Fibril Growth Research Articles

Characterization and kinetics of whey protein isolate amyloid fibril aggregates under moderate electric fields

Food proteins solutions enable the controlled flow of electrical current when subjected to a Moderate electric field (MEF). This application results in internal heat generation, known as ohmic heating, accompanied by electrochemical reactions. These effects play a significant role in influencing the formation of protein amyloid fibril aggregates (AFA), which have broad applications in food and biomedical fields, ranging from improving food texture to biomedical applications such as drug delivery and disease treatment. This study focused on investigating the formation of β-Lactoglobulin (β-lg) AFA under MEF conditions using WPI as protein source and various characterization techniques (e.g., Thioflavin T fluorescence, circular dichroism, and electron microscopy). The results indicated that MEF prolonged the lag phase of AFA formation. Furthermore, nucleation and fibril growth showed higher activation coefficients and cooperativity level (n > 2) compared to conventional heating method. MEF treatment resulted in unique fibril clustering and the presence of unexpected higher-order AFA networks confirmed by electron microscopy. These findings highlight the significant role of MEF in influencing the aggregation kinetics of β-lg AFA. Exploring further into the electrochemical and thermal effects during protein aggregation processes holds the key to unlocking deeper insights fibril formation process.

Examining the inhibitory potency of metal polyphenolic network–coated silver nanoparticles against amyloid fibrillogenesis of lysozyme

There are currently over forty degenerative diseases that are correlated with abnormal accumulation of peptide/protein aggregates in the human body, such as Alzheimer’s disease. Due to their unique physiochemical properties (e.g., small size, large surface-to-volume ratio, and facile surface modification), silver nanoparticles (AgNPs) have been considered potential substrates for designing inhibitors against amyloid fibrillogenesis. Metal polyphenolic network (MPN) that combines the characteristics of organic and inorganic components has been used to suppress amyloid fibril formation. This study is aimed at investigating the effects of MPN–coated AgNPs (MPN–AgNPs) on the in vitro amyloid fibrillogenesis of hen lysozyme (HEWL). The two types of MPN–AgNPs (Zn/MPN–AgNPs and Co/MPN–AgNPs) were synthesized through chemical reduction and metal chelation, and their particle sizes were determined to be in the range of 40–60 nm. The characterization of MPN–AgNPs by ζ–potential and transmission electron microscopy showed that the MPN–AgNPs had negative surface charge and spherical-shaped morphology. Furthermore, the elemental analysis demonstrated that the MPN was uniformly coated on the surface of AgNPs. The thioflavin T fluorescence results revealed that the Co/MPN–AgNPs showed a better percent of inhibition compared to Zn/MPN–AgNPs and TA–AgNPs. The kinetics data of amyloid fibril formation in the presence of MPN–AgNPs were analyzed using the sigmoidal curve, showing that the MPN–AgNPs reduced fibril growth rate and prolonged lag time. Our findings also demonstrated that MPN–AgNPs could effectively suppress HEWL aggregation upon binding to aggregation-prone regions. The quenching of intrinsic fluorescence of HEWL by MPN–AgNPs was found to be a static type. Moreover, the fluorescence quenching data were analyzed using the modified Stern-Volmer equation to determine the number of binding sites. Notably, our findings indicated that the binding between HEWL and MPN–AgNPs was mainly governed by hydrophobic interaction. This work offers an excellent example of utilizing MPN–based materials as anti-aggregating/anti-fibrillogenic nano-drugs for the treatment of amyloidosis.

Single-Molecule Kinetic Observation of Antibody Interactions with Growing Amyloid β Fibrils.

Understanding the dynamic assembly process of amyloid β (Aβ) during fibril formation is essential for developing effective therapeutic strategies against Alzheimer's disease. Here, we employed high-speed atomic force microscopy to observe the growth of Aβ fibrils at the single-molecule level, focusing specifically on their interaction with anti-Aβ antibodies. Our findings show that fibril growth consists of intermittent periods of elongation and pausing, which are dictated by the alternating addition of Aβ monomers to protofilaments. We highlight the distinctive interaction of antibody 4396C, which specifically binds to the fibril ends in the paused state, suggesting a unique mechanism to hinder fibril elongation. Through real-time visualization of fibril growth and antibody interactions combined with molecular simulation, this study provides a refined understanding of Aβ assembly during fibril formation and suggests novel strategies for Alzheimer's therapy aimed at inhibiting the fibril elongation.

