Inhibition and disaggregation effect of flavonoid-derived carbonized polymer dots on protein amyloid aggregation.

In this research, four water-insoluble flavonoid compounds were utilized and reacted with arginine to prepare four carbonized polymer dots with good water-solubility in a hydrothermal reactor. Structural characterization demonstrated that the prepared carbonized polymer dots were classic core-shell structure. Effect of the prepared carbonized polymer dots on protein amyloid aggregation was further investigated using hen egg white lysozyme and human lysozyme as model protein in aqueous solution. All of the prepared carbonized polymer dots could retard the amyloid aggregation of hen egg white lysozyme and human lysozyme in a dose-depended manner. All measurements displayed that the inhibition ratio of luteolin-derived carbonized polymer dots (CPDs-1) was higher than that of the other three carbonized polymer dots under the same dosage. This result may be interpreted by the highest content of phenolic hydroxyl groups on the periphery. The inhibition ratio of CPDs-1 on hen egg white lysozyme and human lysozyme reached 88 % and 83 % at the concentration of 0.5 mg/mL, respectively. CPDs-1 also could disaggregate the formed mature amyloid fibrils into short aggregates.

Co-Assembled Nanoparticles toward Multi-Target Combinational Therapy of Alzheimer's Disease by Making Full Use of Molecular Recognition and Self-Assembly.

The complex pathologies in Alzheimer's disease (AD) severely limit the effectiveness of single-target pharmic interventions, thus necessitating multi-pronged therapeutic strategies. While flexibility is essentially demanded in constructing such multi-target systems, for achieving optimal synergies and also accommodating the inherent heterogeneity within AD. Utilizing the dynamic reversibility of supramolecular strategy for conferring sufficient tunability in component substitution and proportion adjustment, amphiphilic calixarenes are poised to be a privileged molecular tool for facilely achieving function integration. Herein, taking ß-amyloid (Aß) fibrillation and oxidative stress as model combination pattern, a supramolecular multifunctional integration is proposed by co-assembling guanidinium-modified calixarene with ascorbyl palmitate and loading dipotassium phytate within calixarene cavity. Serial pivotal events can be simultaneously addressed by this versatile system, including 1) inhibition of Aß production and aggregation, 2) disintegration of Aß fibrils, 3) acceleration of Aß metabolic clearance, and 4) regulation of oxidative stress, which is verified to significantly ameliorate the cognitive impairment of 5×FAD mice, with reduced Aß plaque content, neuroinflammation, and neuronal apoptosis. Confronted with the extremely intricate clinical realities of AD, the strategy presented here exhibits ample adaptability for necessary alterations on combinations, thereby may immensely expedite the advancement of AD combinational therapy through providing an exceptionally convenient platform.

The food and pharmaceutical additive benzoic acid induces amyloid fibrillation of an intrinsically disordered protein.

Benzoic acid (BA) is a microbe-inhibiting flavoring agent used extensively as an additive in foods, pharmaceuticals, and hygiene and cosmetic products. The level of BA in foodstuffs prescribed by world bodies and governmental agencies is assumed to be safe so as to prevent adverse health effects. The safety level of BA is however controversial, and whether different conditions of its use would be generally regarded as safe (GRAS) has been rarely determined. In the quest of how food additives affect the structure and conformation of proteins, this study evaluates the interaction of BA with an intrinsically disordered protein (IDP) at pH 4.2 that matches the pH conditions applicable for the commercial use of benzoate preservatives, and examines its structural transformation by NMR, fluorescence, and high-resolution microscopy. The interaction with BA transforms the protein to a denatured aggregated mesophase that undergoes reconfiguration to yield rigid amyloid fibrils. Significantly, fibrils are observed even with 0.1 mM BA while the recommended level of its use as a preservative is in the 0.4-8 mM range. The discussion refrains from safety comments with no projection of the BA level that could be GRAS.

Protein Compartments Modulate Fibrillar Self-Assembly.

