation of protein-protein interfaces of otherwise repulsive surfaces, and thereby the realisation of a stable crystal lattice. In addition to retaining the isomorphicity of the protein structure, the anomalous scattering of the POMs was used for macromolecular phasing. The results suggest that hexatungstotellurate(VI) has great potential as a crystallisation additive to promote both protein crystallisation and structure elucidation.

The active site of hen egg-white lysozyme: flexibility and chemical bonding.

Chemical bonding at the active site of hen egg-white lysozyme (HEWL) is analyzed on the basis of Bader's quantum theory of atoms in molecules [QTAIM; Bader (1994), Atoms in Molecules: A Quantum Theory. Oxford University Press] applied to electron-density maps derived from a multipole model. The observation is made that the atomic displacement parameters (ADPs) of HEWL at a temperature of 100 K are larger than ADPs in crystals of small biological molecules at 298 K. This feature shows that the ADPs in the cold crystals of HEWL reflect frozen-in disorder rather than thermal vibrations of the atoms. Directly generalizing the results of multipole studies on small-molecule crystals, the important consequence for electron-density analysis of protein crystals is that multipole parameters cannot be independently varied in a meaningful way in structure refinements. Instead, a multipole model for HEWL has been developed by refinement of atomic coordinates and ADPs against the X-ray diffraction data of Wang and coworkers [Wang et al. (2007), Acta Cryst. D63, 1254-1268], while multipole parameters were fixed to the values for transferable multipole parameters from the ELMAM2 database [Domagala et al. (2012), Acta Cryst. A68, 337-351] . Static and dynamic electron densities based on this multipole model are presented. Analysis of their topological properties according to the QTAIM shows that the covalent bonds possess similar properties to the covalent bonds of small molecules. Hydrogen bonds of intermediate strength are identified for the Glu35 and Asp52 residues, which are considered to be essential parts of the active site of HEWL. Furthermore, a series of weak C-H...O hydrogen bonds are identified by means of the existence of bond critical points (BCPs) in the multipole electron density. It is proposed that these weak interactions might be important for defining the tertiary structure and activity of HEWL. The deprotonated state of Glu35 prevents a distinction between the Phillips and Koshland mechanisms.

Weak protein-cationic co-ion interactions addressed by X-ray crystallography and mass spectrometry.

The adsorption of Rb(+), Cs(+), Mn(2+), Co(2+) and Yb(3+) onto the positively charged hen egg-white lysozyme (HEWL) has been investigated by solving 13 X-ray structures of HEWL crystallized with their chlorides and by applying electrospray ionization mass spectrometry (ESI-MS) first to dissolved protein crystals and then to the protein in buffered salt solutions. The number of bound cations follows the order Cs(+) < Mn(2+) ≃ Co(2+) < Yb(3+) at 293 K. HEWL binds less Rb(+) (qtot = 0.7) than Cs(+) (qtot = 3.9) at 100 K. Crystal flash-cooling drastically increases the binding of Cs(+), but poorly affects that of Yb(3+), suggesting different interactions. The addition of glycerol increases the number of bound Yb(3+) cations, but only slightly increases that of Rb(+). HEWL titrations with the same chlorides, followed by ESI-MS analysis, show that only about 10% of HEWL binds Cs(+) and about 40% binds 1-2 Yb(3+) cations, while the highest binding reaches 60-70% for protein binding 1-3 Mn(2+) or Co(2+) cations. The binding sites identified by X-ray crystallography show that the monovalent Rb(+) and Cs(+) preferentially bind to carbonyl groups, whereas the multivalent Mn(2+), Co(2+) and Yb(3+) interact with carboxylic groups. This work elucidates the basis of the effect of the Hofmeister cation series on protein solubility.

Capillary Flow-Based One-Minute Quantification of Amyloid Proteolysis.

Quantifying the formation and decomposition of amyloid is a crucial issue in the development of new drugs and therapies for treating amyloidosis. The current technologies for grasping amyloid formation and decomposition include fluorescence analysis using thioflavin-T, secondary structure analysis using circular dichroism, and image analysis using atomic force microscopy or transmission electron microscopy. These technologies typically require spectroscopic devices or expensive nanoscale imaging equipment and involve lengthy analysis, which limits the rapid screening of amyloid-degrading drugs. In this study, we introduce a technology for rapidly assessing amyloid decomposition using capillary flow-based paper (CFP). Amyloid solutions exhibit gel-like physical properties due to insoluble denatured polymers, resulting in a shorter flow distance on CFP compared to pure water. Experimental conditions were established to consistently control the flow distance based on a hen-egg-white lysozyme amyloid solution. It was confirmed that as amyloid is decomposed by trypsin, the flow distance increases on the CFP. Our method is highly useful for detecting changes in the gel properties of amyloid solutions within a minute, and we anticipate its use in the rapid, large-scale screening of anti-amyloid agents in the future.

Carboxylic Group Functionalized Carbon Quantum Dots inhibit Hen Egg White Lysozyme Amyloidogenesis, leading to the Formation of Spherical Aggregates with Reduced Toxicity and ROS Generation.

INTRODUCTION: Proteinopathies are a group of diseases where the protein structure has been altered. These alterations are linked to the production of amyloids, which are persistent, organized clumps of protein molecules through inter-molecular interactions. Several disorders, including Alzheimer's and Parkinson's, have been related to the presence of amyloids. Highly ordered beta sheets or beta folds are characteristic of amyloids; these structures can further self- assemble into stable fibrils. METHODS: Protein aggregation is caused by a wide variety of environmental and experimental factors, including mutations, high pH, high temperature, and chemical modification. Despite several efforts, a cure for amyloidosis has yet to be found. Due to its advantageous semi-conducting characteristics, unique optical features, high surface area-to-volume ratio, biocompatibility, etc., carbon quantum dots (CQDs) have lately emerged as key instruments for a wide range of biomedical applications. To this end, we have investigated the effect of CQDs with a carboxyl group on their surface (CQD-CA) on the in vitro amyloidogenesis of hen egg white lysozyme (HEWL). RESULTS: By generating a stable compound that is resistant to fibrillation, our findings show that CQD-CA can suppress amyloid and disaggregate HEWL. In addition, CQD-CA caused the creation of non-toxic spherical aggregates, which generated much less reactive oxygen species (ROS). CONCLUSION: Overall, our results show that more research into amyloidosis treatments, including surface functionalized CQDs, is warranted.