Deciphering the Morphological Difference of Amyloid-β Fibrils in Familial and Sporadic Alzheimer's Diseases.

The aggregation of amyloid-β (Aβ) into amyloid fibrils is the major pathological hallmark of Alzheimer's disease (AD). Aβ fibrils can adopt a variety of morphologies, the relative populations of which are recently found to be associated with different AD subtypes such as familial and sporadic AD (fAD and sAD, respectively). The two AD subtypes differ in their ages of onset, AD-related genetic predispositions, and dominant Aβ fibril morphologies. We postulate that these disease subtype-dependent fibril morphology differences can be attributed to the intrinsic fibril properties and interacting molecules in the environment. Using atomistic discrete molecular dynamics simulations, we demonstrated that the fAD-dominant morphology exhibited a lower free-energy barrier for fibril growth but also a lower stability compared with the sAD-dominant fibril morphology, resulting in the time-dependent population change consistent with experimental observations. Additionally, we studied the effect of the Bri2 BRICHOS domain, an endogenous protein that has been reported to inhibit Aβ aggregation by preferential binding to fibrils, as one of the possible environmental factors. The Bri2 BRICHOS domain showed stronger binding to the fAD-dominant fibril than the sAD-dominant fibril in silico, suggesting a more effective suppression of fAD-dominant fibril formation. This result explains the high population of the sAD-dominant fibril morphology in sporadic cases with normal Bri2 functions. Genetic predisposition in fAD, on the other hand, might impair or overwhelm Bri2 functions, leading to a high population of fAD-associated fibril morphology. Together, our computational findings provide a theoretical framework for elucidating the AD subtypes entailed by distinct dominant amyloid fibril morphologies.

Modulation of AIE and Intramolecular Charge Transfer of a Pyrene-Based Probe for Discriminatory Detection and Imaging of Oligomers and Amyloid Fibrils.

Oligomers and amyloid fibrils formed at different stages of protein aggregation are important biomarkers for a variety of neurodegenerative diseases including Alzheimer's and Parkinson's diseases. The development of probes for the sensitive detection of oligomeric species is important for early stage diagnosis of amyloidogenic diseases. Many small molecular dyes have been developed to probe the dynamic growth of amyloid fibrils. However, there is a lack of discriminatory detection strategies to monitor the dynamics of both oligomers and amyloid fibrils based on the differential modulation of the photophysical properties of a single dye. Here we report a pyrene-based intramolecular charge transfer (ICT) dye with large Stokes shifted red-emitting aggregation induced emission (AIE) for monitoring the dynamic populations of both oligomers and fibrils during the aggregation of hen egg white lysozyme (HEWL) protein. At the early stage of protein aggregation, the accumulation of HEWL oligomers results in a rapid and substantial increase in the red AIE intensity at 660 nm. Later, as the oligomers transform into mature fibrils, the dye exhibits a distinct photophysical change. Binding of the dye to HEWL fibrils strongly suppresses the red AIE and enhances ICT emission. This is evidenced by a gradual decrease in the AIE intensity (∼660 nm) and an increase in LE (∼490 nm) and ICT (∼540 nm) emission intensities during the later stages of protein aggregation. Thus, the dye provides simultaneous measurements of the population dynamics of both HEWL oligomers and fibrils during protein aggregation based on the discriminatory modulation of AIE and ICT of the dye. The dye also enables imaging of both HEWL oligomers and fibrils simultaneously using different emission channels in super-resolution confocal fluorescence microscopy.

Dendrimers-Novel Therapeutic Approaches for Alzheimer's Disease.