A notable feature of complex cellular environments is protein-rich compartments that are formed via liquid-liquid phase separation. Recent studies have shown that these biomolecular condensates can play both promoting and inhibitory roles in fibrillar protein self-assembly, a process that is linked to Alzheimer's, Parkinson's, Huntington's, and various prion diseases. Yet, the exact regulatory role of these condensates in protein aggregation remains unknown. By employing microfluidics to create artificial protein compartments, the self-assembly behavior of the fibrillar protein lysozyme within them can be characterized. It is observed that the volumetric parameters of protein-rich compartments can change the kinetics of protein self-assembly. Depending on the change in compartment parameters, the lysozyme fibrillation process either accelerated or decelerated. Furthermore, the results confirm that the volumetric parameters govern not only the nucleation and growth phases of the fibrillar aggregates but also affect the crosstalk between the protein-rich and protein-poor phases. The appearance of phase-separated compartments in the vicinity of natively folded protein complexes triggers their abrupt percolation into the compartments' core and further accelerates protein aggregation. Overall, the results of the study shed more light on the complex behavior and functions of protein-rich phases and, importantly, on their interaction with the surrounding environment.

Towards Optimal Control of Amyloid Fibrillation.

Epigallocatechin-3-gallate, as a representative amyloid inhibitors, has shown a promising ability against A[Formula: see text] fibrillation by directly degradating the mature fibrils. Most previous studies have been focusing on its functional mechanisms, meanwhile its optimal dosage has been seldom considered. To solve this critical issue, we refer to the generalized Logistic model for amyloid fibrillation and inhibition and adopt the optimal control theory to balance the effectiveness and cost (or toxicity) of inhibitors. The optimal control trajectory of inhibitors is analytically solved, based on which the influence of model parameters, the difference between the optimal control strategy and several other traditional drug dosing strategies are systematically compared and validated through experiments. It is found that the strategy of multiple-times adding is more suitable for a long-term disease treatment, while single high-dose therapy is preferred for a short-term treatment. We hope our findings can shed light on the rational usage of amyloid inhibitors in clinic.

Inherent Adaptivity of Alzheimer Peptides to Crowded Environments.

Amyloid ß (Aß) is the major constituent in senile plaques of Alzheimer's disease in which peptides initially undergo structural conversions to form elongated fibrils. The impact of crowding on the fibrillation pathways of Aß40 and Aß42 , the most common peptide isoforms are studied. PEG and Ficoll are used as model crowders to mimic a macromolecular enriched surrounding. The fibrillar growth is monitored with the help of ThT-fluorescence assays in order to extract two rates describing primary and secondary processes of nucleation and growth. Techniques as fluorescence correlation spectroscopy and analytical ultracentrifugation are used to discuss oligomeric states; fibril morphologies are investigated using negative-staining transmission electron microscopy. While excluded volume effects imposed by macromolecular crowding are expected to always increase rates of intermolecular interactions and structural conversion, a vast variety of effects are found depending on the peptide, the crowder, or ionic strength of the solution. While investigations of the obtained rates with respect to a reactant-occluded model are capable to display specific surface interactions with the crowder, the employment of crystallization-like models reveal the crowder-induced entropic gain with Δ Δ G fib crow = - 116 ± 21 k $\Delta \Delta G_{\text{fib}}^{\text{crow}}=-116\pm 21\; k$ J mol-1 per volume fraction of the crowder.

Coiled-coil structure mediated inhibition of the cytotoxic huntingtin amyloid fibrils by an IP3 receptor fragment.

Disruption of cellular homeostasis by the aggregation of polyglutamine (polyQ) in the huntingtin protein (Htt) leads Huntington's disease (HD). Effective drugs for treating HD have not been developed, as the molecular mechanism underlying HD pathogenesis remains unclear. To develop strategies for inhibiting HD pathogenesis, the intermolecular interaction of Htt with IP3 receptor 1 (IP3R1) was investigated. Peptide (termed ICT60) corresponding to a coiled-coil motif in the C-terminus of IP3R1 was designed. Several biophysical approaches revealed the strong and specific binding of ICT60 to the N-terminal part of HttEx1. ICT60 inhibited not only amyloid formation by HttEx1, but also the cytotoxicity and cell-penetration ability of the amyloid fibrils of HttEx1. The importance of coiled-coil structure was verified by charge-manipulated variants. The coiled-coil structures of ICT60-KK and -EE were partially and largely disrupted, respectively. ICT60 wild-type and -KK inhibited amyloid formation by HttEx1-46Q, whereas ICT60-EE did not block amyloidogenesis. Similarly, the cytotoxicity and cell-penetration ability of the amyloid fibrils of HttEx1-46Q were efficiently inhibited by ICT60 wild-type and ICT60-KK, but not by ICT60-EE. We propose a mechanical model explaining how an IP3 receptor-inspired molecule can modulate cytotoxic amyloid formation by Htt, providing a molecular basis for developing therapeutics to treat HD.

