Straight Fibrils Research Articles

Modification of rice protein and its components: Enhanced fibrils formation and improved foaming properties

Plant-derived proteins like rice protein are considered an ideal source for yielding amyloid fibrils due to sustainability and affordability. However, the inherent low solubility of rice protein often restricts its self-assembly capability under common fibrillization protocols. Hence, this study comprehensively investigated the effect of modification on the self-assembly behavior, protein structure and foaming properties of rice protein and its components. The modification facilitated the transformation of α-helix to β-sheet, promoting fibrils formation. All modified proteins, except rice protein, exhibited significantly increased zeta potential values. AFM results revealed that the modified rice protein and globulin fibrils exhibited entangled aggregation behavior, while the glutelin fibril was notably extended, with several micrometers in length. Additionally, modification promoted the aggregation of albumin and prolamin particles to form short and straight fibrils. Amino acid analysis indicated that the content difference of amino acids with different characteristics and the change of aromatic amino acid interaction caused by modification may lead to different self-assembly behavior of the same protein. Furthermore, the foaming capability and stability of most protein fibrils were significantly improved by the modification. These findings provide valuable insights into the regulation of plant-derived protein fibrillation through modification.

Controlling Solvent Polarity to Regulate Protein Self-Assembly Morphology and Its Universal Insight for Fibrillation Mechanism.

The mechanism of ethanol-induced fibrillation of β-lactoglobulin (β-lg) in the acidic aqueous solution upon heating was investigated using various techniques, mainly thioflavin T fluorescence, atomic force microscopy, nonreducing electrophoresis, mass spectrometry, Fourier transform infrared spectroscopy, and circular dichroism spectroscopy. The results showed that fibrillation occurred with a heating time increase, but high ethanol content slowed down the process. At a low ethanol volume fraction, peptides existed after heating for 2 h, with long and straight fibrils formed after 4-6 h, while at a high ethanol volume fraction, the proteins aggregated with very few peptides appeared at the early stage of heating, and short and curved fibrils formed after heating for 8 h. Ethanol weakened the hydrophobic interactions between proteins in the aqueous solution; therefore the latter could not completely balance the electrostatic repulsion, and thus suppressing the fibrillation process. It is believed that the fibrillation of β-lg in the acidic solution upon heating is mainly dominated by the polypeptide model; however, ethanol inhibited the hydrolysis of proteins, and the self-assembly mechanism changed to the monomer model.

Fibrillization of lentil proteins is impacted by the protein extraction conditions and co-extracted phenolics

Legume proteins can be induced to form amyloid-like fibrils upon heating at low pH, with the exact conditions greatly impacting the fibril characteristics. The protein extraction method may also impact the resulting fibrils, although this effect has not been carefully examined. Here, the fibrillization of lentil protein prepared using various extraction methods and the corresponding fibril morphology were characterized. It was found that an acidic, rather than alkaline, protein extraction method was better suited for producing homogeneous, long, and straight fibrils from lentil proteins. During alkaline extraction, co-extracted phenolic compounds bound proteins through covalent and non-covalent interactions, contributing to the formation of heterogeneous, curly, and tangled fibrils. Recombination of isolated phenolics and proteins (from acidic extracts) at alkaline pH resulted in a distinct morphology, implicating a role for polyphenol oxidase also in modifying proteins during alkaline extraction. These results help disentangle the complex factors affecting legume protein fibrillization.

Resolving the Nanoscale Structure of β-Sheet Peptide Self-Assemblies Using Single-Molecule Orientation-Localization Microscopy.

Synthetic peptides that self-assemble into cross-β fibrils are versatile building blocks for engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarities to amyloid species have been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize by using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize Nile red (NR), an amyloidophilic fluorogenic probe, and single-molecule orientation-localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers KFE8L and KFE8D and the pathological amyloid-beta peptide Aβ42. Importantly, NR SMOLM reveals the helical (bilayer) ribbon structure of both KFE8 and Aβ42 and quantifies the precise tilt of the fibrils' inner and outer backbones in relevant buffer conditions without the need for covalent labeling or sequence mutations. SMOLM also distinguishes polymorphic branched and curved morphologies of KFE8, whose backbones exhibit much more heterogeneity than those of typical straight fibrils. Thus, SMOLM is a powerful tool to interrogate the structural differences and polymorphism between engineered and pathological cross-β-rich fibrils.