Dendrimers are covalently bonded globular nanostructures that may be used in the treatment of Alzheimer's disease (AD). Nowadays, AD therapies are focused on improving cognitive functioning and not causal treatment. However, this may change with the use of dendrimers, which are being investigated as a drug-delivery system or as a drug per se. With their ability to inhibit amyloid formation and their anti-tau properties, they are a promising therapeutic option for AD patients. Studies have shown that dendrimers may inhibit amyloid formation in at least two ways: by blocking fibril growth and by breaking already existing fibrils. Neurofibrillary tangles (NFTs) are abnormal filaments built by tau proteins that can be accumulated in the cell, which leads to the loss of cytoskeletal microtubules and tubulin-associated proteins. Cationic phosphorus dendrimers, with their anti-tau properties, can induce the aggregation of tau into amorphous structures. Drug delivery to mitochondria is difficult due to poor transport across biological barriers, such as the inner mitochondrial membrane, which is highly negatively polarized. Dendrimers may be potential nanocarriers and increase mitochondria targeting. Another considered use of dendrimers in AD treatment is as a drug-delivery system, for example, carbamazepine (CBZ) or tacrine. They can also be used to transport siRNA into neuronal tissue and to carry antioxidants and anti-inflammatory drugs to act protectively on the nervous system.

Sound-mediated nucleation and growth of amyloid fibrils

Mechanical energy, specifically in the form of ultrasound, can induce pressure variations and temperature fluctuations when applied to an aqueous media. These conditions can both positively and negatively affect protein complexes, consequently altering their stability, folding patterns, and self-assembling behavior. Despite much scientific progress, our current understanding of the effects of ultrasound on the self-assembly of amyloidogenic proteins remains limited. In the present study, we demonstrate that when the amplitude of the delivered ultrasonic energy is sufficiently low, it can induce refolding of specific motifs in protein monomers, which is sufficient for primary nucleation; this has been revealed by MD. These ultrasound-induced structural changes are initiated by pressure perturbations and are accelerated by a temperature factor. Furthermore, the prolonged action of low-amplitude ultrasound enables the elongation of amyloid protein nanofibrils directly from natively folded monomeric lysozyme protein, in a controlled manner, until it reaches a critical length. Using solution X-ray scattering, we determined that nanofibrillar assemblies, formed either under the action of sound or from natively fibrillated lysozyme, share identical structural characteristics. Thus, these results provide insights into the effects of ultrasound on fibrillar protein self-assembly and lay the foundation for the potential use of sound energy in protein chemistry.

Real-time single-molecule observation of incipient collagen fibrillogenesis and remodeling

The hierarchic assembly of fibrillar collagen into an extensive and ordered supramolecular protein fibril is critical for extracellular matrix function and tissue mechanics. Despite decades of study, we still know very little about the complex process of fibrillogenesis, particularly at the earliest stages where observation of rapidly forming, nanoscale intermediates challenges the spatial and temporal resolution of most existing microscopy methods. Using video rate scanning atomic force microscopy (VRS-AFM), we can observe details of the first few minutes of collagen fibril formation and growth on a mica surface in solution. A defining feature of fibrillar collagens is a 67-nm periodic banding along the fibril driven by the organized assembly of individual monomers over multiple length scales. VRS-AFM videos show the concurrent growth and maturation of small fibrils from an initial uniform height to structures that display the canonical banding within seconds. Fibrils grow in a primarily unidirectional manner, with frayed ends of the growing tip latching onto adjacent fibrils. We find that, even at extremely early time points, remodeling of growing fibrils proceeds through bird-caging intermediates and propose that these dynamics may provide a pathway to mature hierarchic assembly. VRS-AFM provides a unique glimpse into the early emergence of banding and pathways for remodeling of the supramolecular assembly of collagen during the inception of fibrillogenesis.

TDP-43 Promotes Amyloid-Beta Toxicity by Delaying Fibril Maturation via Direct Molecular Interaction.