Effect of mono- and diketone group in curcumin analogues on amyloid fibrillation of hen egg white lysozyme.

Curcumin has attracted more attention because of its inhibition efficacy on protein amyloid fibrillation. However, the inhibition mechanism was still ambiguous and the clinical application of curcumin was greatly limited because of its poor stability at physiological conditions for the presence of ß-diketone moiety. In this paper, a new mono-ketone-containing curcumin analogue (MDHC) was designed and synthesized to realize the possible inhibition mechanism and unveil the important role of ß-diketone moiety of curcumin in the inhibition process of amyloid fibrillation using hen egg white lysozyme (HEWL) as model protein. Although all experiment results (ThT, CR, ANS and TEM) showed that the inhibitory capacity of curcumin was better than MDHC, MDHC still could show obvious inhibition effect. Molecular docking showed that both curcumin and MDHC could bind with HEWL by hydrogen bond of phenloic hydroxyl and the binding energy of MDHC was higher than that of curcumin. All the findings inferred that ß-diketone group was one of great important groups in the inhibition process of HEWL amyloid fibrillation, which provided more room to construct novel inhibition reagents.

The anti-platelet drug ticlopidine inhibits FapC fibrillation and biofilm production: Highlighting its antibiotic activity.

Multidrug resistance of bacteria and persistent infections related to biofilms, as well as the low availability of new antibacterial drugs, make it urgent to develop new antibiotics. Here, we evaluate the antibacterial and anti-biofilm properties of ticlopidine (TP), an anti-platelet aggregation drug, TP showed antibacterial activity against both gram-positive (MRSA) and gram-negative (E. coli, and P. aeruginosa) bacteria over a long treatment period. TP significantly reduced the survival of gram-negative bacteria in human blood though impact on gram-positives was more limited. TP may cause death in MRSA by inhibiting staphyloxanthin pigment synthesis, leading to oxidative stress, while scanning electron microscopy imaging indicate a loss of membrane integrity, damage, and consequent death due to lysis in gram-negative bacteria. TP showed good anti-biofilm activity against P. aeruginosa and MRSA, and a stronger biofilm degradation activity on P. aeruginosa compared to MRSA. Measuring fluorescence of the amyloid-reporter Thioflavin T (ThT) in biofilm implicated inhibition of amyloid formation as part of TP activity. This was confirmed by assays on the purified protein in P. aeruginosa, FapC, whose fibrillation kinetics was inhibited by TP. TP prolonged the lag phase of aggregation and reduced the subsequent growth rate and prolonging the lag phase to very long times provides ample opportunity to exert TP's antibacterial effect. We conclude that TP shows activity as an antibiotic against both gram-positive and gram-negative bacteria thanks to a broad range of activities, targeting bacterial metabolic processes, cellular structures and the biofilm matrix.

Unveiling the Metal-Dependent Aggregation Properties of the C-terminal Region of Amyloidogenic Intrinsically Disordered Protein Isoforms DPF3b and DPF3a.

Double-PHD fingers 3 (DPF3) is a BAF-associated human epigenetic regulator, which is increasingly recognised as a major contributor to various pathological contexts, such as cardiac defects, cancer, and neurodegenerative diseases. Recently, we unveiled that its two isoforms (DPF3b and DPF3a) are amyloidogenic intrinsically disordered proteins. DPF3 isoforms differ from their C-terminal region (C-TERb and C-TERa), containing zinc fingers and disordered domains. Herein, we investigated the disorder aggregation properties of C-TER isoforms. In agreement with the predictions, spectroscopy highlighted a lack of a highly ordered structure, especially for C-TERa. Over a few days, both C-TERs were shown to spontaneously assemble into similar antiparallel and parallel ß-sheet-rich fibrils. Altered metal homeostasis being a neurodegeneration hallmark, we also assessed the influence of divalent metal cations, namely Cu2+, Mg2+, Ni2+, and Zn2+, on the C-TER aggregation pathway. Circular dichroism revealed that metal binding does not impair the formation of ß-sheets, though metal-specific tertiary structure modifications were observed. Through intrinsic and extrinsic fluorescence, we found that metal cations differently affect C-TERb and C-TERa. Cu2+ and Ni2+ have a strong inhibitory effect on the aggregation of both isoforms, whereas Mg2+ impedes C-TERb fibrillation and, on the contrary, enhances that of C-TERa. Upon Zn2+ binding, C-TERb aggregation is also hindered, and the amyloid autofluorescence of C-TERa is remarkably red-shifted. Using electron microscopy, we confirmed that the metal-induced spectral changes are related to the morphological diversity of the aggregates. While metal-treated C-TERb formed breakable and fragmented filaments, C-TERa fibrils retained their flexibility and packing properties in the presence of Mg2+ and Zn2+ cations.