Comparison of the self-assembly and cytocompatibility of conjugates of Fmoc (9-fluorenylmethoxycarbonyl) with hydrophobic, aromatic, or charged amino acids.

The self-assembly in aqueous solution of three Fmoc-amino acids with hydrophobic (aliphatic or aromatic, alanine or phenylalanine) or hydrophilic cationic residues (arginine) is compared. The critical aggregation concentrations were obtained using intrinsic fluorescence or fluorescence probe measurements, and conformation was probed using circular dichroism spectroscopy. Self-assembled nanostructures were imaged using cryo-transmission electron microscopy and small-angle X-ray scattering (SAXS). Fmoc-Ala is found to form remarkable structures comprising extended fibril-like objects nucleating from spherical cores. In contrast, Fmoc-Arg self-assembles into plate-like crystals. Fmoc-Phe forms extended structures, in a mixture of straight and twisted fibrils coexisting with nanotapes. Spontaneous flow alignment of solutions of Fmoc-Phe assemblies is observed by SAXS. The cytocompatibility of the three Fmoc-amino acids was also compared via MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] mitochondrial activity assays. All three Fmoc-amino acids are cytocompatible with L929 fibroblasts at low concentration, and Fmoc-Arg shows cell viability up to comparatively high concentration (0.63 mM).

Amyloidogenic 60–71 deletion/ValThr insertion mutation of apolipoprotein A-I generates a new aggregation-prone segment that promotes nucleation through entropic effects

The N-terminal fragment of apolipoprotein A-I (apoA-I), comprising residues 1–83, contains three segments prone to aggregation: residues 14–22, 53–58, and 67–72. We previously demonstrated that residues 14–22 are critical in apoA-I fibril formation while residues 53–58 entropically drove the nucleation process. Here, we investigated the impact of amyloidogenic mutations (Δ60–71/VT, Δ70–72, and F71Y) located around residues 67–72 on fibril formation by the apoA-I 1–83 fragment. Thioflavin T fluorescence assay demonstrated that the Δ60–71/VT mutation significantly enhances both nucleation and fibril elongation rates, whereas the Δ70–72 and F71Y mutations had minimal effects. Circular dichroism measurements and microscopic observations revealed that all variant fragments formed straight fibrils, transitioning from random coils to β-sheet structures. Kinetic analysis demonstrated that primary nucleation is the dominant step in fibril formation, with fibril elongation reaching saturation at high protein concentrations. Thermodynamically, both nucleation and fibril elongation were enthalpically and entropically unfavorable in all apoA-I 1–83 variants, in which the entropic barrier of nucleation was almost eliminated for the Δ60–71/VT variant. Taken together, our results suggest the presence of new aggregation-prone segment in the Δ60–71/VT variant that promotes nucleation through entropic effects.

Concentration-Regulated Fibrillation of Soy Protein: Structure and In Vitro Digestion.

The impact of protein types, heating temperatures, and times on protein fibrillation has been widely studied. However, there is little understanding of the influence of protein concentration (PC) on the protein fibril assembly. In this work, the structure and in vitro digestibility of soy protein amyloid fibrils (SAFs) were investigated at pH 2.0 and different PCs. Significant increases in fibril conversion rate and parallel β-sheets proportion were observed in SAFs upon increasing the PC from 2 to 8% (w/v). The AFM images showed that curly fibrils were prone to form at 2-6% PCs, while rigid, straight fibrils developed at higher PCs (≥8%). As evidenced in XRD results, increasing PC led to a more stable structure of SAFs with enhanced thermal stability and lower digestibility. Moreover, positive correlations among PC, β-sheet content, persistence length, enthalpy, and total hydrolysis were established. These findings would provide valuable insights into concentration-regulated protein fibrillation.

Morphology, Formation Kinetics and Core Composition of Pea and Soy 7S and 11S Globulin Amyloid Fibrils.