Amyloid-β (Aβ) is a peptide that undergoes self-assembly into amyloid fibrils, which compose the hallmark plaques observed in Alzheimer's disease (AD). TAR DNA-binding protein 43 (TDP-43) is a protein with mislocalization and aggregation implicated in amyotrophic lateral sclerosis and other neurodegenerative diseases. Recent work suggests that TDP-43 may interact with Aβ, inhibiting the formation of amyloid fibrils and worsening AD pathology, but the molecular details of their interaction remain unknown. Using all-atom discrete molecular dynamics simulations, we systematically investigated the direct molecular interaction between Aβ and TDP-43. We found that Aβ monomers were able to bind near the flexible nuclear localization sequence of the N-terminal domain (NTD) of TDP-43, adopting β-sheet rich conformations that were promoted by the interaction. Furthermore, Aβ associated with the nucleic acid binding interface of the tandem RNA recognition motifs of TDP-43 via electrostatic interactions. Using the computational peptide array method, we found the strongest C-terminal domain interaction with Aβ to be within the amyloidogenic core region of TDP-43. With experimental evidence suggesting that the NTD is necessary for inhibiting Aβ fibril growth, we also simulated the NTD with an Aβ40 fibril seed. We found that the NTD was able to strongly bind the elongation surface of the fibril seed via extensive hydrogen bonding and could also diffuse along the lateral surface via electrostatic interactions. Our results suggest that TDP-43 binding to the elongation surface, thereby sterically blocking Aβ monomer addition, is responsible for the experimentally observed inhibition of fibril growth. We conclude that TDP-43 may promote Aβ toxicity by stabilizing the oligomeric state and kinetically delaying fibril maturation.

Histidine Protonation Behaviors on Structural Properties and Aggregation Properties of Aβ(1-42) Mature Fibril: Approaching by Edge Effects.

The histidine behavior plays a crucial role in the structural and aggregation properties of protein folding and misfolding. Understanding the histidine behavior at the edge of the protein structure is critical for finding ways to disrupt fibril elongation and growth, but this impact remains poorly understood. In the current study, we used molecular dynamics simulations to investigate the edge substitution effect of histidine protonation on the structural and aggregation properties. Our data showed that ΔG1 contributed the most to binding affinity compared to ΔG2 and ΔG3. The different protonation states at the edge chain significantly impacted the secondary structure properties of the edge chain. Specifically, we found that such protonation behavior significantly affected specific regions, particularly the N-terminus (G9-Q15) and C-terminus (K28-A30). Further analysis confirmed that H6, H13, and H14 were directly involved in H-bonding networks with the C1_H14//C2_H13 interchain interactions critical for maintaining the interchain stability. Furthermore, we confirmed that H6, H13, and H14 were directly involved in the loss of the carbon skeleton contact in the N-terminus. Our findings indicate that the edge condition is more susceptible to changes in structural properties than the middle condition. The current study is helpful for understanding the histidine behavior hypothesis in related misfolding diseases.

Catechol-induced covalent modifications modulate the aggregation tendency of α-synuclein: An in-solution and in-silico study.

Parkinson's disease (PD) stands as a challenging neurodegenerative condition characterized by the emergence of Lewy Bodies (LBs), intracellular inclusions within dopaminergic neurons. These LBs harbor various proteins, prominently including α-Synuclein (Syn) aggregates, implicated in disease pathology. A promising avenue in PD treatment involves targeting Syn aggregation. Recent findings from our research have shown that 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET) possess the ability to impede the formation of Syn fibrils by disrupting the aggregation process. Notably, these compounds primarily engage in noncovalent interactions with the protein, leading to the formation of off-pathway oligomers that deter fibril growth. Through proteolysis studies and mass spectrometry (MS) analysis, we have identified potential covalent modifications of Syn in the presence of DOPAC, although the exact site remains elusive. Employing molecular dynamics simulations, we delved into how DOPAC-induced covalent alterations might affect the mechanism of Syn aggregation. Our findings indicate that the addition of a covalent adduct on certain residues enhances fibril flexibility without compromising its secondary structure stability. Furthermore, in the monomeric state, the modified residue fosters novel bonding interactions, thereby influencing long-range interactions between the N- and C-termini of the protein.

Controlled Supramolecular Polymerization via Bioinspired, Liquid-Liquid Phase Separation of Monomers.