A penetratin-derived peptide reduces the membrane permeabilization and cell toxicity of α-synuclein oligomers.

Parkinson's disease is a neurodegenerative movement disorder associated with the intracellular aggregation of α-synuclein (α-syn). Cytotoxicity is mainly associated with the oligomeric species (αSOs) formed at early stages in α-syn aggregation. Consequently, there is an intense focus on the discovery of novel inhibitors such as peptides to inhibit oligomer formation and toxicity. Here, using peptide arrays, we identified nine peptides with high specificity and affinity for αSOs. Of these, peptides p194, p235, and p249 diverted α-syn aggregation from fibrils to amorphous aggregates with reduced ß-structures and increased random coil content. However, they did not reduce αSO cytotoxicity and permeabilization of large anionic unilamellar vesicles. In parallel, we identified a non-self-aggregating peptide (p216), derived from the cell-penetrating peptide penetratin, which showed 12-fold higher binding affinity to αSOs than to α-syn monomers (Kdapp 2.7 and 31.2 µM, respectively). p216 reduced αSOs-induced large anionic unilamellar vesicle membrane permeability at 10-1 to 10-3 mg/ml by almost 100%, was not toxic to SH-SY5Y cells, and reduced αSOs cytotoxicity by about 20%. We conclude that p216 is a promising starting point from which to develop peptides targeting toxic αSOs in Parkinson's disease.

From Small Peptides to Large Proteins against Alzheimer'sDisease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly. The two cardinal neuropathological hallmarks of AD are the senile plaques, which are extracellular deposits mainly constituted by beta-amyloids, and neurofibrillary tangles formed by abnormally phosphorylated Tau (p-Tau) located in the cytoplasm of neurons. Although the research has made relevant progress in the management of the disease, the treatment is still lacking. Only symptomatic medications exist for the disease, and, in the meantime, laboratories worldwide are investigating disease-modifying treatments for AD. In the present review, results centered on the use of peptides of different sizes involved in AD are presented.

Orange G is a potential inhibitor of human insulin amyloid fibrillation and can be used as a probe to study mechanism of amyloid fibrillation and its inhibition.

The extracellular insoluble deposits of highly ordered cross-ß-structure-containing amyloid fibrils form the pathological basis for protein misfolding diseases. As amyloid fibrils are cytotoxic, inhibition of the process is a therapeutic strategy. Several small molecules have been identified and used as fibrillation inhibitors in the recent past. In this work, we investigate the effect of Orange G on insulin amyloid formation using fluorescence-based assays and negative-stain electron microscopy (EM). We show that Orange G effectively attenuates nucleation, thereby inhibiting amyloid fibrillation in a dose-dependent manner. Fluorescence quenching titrations of Orange G showed a reasonably strong binding affinity to native insulin. Binding isotherm measurements revealed the binding of Orange G to pre-formed insulin fibrils too, indicating that Orange G likely binds and stabilizes the mature fibrils and prevents the release of toxic oligomers which could be potential nuclei or templates for further fibrillation. Molecular docking of Orange G with native insulin and amyloid-like peptide structures were also carried out to analyse the contributing interactions and binding free energy. The findings of our study emphasize the use of Orange G as a molecular probe to identify and design inhibitors of amyloid fibrillation and to investigate the structural and toxic mechanisms underlying amyloid formation.

Heparin promotes rapid fibrillation of the basic parathyroid hormone at physiological pH.

In acidic secretory granules of mammalian cells, peptide hormones including the parathyroid hormone are presumably stored in the form of functional amyloid fibrils. Mature PTH, however, is considerably positively charged in acidic environments, a condition known to impede unassisted self-aggregation into fibrils. Here, we studied the role of the polyanion heparin on promoting fibril formation of PTH. Employing ITC, CD spectroscopy, NMR, SAXS, and fluorescence-based assays, we could demonstrate that heparin binds PTH with submicromolar affinity and facilitates its conversion into fibrillar seeds, enabling rapid formation of amyloid fibrils under acidic conditions. In the absence of heparin, PTH remained in a soluble monomeric state. We suspect that heparin-like surfaces are required in vivo to convert PTH efficiently into fibrillar deposits.

