Fibril Growth Research Articles

Mechanisms of Protein Fibril Formation in Amyloid Beta and Lysozyme Proteins

Rigid fibril (RFs) formation of amyloid proteins is a precursor to many degenerative disorders, thus understanding the kinetics behind their generation could yield important information with respect to disease pathology. These amyloid proteins exhibit what is known as the “critical oligomer concentration” (COC), exhibiting similar behavior as that of micelle formation by surfactants. At low concentrations these proteins generate only smaller oligomeric species and RFs, upon crossing the COC the growth of globular oligomers (gOs) and curvilinear fibrils (CFs) begin, aggregate species that have been shown to be strongly correlated with disease pathology. Below the COC, RFs result in a sigmoidal rise with an initial lag period generated by delayed polymer nucleation. Above the COC, the rapid generation of gOs/CFs result in the elimination of the initial lag period, followed by a secondary rise due to RF growth, producing a biphasic onset. Here, we simulate the growth kinetics of amyloid beta and lysozyme proteins utilizing the theory of nucleated polymerization with off-pathway aggregation, with the assumption that on-pathway nucleation and off-pathway aggregation occur so rapidly that they can be represented as a single step. We show that in order to simulate experimental fibril formation, the primary nucleation rate must be significantly decreased at initial monomer concentration where there is a significant amount of off-pathway aggregates, resulting in a corresponding increase in initial lag periods. In addition, attempting similar fitting with models of the same type produce comparable trends, further supporting our initial findings, and hinting at a possibly inhibitory effect on RF growth by gOs/CFs.

Induced Lanthanide Circularly Polarized Luminescence as a Probe of Protein Fibrils.

Protein fibrils are involved in a number of biological processes. Because their structure is very complex and not completely understood, different spectroscopic methods are used to monitor different aspects of fibril structure. We have explored circularly polarized luminescence (CPL) induced in lanthanide compounds to indicate fibril growth and discriminate among fibril types. For hen egg-white lysozyme and polyglutamic acid-specific CPL, spectral patterns were obtained and could be correlated with vibrational circular dichroism (VCD) spectra and thioflavin T fluorescence. The CPL spectra were measured on a Raman optical activity spectrometer, and its various polarization modes are discussed. The experiments indicate that the induced CPL is sensitive to more local aspects of the fibril structure than VCD. For CPL, smaller amounts of the sample are required for the analysis, and thus this method appears to be a good candidate for future spectroscopic characterization of these peptide and protein aggregates.

Modulation of amyloid fibril formation of plasma protein by saffron constituent “safranal”: Spectroscopic and imaging analyses

Anti-amyloidogenic activity of safranal towards induced HSA amyloids has been observed using a variety of techniques including fluorescence, UV–visible, CD, DLS and microscopies. The HSA solution was pre-incubated at 65 °C for 120 h and, in between, the growth of amyloid fibrils, using ThT aggregation kinetics, was monitored at different time intervals. It was found that the amyloid fibril formation of HSA diminishes in presence of safranal and the inhibition was concentration dependent. The surface hydrophobicity of HSA amyloid fibrils also decreased in presence of safranal. The increased CR binding of HSA fibrils also decreased and high concentration of safranal causes the CR binding to resemble like that of native HSA. Both RLS and turbidity intensities were also in inverse relation to the safranal concentration. Safranal also has a good impact to protect the secondary structure of incubated HSA. From the electron microscopy it was seen that the fibrillar network of HSA amyloids gradually vanishes as the concentration of safranal increased. The largely decreased population of HSA aggregates in safranal containing solution as compared to the one without it also suggests the inhibition of formation of large fibrillar aggregates.

Governing the Inhibition of Reconstituted Collagen Type I Assemblies Mediated Through Noncovalent Forces of (±)-α Lipoic Acid.