Dynamic supramolecular assemblies, driven by noncovalent interactions, pervade the biological realm. In the synthetic domain, their counterparts, supramolecular polymers, endowed with remarkable self-repair and adaptive traits, are often realized through bioinspired designs. Recently, controlled supramolecular polymerization strategies have emerged, drawing inspiration from protein self-assembly. A burgeoning area of research involves mimicking the liquid-liquid phase separation (LLPS) observed in proteins to create coacervate droplets and recognizing their significance in cellular organization and diverse functions. Herein, we introduce a novel perspective on synthetic coacervates, extending beyond their established role in synthetic biology as dynamic, membraneless phases to enable structural control in synthetic supramolecular polymers. Drawing parallels with the cooperative growth of amyloid fibrils through LLPS, we present metastable coacervate droplets as dormant monomer phases for controlled supramolecular polymerization. This is achieved via a π-conjugated monomer design that combines structural characteristics for both coacervation through its terminal ionic groups and one-dimensional growth via a π-conjugated core. This design leads to a unique temporal LLPS, resulting in a metastable coacervate phase, which subsequently undergoes one-dimensional growth via nucleation within the droplets. In-depth spectroscopic and microscopic characterization provides insights into the temporal evolution of disordered and ordered phases. Furthermore, to modulate the kinetics of liquid-to-solid transformation and to achieve precise control over the structural characteristics of the resulting supramolecular polymers, we invoke seeding in the droplets, showcasing living growth characteristics. Our work thus opens up new avenues in the exciting field of supramolecular polymerization, offering general design principles and controlled synthesis of precision self-assembled structures in confined environments.

Molecular Dynamics Insights into the Aggregation Behavior of N-Terminal β-Lactoglobulin Peptides.

β-lactoglobulin (BLG) forms amyloid-like aggregates at high temperatures, low pH, and low ionic strengths. At a pH below 2, BLG undergoes hydrolysis into peptides, with N-terminal peptides 1-33 and 1-52 being prone to fibrillization, forming amyloid-like fibrils. Due to their good mechanical properties, BLG amyloids demonstrate great potential for diverse applications, including biosensors, nanocomposites, and catalysts. Consequently, further studies are essential to comprehensively understand the factors governing the formation of BLG amyloid-like morphologies. In this study, all-atom molecular dynamics simulations were employed to explore the aggregation of N-terminal 1-33 and 1-52 BLG peptides under conditions of pH 2 and at 10 mM NaCl concentration. The simulations revealed that the peptides spontaneously assembled into aggregates of varying sizes. The aggregation process was enabled by the low charge of peptides and the presence of hydrophobic residues within them. As the peptides associated into aggregates, there was a concurrent increase in β-sheet structures and the establishment of hydrogen bonds, enhancing the stability of the aggregates. Notably, on average, 1-33 peptides formed larger aggregates compared to their 1-52 counterparts, while the latter exhibited a slightly higher content of β-sheets and higher cluster orderliness. The applied approach facilitated insights into the early stages of amyloid-like aggregation and molecular-level insight into the formation of β-sheets, which serve as nucleation points for further fibril growth.

Insights into the mechanism of peptide fibril growth on gold surface

Understanding the formation of β-fibrils over the gold surface is of paramount interest in nano-bio-medicinal Chemistry. The intricate mechanism of self-assembly of neurofibrillogenic peptides and their growth over the gold surface remains elusive, as experiments are limited in unveiling the microscopic dynamic details, in particular, at the early stage of the peptide aggregation. In this work, we carried out equilibrium molecular dynamics and enhanced sampling simulations to elucidate the underlying mechanism of the growth of an amyloid-forming sequence of tau fragments over the gold surface. Our results disclose that the collective intermolecular interactions between the peptide chains and peptides with the gold surface facilitate the peptide adsorption, followed by integration, finally leading to the fibril formation.

Terahertz Spectroscopy Analysis of Amyloid Fibrils Derived from Human Serum Albumin