Insulin Complexation with Cyclodextrins-A Molecular Modeling Approach.

Around 5% of the population of the world is affected with the disease called diabetes mellitus. The main medication of the diabetes is the insulin; the active form is the insulin monomer, which is an instable molecule, because the long storage time, or the high temperature, can cause the monomer insulin to adapt an alternative fold, rich in ß-sheets, which is pharmaceutically inactive. The aim of this study is to form different insulin complexes with all the cyclodextrin used for pharmaceutical excipients (native cyclodextrin, methyl, hydroxyethyl, hydroxypropyl and sulfobutylether substituted ß-cyclodextrin), in silico condition, with the AutoDock molecular modeling program, to determine the best type of cyclodextrin or cyclodextrin derivate to form a complex with an insulin monomer, to predict the molar ratio, the conformation of the complex, and the intermolecular hydrogen bonds formed between the cyclodextrin and the insulin. From the results calculated by the AutoDock program it can be predicted that insulin can make a stable complex with 5-7 molecules of hydroxypropyl-ß-cyclodextrin or sulfobutylether-ß-cyclodextrin, and by forming a complex potentially can prevent or delay the amyloid fibrillation of the insulin and increase the stability of the molecule.

Inhibition potential evaluation of two synthetic bis-indole compounds on amyloid fibrillation: a molecular simulation study.

Protein aggregation is known as the main mechanism of amyloid fibrillation in amyloidosis diseases. Recent studies confirmed that compounds with one or two indole rings have inhibitory potential against amyloid fibrillation. Herein, the interaction of two similar compounds 'bis(indolyl)-2-methyl-phenyl-methene' and 'bis(indolyl)-2-chloro-phenyl-methene' with an amyloid core model was investigated. To this aim, molecular docking and all-atom molecular dynamics (MD) simulations were used. Docking results between aggregation-prone region (APR) of hen egg-white lysozyme (HEWL) and either of ligands showed that they interact with different residues of the APR (amyloid fibril nucleus). According to MD results, bis(indolyl)-2-methyl-phenyl-methene made a distance between the two cores, which was 1.5 times greater than that bis(indolyl)-2-chloro-phenyl-methene made. Analysis of RMSD/RMSF values revealed that bis(indolyl)-2-methyl-phenyl-methene stabilized strands of A and B, while destabilized strands C and D. The hydrophobic 'methyl' functional group in bis(indolyl)-2-methyl-phenyl-methene facilitate its deep penetration between core nuclei, via destabilizing outer strands of C and D. Considering this fact that results of this study are in agreement with experimental findings, details of the discovered mechanism of interaction between ligands and HEWL's APR would be inspiring for further anti-fibrillation drug designs.Communicated by Ramaswamy H. Sarma.

Is amyloid fibrillation related to 3D domain swapping for the C-terminal domain of SARS-CoV main protease?

The C-terminal domain of SARS-CoV main protease (Mpro-C) can form 3D domain-swapped dimer by exchanging the α1-helices fully buried inside the protein hydrophobic core, under non-denaturing conditions. Here, we report that Mpro-C can also form amyloid fibrils under the 3D domain-swappable conditions in vitro, and the fibrils are not formed through runaway/propagated domain swapping. It is found that there are positive correlations between the rates of domain swapping dimerization and amyloid fibrillation at different temperatures, and for different mutants. However, some Mpro-C mutants incapable of 3D domain swapping can still form amyloid fibrils, indicating that 3D domain swapping is not essential for amyloid fibrillation. Furthermore, NMR H/D exchange data and molecular dynamics simulation results suggest that the protofibril core region tends to unpack at the early stage of 3D domain swapping, so that the amyloid fibrillation can proceed during the 3D domain swapping process. We propose that 3D domain swapping makes it possible for the unpacking of the amyloidogenic fragment of the protein and thus accelerates the amyloid fibrillation process kinetically, which explains the well-documented correlations between amyloid fibrillation and 3D domain swapping observed in many proteins.

Interfacial Interactions within Amyloid Protein Corona Based on 2D MoS2 Nanosheets.