Legume seed storage proteins can be induced to form amyloid fibrils upon heating at low pH, which could improve their functionality for use in food and materials. However, the amyloidogenic regions of legume proteins are largely unknown. Here, we used LC-MS/MS to determine the amyloid core regions of fibrils formed by enriched pea and soy 7S and 11S globulins at pH 2, 80 °C, and characterized their hydrolysis, assembly kinetics, and morphology. A lag phase was absent from the fibrillation kinetics of pea and soy 7S globulins, while 11S globulins and crude extracts displayed a similar lag time. Pea and soy protein fibrils differed in morphology, with most pea fibrils being straight and soy fibrils being worm-like. Pea and soy globulins were abundant in amyloid-forming peptides, with over 100 unique fibril-core peptides from pea 7S and around 50 unique fibril-core peptides identified from pea 11S, soy 7S, and soy 11S globulins. Amyloidogenic regions derive predominantly from the homologous core region of 7S globulins and the basic subunit of 11S globulins. Overall, pea and soy 7S and 11S globulins are rich in amyloidogenic regions. This study will help understand their fibrillation mechanism and engineer protein fibrils with specific structures and functionality.

Effects of charge residues on α-synuclein fibril formation.

α-Synuclein (α-syn) amyloid fibrils are associated with Parkinson's disease (PD), multiple system atrophy, and dementia with Lewy bodies. α-Syn fibrils derived from ex vivo patient materials as well as those formed in vitro adopt an array of structures or polymorphs. It is important to delineate the structure-function relationship of fibril polymorphs and the mechanisms that give rise to these molecular differences. In particular, salt bridges between Lys and Glu residues are widely observed in all α-syn fibril structures to date. On the one hand, this is not surprising given the preponderance of Lys (14) and Glu (17) residues in its 140 amino acid long sequence; but, because of their large numbers, it is cumbersome and challenging to identify key residues that modulate fibril formation. Here, we performed studies using a smaller PD-related N-terminally truncated α-syn variant, composed of residues 66‒140 (ΔN1-65), that forms amyloid fibrils. Within the proteolytic-resistant core, the number of charged residues reduces to 4 Lys, 4 Glu, and 1 Asp, which is tractable for alanine substitutions to determine their effects. Using atomic force microscopy (AFM) and transmission electron microscopy, we show that fibril structure is strongly impacted by charge neutralization. For example, while ΔN1-65 formed predominantly paired twisted protofilaments with uniform helical periodicity, E83A formed only paired straight fibrils, whereas K80A displayed both twisted and straight fibrils. Interestingly, different heights were seen by AFM for K80A. Of note, structural complexity within a single fibril was revealed in K96A/K97A, where varying helical periodicities were measured along the fibril axis. In conclusion, these results on ΔN1-65 offer residue-specific insights and bolster the importance of electrostatic interactions in fibril formation, which is also involved in the full-length protein.

The influence of substrate stiffness on interfacial stresses for adhesive microfibrils

Bioinspired microfibrillar surfaces adhere by exploiting the presence of intermolecular forces at the contact interface. Experimental work has shown that compliant mushroom shaped fibrils can facilitate optimal adhesion across a range of substrates. This work evaluates numerically how fibril contact tip shape and the degree of elastic mismatch at the fibril-substrate interface influence adhesion. In a finite element simulation of fibrils subject to a remote stress, the interfacial stress distributions that define the detachment behaviour are computed for a range of fibril cap diameters, thicknesses, and substrate stiffnesses. Depending on the substrate stiffness, there is a singular stress field present at the edge of contact for smaller mushroom cap and straight punch fibrils while the stress at the corner for larger mushroom fibrils tends to zero. This variation in fibril-substrate interfacial stress has implications for the most probable defect type to initiate detachment and the stability of this crack growth. For fibrils within this corner singularity regime, the adhesion strength of a patch of fibrils is determined using interfacial fracture mechanics. The simulations are compared with experimental data of pull-off strengths and the biomedical applications of adhesive microfibrillar patches are considered.

Impact of heat and enzymatic treatment on ovalbumin amyloid-like fibril formation and enzyme-induced gelation