Amyloid fibrils, also referred to as protein fibrils, represent irregular assemblies of proteins exhibiting a cross-β configuration. Terahertz radiation, a segment of the electromagnetic spectrum spanning 0.3 to 10 terahertz (THz), is known to interact with these fibrils due to their cylindrical shape. This study's objective was to explore the potential of terahertz spectroscopy as a non-invasive means to detect the progressive growth of protein fibrils. In this investigation, human serum albumin (HSA) was synthetically induced to form protein fibrils. Confirmation of fibril formation was done by fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, UV-Vis spectroscopy, and Terahertz time-domain spectroscopy (THz-TDS). To initiate the process, HSA was dissolved in distilled water at a concentration of 160 µM, followed by the addition of 60% ethanol to both samples for dilution. The HSA to Cu (II) ratio was maintained at 1:1 (160 µM) for stock solutions containing Cu (II). Subsequently, each set underwent a 6-hour heating phase at 65 °C to cultivate fibrils, which were then stored at room temperature for 30, 60, and 90 days before evaluation. Incubation of human serum albumin yielded amyloid fibrils as evidenced by ThT fluorescence, FT-IR spectroscopy revealed an emergence of fibrils within the amide I bands, extending from 1630 cm⁻¹ to 1650 cm⁻¹ and UV-Vis spectroscopy disclosed the augmentation of protein fibril absorbance over the incubation period. THz spectroscopy absorbance progressively heightened with prolonged heating of the protein fibrils due to the hydration shell surrounding them. Through these spectroscopic methods, it was elucidated that the fibrillation process led to the gradual development of β-sheet and unordered helix structures as well as the potential of THz spectroscopy as non-invasive tool for detection and monitoring of amyloidal diseases, promising insights into early diagnosis and treatment strategies.

Experimental and computational investigation of the effect of Hsc70 structural variants on inhibiting amylin aggregation

The misfolding and aggregation of human islet amyloid polypeptide (hIAPP), also known as amylin, have been implicated in the pathogenesis of type 2 diabetes (T2D). Heat shock proteins, specifically, heat shock cognate 70 (Hsc70), are molecular chaperones that protect against hIAPP misfolding and inhibits its aggregation. Nevertheless, there is an incomplete understanding of the mechanistic interactions between Hsc70 domains and hIAPP, thus limiting their potential therapeutic role in diabetes. This study investigates the inhibitory capacities of different Hsc70 variants, aiming to identify the structural determinants that strike a balance between efficacy and cytotoxicity. Our experimental findings demonstrate that the ATPase activity of Hsc70 is not a pivotal factor for inhibiting hIAPP misfolding. We underscore the significance of the C-terminal substrate-binding domain of Hsc70 in inhibiting hIAPP aggregation, emphasizing that the removal of the lid subdomain diminishes the inhibitory effect of Hsc70. Additionally, we employed atomistic discrete molecular dynamics simulations to gain deeper insights into the interaction between Hsc70 variants and hIAPP. Integrating both experimental and computational findings, we propose a mechanism by which Hsc70's interaction with hIAPP monomers disrupts protein-protein connections, primarily by shielding the β-sheet edges of the Hsc70-β-sandwich. The distinctive conformational dynamics of the alpha helices of Hsc70 potentially enhance hIAPP binding by obstructing the exposed edges of the β-sandwich, particularly at the β5-β8 region along the alpha helix interface. This, in turn, inhibits fibril growth, and similar results were observed following hIAPP dimerization. Overall, this study elucidates the structural intricacies of Hsc70 crucial for impeding hIAPP aggregation, improving our understanding of the potential anti-aggregative properties of molecular chaperones in diabetes treatment.

Hairpin trimer transition state of amyloid fibril

Protein fibril self-assembly is a universal transition implicated in neurodegenerative diseases. Although fibril structure/growth are well characterized, fibril nucleation is poorly understood. Here, we use a computational-experimental approach to resolve fibril nucleation. We show that monomer hairpin content quantified from molecular dynamics simulations is predictive of experimental fibril formation kinetics across a tau motif mutant library. Hairpin trimers are predicted to be fibril transition states; one hairpin spontaneously converts into the cross-beta conformation, templating subsequent fibril growth. We designed a disulfide-linked dimer mimicking the transition state that catalyzes fibril formation, measured by ThT fluorescence and TEM, of wild-type motif - which does not normally fibrillize. A dimer compatible with extended conformations but not the transition-state fails to nucleate fibril at any concentration. Tau repeat domain simulations show how long-range interactions sequester this motif in a mutation-dependent manner. This work implies that different fibril morphologies could arise from disease-dependent hairpin seeding from different loci.