The interfacial interaction within the amyloid protein corona based on MoS2 nanomaterial is crucial, both for understanding the biological effects of MoS2 nanomaterial and the evolution of amyloid diseases. The specific nano-bio interface phenomenon of human islet amyloid peptide (hIAPP) and MoS2 nanosheet was investigated by using theoretical and experimental methods. The MoS2 nanosheet enables the attraction of hIAPP monomer, dimer, and oligomer on its surface through van der Waals forces. Especially, the means of interaction between two hIAPP peptides might be changed by MoS2 nanosheet. In addition, it is interesting to find that the hIAPP oligomer can stably interact with the MoS2 nanosheet in one unique "standing" binding mode with an entire exposed ß-sheet surface. All the interaction modes on the surface of MoS2 nanosheet can be the essence of amyloid protein corona that may provide the venue to facilitate the fibrillation of hIAPP proteins. Further, it was verified experimentally that MoS2 nanosheets could accelerate the fibrillation of hIAPP at a certain concentration mainly based on the newly formed nano-bio interface. In general, our results provide insight into the molecular interaction mechanism of the nano-bio interface within the amyloid protein corona, and shed light on the pathway of amyloid protein aggregation that is related to the evolution of amyloid diseases.

Interest of novel N-alkylpyridinium-indolizine hybrids in the field of Alzheimer's disease: Synthesis, characterization and evaluation of antioxidant activity, cholinesterase inhibition, and amyloid fibrillation interference.

A small library of molecules combining indolizine and N-alkyl pyridinium was synthesized and evaluated in a multi-target-directed-ligand strategy for Alzheimer's disease (AD) treatment. The new compounds were classified in three series depending on the number of methylene residues linking the two heterocycles (Ind-PyCx with x = 0, 2 or 3). The molecules were synthesized from the corresponding bis-pyridines by two-step formation of the indolizine core including mono-alkylation of pyridine and 1,3-dipolar cycloaddition with an alkylpropiolate. Their activities against AD's key-targets were evaluated in vitro: acetyl- and butyrylcholinesterase (AChE and BChE) inhibition, antioxidant properties and inhibition of amyloid fibril formation. None of the three series showed significant activities against all the targets. The Ind-PyC2 and Ind-PyC3 series are active on eeAChE and hAChE (µM IC50 values). Most of the positively charged molecules from these two series also appeared active against eqBChE, however they lost their activity on hBChE. Comparative molecular modeling of 13 and 15 docked in hAChE and hBChE highlighted the importance of the substituent (p-methoxybenzoyl or methyloxycarbonyl, respectively) located on the indolizine C-3 for the binding. The larger molecule 13 fits more tightly at the active site of the two enzymes than 15 that shows a larger degree of freedom. The Ind-PyC2 and Ind-PyC3 hybrids displayed some antioxidant activity when tested at 750 µg/mL (up to 95% inhibition of DPPH radical scavenging for 10). In both series, most hybrids were also able to interact with amyloid fibers, even if the inhibitory effect was observed at a high 100 µM concentration. The Ind-PyC0 molecules stand out completely due to their spectroscopic properties which prevent their evaluation by Ellman's and ThT assays. However, these molecules showed interesting features in the presence of preformed fibers. In particular, the strong increase in fluorescence of 3 in the presence of amyloid fibers is very promising for its use as a fibrillation fluorescent reporter dye.

On the Protein Fibrillation Pathway: Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy.

Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods such as Thioflavin T (ThT) fluorescence, a more sensitive method is needed for their detection. Here we apply Fourier transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode for fast and cheap analysis of destabilized hen-egg-white lysozyme solution and detection of oligomer intermediates of amyloid fibrillation. Standard methods of protein aggregation analysis- Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), and 8-anilinonaphthalene-1-sulphonic acid (ANS) fluorescence were applied and compared to FTIR spectroscopy data. Results show the great potential of FTIR for both, qualitative and quantitative monitoring of oligomer formation based on the secondary structure changes. While oligomer intermediates do not induce significant changes in ThT fluorescence, their secondary structure changes were very prominent. Normalization of specific Amide I region peak intensities by using Amide II peak intensity as an internal standard provides an opportunity to use FTIR spectroscopy for both qualitative and quantitative analysis of biological samples and detection of potentially toxic oligomers, as well as for screening of efficiency of fibrillation procedures.