Heating aqueous solutions of ovalbumin (OVA) may cause gel formation. When heated at pH conditions close to the protein's isoelectric point (towards neutral pH), turbid particulate gels are formed, whereas at acidic pH fine-stranded, transparent gels are formed already at lower concentrations. Here, transparent gels were formed when subjecting 2.0% OVA to a combined heat (78 °C for 22 h at pH 7) and trypsin (37 °C for 48 h) treatment. Transmission electron microscopy clearly revealed the presence of long, straight OVA fibrils which contributed to the gel formation. Quartz crystal microbalance with dissipation (adsorption of small structures), size exclusion – HPLC (presence of both structures larger and smaller than native OVA) and atomic force microscopy (presence of long fibrils with a higher thickness, whereas heated OVA mainly showed amorphous aggregates) analyses confirmed that the additional enzymatic treatment was able to break down the amorphous aggregates formed by heating OVA into peptides, which then partly re-assembled into longer OVA fibrils. The above mentioned heat and enzymatic treatment conditions brought about gelation after 17 h with a gel strength of 68 Pa which broke at a stress of 38 Pa. By varying the temperature during heat (58–88 °C) and enzymatic (27–67 °C) treatments, gels were formed the fastest when heated at 78 °C followed by enzymatic treatment at 57 °C. A design of experiments for evaluating the impact of OVA and trypsin concentration revealed that the fastest gelation occurred at the higher considered OVA and trypsin concentrations. Additionally, the gel strength was also higher under the latter conditions. It is clear that different gel characteristics can be reached when varying the different process conditions, creating the opportunity for reconsidering the formulation of various foods such as jellies, marmalades and desserts. • Combined heat and tryptic treatment of ovalbumin (OVA) induces gelation. • Trypsin hydrolyzes amorphous aggregates of OVA. • The resultant peptides can form amyloid-like fibrils. • Preheating and trypsin-incubation temperatures influence the gelation. • Increasing OVA and trypsin concentrations result in rapid formation of strong gels.

Investigating the Sequence Determinants of the Curling of Amyloid Fibrils Using Ovalbumin as a Case Study.

Highly ordered, straight amyloid fibrils readily lend themselves to structure determination techniques and have therefore been extensively characterized. However, the less ordered curly fibrils remain relatively understudied, and the structural organization underlying their specific characteristics remains poorly understood. We found that the exemplary curly fibril-forming protein ovalbumin contains multiple aggregation prone regions (APRs) that form straight fibrils when isolated as peptides or when excised from the full-length protein through hydrolysis. In the context of the intact full-length protein, however, the regions separating the APRs facilitate curly fibril formation. In fact, a meta-analysis of previously reported curly fibril-forming proteins shows that their inter-APRs are significantly longer and more hydrophobic when compared to straight fibril-forming proteins, suggesting that they may cause strain in the amyloid state. Hence, inter-APRs driving curly fibril formation may not only apply to our model protein but rather constitute a more general mechanism.

Heating Wheat Gluten Promotes the Formation of Amyloid-like Fibrils.

Amyloid fibrils (AFs) are highly ordered nanofibers composed of proteins rich in β-sheet structures. In this study, the impact of heating conditions relevant in food processing on AF formation of wheat gluten (WG) was investigated. Unheated and heated WG samples were treated with proteinase K and trypsin to solubilize the nonfibrillated protein, while protein fibrils were extracted with 0.05 M sodium phosphate buffer (pH 7.0) from the undissolved fraction obtained by the same enzymatic treatment. Conditions (i.e., heating at 78° for 22 h) resembling those in slow cooking induced the formation of straight fibrils (ca. 700 nm in length), whereas boiling WG for at least 15 min resulted in longer straight fibrils (ca. 1–2 μm in length). The latter showed the typical green birefringence of AFs when stained with Congo red. Their X-ray fiber diffraction patterns showed the typical reflection (4.7 Å) for inter-β-strand spacing. These results combined with those of Fourier transform infrared and thioflavin T spectroscopy measurements validated the identification of β-rich amyloid-like fibrils (ALFs) in dispersions of boiled WG. Boiling for at least 15 min converted approximately 0.1–0.5% of WG proteins into ALFs, suggesting that they can be present in heat-treated WG-containing food products and that food-relevant heating conditions have the potential to induce protein fibrillation.

Effects of Dissolved Gases on the Amyloid Fibril Morphology.

The onset or progression of numerous neurodegenerative diseases occurs due to aggregation of proteins that ultimately form fibrils. The assembly and morphology of fibrils are susceptible to environmental factors. In this work, we used atomic force microscopy (AFM) to investigate the effects of dissolved nitrogen and oxygen molecules on the morphology of fibrils formed by a hydrophobic amyloid peptide implicated in amyotrophic lateral sclerosis, 15 repeats of glycine-alanine, on a highly oriented pyrolytic graphite substrate. We started with preformed fibril solutions that were then diluted with buffers of different gas conditions, resulting in the aggregation of the fibrils into different morphologies that were revealed by AFM after adsorption on the substrate. Straight fibrils were observed in both degassed and ambient buffers, but a stronger lateral association was seen in degassed buffers. Smaller and softer fibrils were observed in O2-supersaturated buffers, and plaque-like fibril aggregates of considerably large size were evident in N2-supersaturated buffers. In overnight incubation experiments, we observed changes in both the morphology and height of the fibril aggregates, and their evolution varied with different gas conditions. These findings indicate that the gas type and concentration affect the aggregation of amyloid fibrils and may facilitate the development of biomaterial applications and treatments for amyloid-related diseases.