Complexes of tubulin oligomers and tau form a viscoelastic intervening network cross-bridging microtubules into bundles

The axon-initial-segment (AIS) of mature neurons contains microtubule (MT) fascicles (linear bundles) implicated as retrograde diffusion barriers in the retention of MT-associated protein (MAP) tau inside axons. Tau dysfunction and leakage outside of the axon is associated with neurodegeneration. We report on the structure of steady-state MT bundles in varying concentrations of Mg2+ or Ca2+ divalent cations in mixtures containing αβ-tubulin, full-length tau, and GTP at 37 °C in a physiological buffer. A concentration-time kinetic phase diagram generated by synchrotron SAXS reveals a wide-spacing MT bundle phase (Bws), a transient intermediate MT bundle phase (Bint), and a tubulin ring phase. SAXS with TEM of plastic-embedded samples provides evidence of a viscoelastic intervening network (IN) of complexes of tubulin oligomers and tau stabilizing MT bundles. In this model, αβ-tubulin oligomers in the IN are crosslinked by tau’s MT binding repeats, which also link αβ-tubulin oligomers to αβ-tubulin within the MT lattice. The model challenges whether the cross-bridging of MTs is attributed entirely to MAPs. Tubulin-tau complexes in the IN or bound to isolated MTs are potential sites for enzymatic modification of tau, promoting nucleation and growth of tau fibrils in tauopathies.

Edge Substitution Effects of Histidine Tautomerization Behaviors on the Structural Properties and Aggregation Properties of Aβ(1-42) Mature Fibril.

Histidine behaviors play critical roles in folding and misfolding processes due to the changes in net charge and the various N/N-H orientations on imidazole rings. However, the effect of histidine tautomerization (HIE (Nε-H, ε) and HID (Nδ-H, δ) states) behaviors on the edge chain of Aβ mature fibrils remains inadequately understood, which is critical for finding a strategy to disturb fibril elongation and growth. In the current study, eight independent molecular dynamics simulations were conducted to investigate such impacts on the structural and aggregation properties. Our results from three different binding models revealed that the binding contributions of edge substitution effects are primarily located between chains 1 and 2. Histidine states significantly influence the secondary structure of each domain. Further analysis confirmed that the C1_H6//C1_E11 intrachain interaction is essential in maintaining the internal stability of chain 1, while the C1_H13//C2_H13 and C1_H14//C2_H13 interchain interactions are critical in maintaining the interchain stability of the fibril structure. Our subsequent analysis revealed that the current edge substitution leads to the loss of the C1_H13//C1_E11 intrachain and C1_H13//C2_H14 interchain interactions. The N-terminal regularity was significantly directly influenced by histidine states, particularly by the residue of C1_H13. Our study provides valuable insights into the effect of histidine behaviors on the edge chain of Aβ mature fibril, advancing our understanding of the histidine behavior hypothesis in misfolding diseases.

Growth kinetics of amyloid-like fibrils: An integrated atomistic simulation and continuum theory approach.

Amyloid fibrils have long been associated with many neurodegenerative diseases. The conventional picture of the formation and proliferation of fibrils from unfolded proteins comprises primary and secondary nucleation of oligomers followed by elongation and fragmentation thereof. In this work, we first employ extensive all-atom molecular dynamics (MD) simulations of short peptides to investigate the governing processes of fibril growth at the molecular scale. We observe that the peptides in the bulk solution can bind onto and subsequently diffuse along the fibril surface, which leads to fibril elongation via either bulk- or surface-mediated docking mechanisms. Then, to guide the quantitative interpretation of these observations and to provide a more comprehensive picture of the growth kinetics of single fibrils, a continuum model which incorporates the key processes observed in the MD simulations is formulated. The model is employed to investigate how relevant physical parameters affect the kinetics of fibril growth and identify distinct growth regimes. In particular, it is shown that fibrils which strongly bind peptides may undergo a transient exponential growth phase in which the entire fibril surface effectively acts as a sink for peptides. We also demonstrate how the relevant model parameters can be estimated from the MD trajectories. Our results provide compelling evidence that the overall fibril growth rates are determined by both bulk and surface peptide fluxes, thereby contributing to a more fundamental understanding of the growth kinetics of amyloid-like fibrils.