Type I collagen is a fibrous protein, which is highly biocompatible and biodegradable and exhibits low immunogenicity with its unique feature of undergoing a spontaneous self-assembly process. However, the excessive accumulation of collagen may lead to a condition known as fibrosis in vertebrates. Recently, saturated fatty acids have gained much attention as biomedical and therapeutic agents. Therefore, drawing inspiration from the biological and structural tunability of these fatty acids, this work aims to inhibit the self-assembly of type I collagen using (±)-α-lipoic acid (ALA). Reconstituted collagen and its blends with (±)-ALA under physiological conditions were subjected to fibril growth kinetics measurements, which exhibited the decrease in the rate of fibrillogenesis ( t1/2) with an increase in the concentration of ALA. Variations in the viscoelasticity of collagen and ALA blend with respect to rate and frequency showed significant changes. Further, the frequency shifts of different functional groups via FT-IR (ATR) and the morphological changes associated with fibril inhibition were visualized using a cryoscanning electron microscope. Molecular dynamics simulation of the collagen-like peptide with the (±)-ALA molecule at different molar ratios proved that (±)-ALA had a strong potential to bind at various sites of collagen mediated by conventional secondary or noncovalent forces. Thus, the protein-small molecule interaction dominates the forces prevailing between protein-protein binding, leading to the inhibition of the self-assembly process. Such inhibitory effects by a fatty acid may unfold newer avenues for development of targeted and sustainable drug delivery systems for fibrotic diseases.

Thermodynamics of amyloid fibril formation from chemical depolymerization.

Amyloid fibrils are homo-molecular protein polymers that play an important role in disease and biological function. While much is known about their kinetics and mechanisms of formation, the origin and magnitude of their thermodynamic stability has received significantly less attention. This is despite the fact that the thermodynamic stability of amyloid fibrils is an important determinant of their lifetimes and processing in vivo. Here we use depolymerization by chemical denaturants of amyloid fibrils of two different proteins (PI3K-SH3 and glucagon) at different concentrations and show that the previously applied isodesmic linear polymerization model is an oversimplification that does not capture the concentration dependence of chemical depolymerization of amyloid fibrils. We show that cooperative polymerization, which is compatible with the picture of amyloid formation as a nucleated polymerization process, is able to quantitatively describe the thermodynamic data. We use this combined experimental and conceptual framework in order to probe the ionic strength dependence of amyloid fibril stability. In combination with previously published data on the ionic strength dependence of amyloid fibril growth kinetics, our results provide strong evidence for the product-like nature of the transition state of amyloid fibril growth.

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.

In Situ Observation of Amyloid Nucleation and Fibrillation by FastScan Atomic Force Microscopy.

Amyloidogenic proteins are key components in various amyloid diseases. The aggregation process and the local structural changes of the toxic species from toxic oligomers to protofibrils and subsequently to mature fibrils are crucial for understanding the molecular mechanism of the amyloidgenic process and also for developing a treatment strategy. Exploration on amyloid aggregation dynamics in situ under real liquid condition is feasible for reflection of the whole process with biological correlations. Herein we report the in situ dynamic study and structure exploration of Amylin1-37 aggregation by FastScan atomic force microscopy. Amylin1-37 nucleation process was observed in which smaller oligomers or monomers were assimilated by the surrounding big oligomers. Amylin1-37 protofibril aggregation was positively correlated with monomer concentration, whereas no direct relationship was observed between fibril elongation and monomer concentration. Growing end and passivated end were found during Amylin1-37 fibrillation. In the assembly process, the growing end kept its structure, and its stiffness was lower than the aggregate body, whereas the passivated end might experience rearrangements of β-structures, which eventually enabled fibril growth from this end. This work is beneficial to the insights of amyloid fibrillation and may shed light on the development of drugs targeting the specific phase of amyloid aggregation.

Mkx-Deficient Mice Exhibit Hedgehog Signaling-Dependent Ectopic Ossification in the Achilles Tendons.