Measurement of length distribution of beta-lactoglobulin fibrils by multiwavelength analytical ultracentrifugation

The whey protein beta-lactoglobulin is the building block of amyloid fibrils which exhibit a great potential in various applications. These include stabilization of gels or emulsions. During biotechnological processing, high shear forces lead to fragmentation of fibrils and therefore to smaller fibril lengths. To provide insight into such processes, pure straight amyloid fibril dispersions (prepared at pH 2) were produced and sheared using the rotor stator setup of an Ultra Turrax. In the first part of this work, the sedimentation properties of fragmented amyloid fibrils sheared at different stress levels were analyzed with mulitwavelength analytical ultracentrifugation (AUC). Sedimentation data analysis was carried out with the boundary condition that fragmented fibrils were of cylindrical shape, for which frictional properties are known. These results were compared with complementary atomic force microscopy (AFM) measurements. We demonstrate how the sedimentation coefficient distribution from AUC experiments is influenced by the underlying length and diameter distribution of amyloid fibrils.In the second part of this work, we show how to correlate the fibril size reduction kinetics with the applied rotor revolution and the resulting energy density, respectively, using modal values of the sedimentation coefficients obtained from AUC. Remarkably, the determined scaling laws for the size reduction are in agreement with the results for other material systems, such as emulsification processes or the size reduction of graphene oxide sheets.

Influence of the polydispersity of pH 2 and pH 3.5 beta-lactoglobulin amyloid fibril solutions on analytical methods

It is well known that amyloid beta-lactoglobulin (BLG) fibril solutions contain a heterogeneous mixture of amyloid aggregates and non-amyloid material. However, few information are available on how strongly separated fractions of different morphologies (straight fibrils at pH 2 and worm-like aggregates at pH 3.5) vary with respect to physicochemical properties and building blocks as most analyses are conducted with unfractionized solutions where superposition effects occur.The pH-value shift resulted in an altered degree of acid hydrolysis which led to dissimilar building blocks of the aggregates (peptides at pH 2, non-hydrolyzed protein at pH 3.5). The respective separated amyloid and non-amyloid fractions showed significantly different size (SAXS, SEC, AUC) and charge properties (Zeta potential) than the whole samples. Strong superposition effects were evident with common analyses such as FTIR, TRP fluorescence and Thioflavin-T. At the same time, structural differences of pH 2 and pH 3.5 aggregates could be presented more clearly.

Modulating amyloid fibrillation in a minimalist model peptide by intermolecular disulfide chemical reduction.

Peptide structural transformation and aggregation is associated with a large number of outsider aetiology diseases, and it is intrinsically linked to amyloid peptide aggregation. Diphenylalanine self-assembled structures are used as robust minimalist beta amyloids not only to elucidate protein aggregation but also to generate hydrogels. Herein, we employed a neutral model peptide Ac-Phe-Phe-Cys-NH2 (Ac-FFC-NH2) to elucidate the role of intermolecular disulfide bonds in protein fibrillation. The Ac-FFC-NH2 peptide initially self-assembles into nanospheres that evolve to amyloid type fibrils under mild oxidative conditions. Incubation of the peptide in the presence of the chemical reduction agent TCEP inhibits the formation of the fibrils, detecting only spherical nanostructures with no secondary structure. Importantly, we triggered the transformation of the preformed linear straight amyloid fibrils to non-fibrillar structures by TCEP treatment. Under this condition, the amyloid bundles are transformed into rings, which evolve to a new spherical microstructure. We showed that the chemical reduction of intermolecular S-S in internal amyloid sequences might favour the off-path intermediates of amyloid fibril growth, even when the fibrils are formed. Our findings demonstrated that in internal amyloid sequences, the formation of intermolecular S-S promotes the formation of amyloid type fibrils; meanwhile, its reduction stabilises non-fibrillar structures. Altogether, this work provides fundamental understanding at the molecular and supramolecular level, thus facilitating the rational design of therapeutic tools for protein aggregation diseases.