Heterotopic ossification is the abnormal formation of mineralized bone in skin, muscle, tendon, or other soft tissues. Tendon ossification often occurs from acute tendon injury or chronic tendon degeneration, for which current treatment relies heavily on surgical removal of the ectopic bony tissues. Unfortunately, surgery creates additional trauma, which often causes recurrence of heterotopic ossification. The molecular mechanisms of heterotopic ossification are not well understood. Previous studies demonstrate that Mkx is a transcription factor crucial for postnatal tendon fibril growth. Here we report that Mkx-/- mutant mice exhibit ectopic ossification in the Achilles tendon within 1 month after birth and the tendon ossification deteriorates with age. Genetic lineage labeling revealed that the tendon ossification in Mkx-/- mice resulted from aberrant differentiation of tendon progenitor cells. Furthermore, tissue-specific inactivation of Mkx in tendon cells postnatally resulted in a similar ossification phenotype, indicating that Mkx plays a key role in tendon tissue homeostasis. Moreover, we show that Hedgehog signaling is ectopically activated at early stages of tendon ossification and that tissue-specific inactivation of Smoothened, which encodes the obligatory transducer of Hedgehog signaling, in the tendon cell lineage prevented or dramatically reduced tendon ossification in Mkx-/- mice. Together, these studies establish a new genetic mouse model of tendon ossification and provide new insight into its pathogenic mechanisms. © 2018 American Society for Bone and Mineral Research.

Repulsive interaction induces fibril formation and their growth

In a type-II diabetes disease, amylin protein takes an incorrect structure that leads to the formation of the amyloid fibril. The conversion mechanism of amyloid fibril is not well understood. We have observed a repulsive interaction, in terms of second virial co-efficient (A2), between protein molecules in their native state in the PBS buffer through laser light scattering technique. The A2 switches from repulsive (positive A2) to attractive (negative A2) interactions with elapsed time favoring the formation and growth of the fibril. We report aggregation and fibril growth kinetics of amylin protein in different environmental conditions. The measurement of shape factor (ρ) through light scattering experiment shows a transition from coil-like structure to rod-like growth. In addition to rod-like growth, sheet-like growth of fibril is also observed through analytical and high-resolution TEM imaging techniques. The nucleation leading to elongation of fibrils as well as stacking of individual fibril perpendicular to the fibril axis is held by hydrogen bonding observed through high-resolution TEM.

Mechanistic and Structural Origins of the Asymmetric Barrier to Prion-like Cross-Seeding between Tau-3R and Tau-4R

The spread and deposition of infectious fibrillar protein aggregates in the brain via a prion-like mechanism is a critical component in the patho-physiology of various neurodegenerative diseases, including the tauopathies. In tauopathies, two isoforms of tau, containing three and four microtubule binding repeats, are found to aggregate, and the type of isoform present in aggregates determines the type of tauopathy. Cross-seeding between the two tau isoforms is limited by an asymmetric barrier similar to the species barrier that restricts prion transmission across species, whose origin has remained unclear. In this study, the growth of the tau fibrils is shown to be describable by a two-step Michaelis–Menten-like model. Delineation of the mechanism as a Michaelis–Menten-like mechanism has enabled a quantitative understanding of the asymmetric seeding barrier that exists between two isoforms of tau, tau-K18 and tau-K19 (which differ in containing four and three microtubule binding repeats, respectively), wherein tau-K18 fibrils cannot seed tau-K19 monomer. Furthermore, high-resolution structural analysis of the two isoforms shows that the structural core is more ordered in tau-K19 than in tau-K18. Hence, the current work provides kinetic and structural rationales for asymmetric seeding barriers in general and for the two tau isoforms in particular.

Comparison of covalently and physically cross-linked collagen hydrogels on mediating vascular network formation for engineering adipose tissue

Timely tissue vascularization and integration of engineered tissues into a patient plays an important role in the successful translation of engineered tissues into clinically relevant therapies. To decrease the time needed to vascularize an engineered adipose tissue, suitable local microenvironments provided by hydrogels to support cell-based functional vascular network formation have been investigated. Using the same biomolecule in solution, two types of hydrogels can be obtained: a “physical hydrogel” which is thermal-induced self-assemble fibril initiation and growth, due to amino and carboxyl telopeptides on collagen chains, and a “chemical hydrogel” which results from the covalently cross-linking of the side chains induced by one step enzyme mediation in aqueous solution. In this paper, we compare the capability of engineering vascular network and large-sized vascularized adipose tissue in vivo in different types of collagen hydrogels, physical and chemical crosslinking. The relationships between vascular network formation and hydrogel properties for the two types of hydrogels are discussed. Finally, we successfully engineered a vascularized adipose tissue construct (∼877.6 adipocytes/mm2; 94% area of a construct) in the absence of exogenous cytokines in chemical covalently crosslinking cell-laden hydrogel. These results show manipulating the polymerized methods of a hydrogel could not only modulate vascular network formation, but also regenerate adipose tissue in vivo.