Alpha-synuclein is highly prone to distribution in the hippocampus and midbrain in tree shrews, and its fibrils seed Lewy body-like pathology in primary neurons.

The Chinese tree shrew (TS) has many unique advantages that make it suitable for use as an experimental animal model for human disease including moderate body size, low cost of feeding, short reproductive cycle and lifespan, and close phylogenetic relationship to primates. Our previous studies have shown that TS treated with the mitochondrial inhibitor MPTP displayed classic Parkinsonian symptoms. Additionally, the structure of TS alpha-synuclein (α-syn) is highly homologous to that found in humans. Previous studies have concluded that misfolded, fibrillar α-syn is a hallmark of α-synucleinopathies. In this study, we examined the distribution and expression levels of α-syn in different TS brain regions. We also obtained recombinant TS α-syn protein to study its aggregation and cytotoxic properties in primary neurons. Our results showed that α-syn was expressed in numerous different brain regions in TS but was most abundant in the hippocampus and midbrain. The recombinant α-syn of TS displayed straight fibrils when incubated for 72 h in vitro, which is very similar to human α-syn. When exposed to primary neurons, the TS and human α-syn fibrils led to cytotoxicity and Lewy-like pathology. Our findings indicated that TS could be a potential animal model to study the pathology of α-synucleinopathies.

Biochemical and morphological classification of disease-associated alpha-synuclein mutants aggregates

Alpha-synuclein (a-syn) aggregation in brain is implicated in several synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Until date, at least six disease-associated mutations in a-syn (namely A30P, E46K, H50Q, G51D, A53T, and A53E) are known to cause dominantly inherited familial forms of synucleinopathies. Previous studies using recombinant proteins have reported that a subset of disease-associated mutants show higher aggregation propensities and form spectroscopically distinguishable aggregates compared to wild-type (WT). However, morphological and biochemical comparison of the aggregates for all disease-associated a-syn mutants have not yet been performed. In this study, we performed electron microscopic examination, guanidinium hydrochloride (GdnHCl) denaturation, and protease digestion to classify the aggregates from their respective point mutations. Using electron microscopy we observed variations of amyloid fibrillar morphologies among the aggregates of a-syn mutants, mainly categorized into two groups: twisted fibrils observed for both WT and E46K while straight fibrils for the other mutants. GdnHCl denaturation experiments revealed the a-syn mutants except for E46K were more resistant than WT against the denaturation. Mass spectrometry analysis of protease-treated aggregates showed a variety of protease-resistant cores, which may correspond to their morphological properties. The difference of their properties could be implicated in the clinicopathological difference of synucleinopathies with those mutations.

Amyloidogenic cross-seeding of Tau protein: Transient emergence of structural variants of fibrils.

Amyloid aggregates of Tau protein have been implicated in etiology of many neurodegenerative disorders including Alzheimer's disease (AD). When amyloid growth is induced by seeding with preformed fibrils assembled from the same protein, structural characteristics of the seed are usually imprinted in daughter generations of fibrils. This so-called conformational memory effect may be compromised when the seeding involves proteins with non-identical sequences leading to the emergence of distinct structural variants of fibrils (amyloid ‘strains’). Here, we investigate cross-seeding of full-length human Tau (FL Tau) with fibrils assembled from K18 and K18ΔK280 fragments of Tau in the presence of poly-L-glutamate (poly-Glu) as an enhancer of Tau aggregation. To study cross-seeding between Tau polypeptides and the role of the conformational memory effect in induction of Tau amyloid polymorphism, kinetic assays, transmission electron microscopy, infrared spectroscopy and limited proteolysis have been employed. The fastest fibrillization was observed for FL Tau monomers seeded with preformed K18 amyloid yielding daughter fibrils with unique trypsin digestion patterns. Morphological features of daughter FL Tau fibrils induced by K18 and K18ΔK280 seeds were reminiscent of the mother fibrils (i.e. straight paired fibrils and paired helical filaments (PHFs), respectively) but disappeared in the following generations which became similar to unpaired FL Tau amyloid fibrils formed de novo. The structural evolution observed in our study was accompanied by disappearance of the unique proteolysis profile originated from K18. Our findings may have implications for understanding molecular mechanisms of the emergence and stability of Tau amyloid strains.