Different effects of lipid on conformational conversion of chicken and murine prion proteins

Prion diseases are characterized by the conformational conversion of the cellular prion protein (PrPC) to the pathogenic isoform (PrPSc). Lipids have been found to interact with PrPC and contribute to the efficient formation of PrPSc. Non-mammalian PrPs are not readily to undergo the conversion process into an infectious isoform, yet the effect of lipid on the conformational conversion of non-mammalian PrPC remains to be explored. Herein, the effects of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) on full-length recombinant chicken PrP (ChPrP) 24–249 and murine PrP (MoPrP) 23–230 were investigated. Firstly, it was found that in the presence of chemical denaturant, POPG remarkably inhibited MoPrP amyloid fibril growth, while had slight effect on that of ChPrP as estimated by amyloid fibril growth and transmissible electronic microscope assays. Secondly, under physiological condition, POPG induced conformation changes in both MoPrP and ChPrP using Thioflavin T (ThT) fluorescence, circular dichroism, proteinase K digestion and transmission electron microscopy assays. With a POPG:PrP molar ratio of 30:1, the ThT fluorescence of MoPrP was found to be lower than that of ChPrP, however, the POPG-induced MoPrP had higher β-sheet content and was more proteinase K-resistant than POPG-induced ChPrP. In summary, the present results suggested that the effects of POPG on conformational conversion of MoPrP and ChPrP were different under both denaturation and physiological conditions.

Electrostatic effects in collagen fibril formation.

Using light scattering and Atomic Force Microscopy techniques, we have studied the kinetics and equilibrium scattering intensity of collagen association, which is pertinent to the vitreous of the human eye. Specifically, we have characterized fibrillization dependence on pH, temperature, and ionic strength. At higher and lower pH, collagen triple helices remain stable in solution without fibrillization. At physiological pH, fibrillization occurs and the fibril growth is slowed upon either an increase in ionic strength or a decrease in temperature. The total light scattering with respect to ionic strength is non-monotonic in these conditions as a result of a competing dependence of fibril concentration and size on ionic strength. Fibril concentration is the highest at lower ionic strengths and rapidly decays for higher ionic strengths. On the other hand, fibril size is larger in solutions with higher ionic strength. We present a theoretical model, based on dipolar interactions in solutions, to describe the observed electrostatic nature of collagen assembly. At extreme pH values, either very low or very high, collagen triple helices carry a large net charge of the same sign preventing their assembly into fibrils. At intermediate pH values, fluctuations in the charge distribution of the collagen triple helices around roughly zero net charge lead to fibrillization. The growth kinetics of fibrils in this regime can be adequately described by dipolar interactions arising from charge fluctuations.

Growth probability and formation time of the individual Oosawa-Kasai protein fibril.

Protein fibrils are currently of great academic and practical interest because of their involvement in scores of severe human diseases and their promising use in various high-technology devices. The Oosawa-Kasai (OK) model of protein self-assembly into fibrils has been widely used to gain mechanistic insight into the process of fibril formation and growth. Here this model is employed to obtain exact and mathematically simple expressions for the probability P_{n} of an individual fibril of n protein monomers to grow to a macroscopically large size and for the mean time τ_{n} that such a fibril needs for its formation. These expressions quantify the increase of P_{n} and the decrease of τ_{n} with increasing the concentration of monomeric protein in the solution. When used for analysis of experimental P_{n} and τ_{n} data, they make it possible to determine the parameters characterizing fibril nucleation and growth in the framework of the OK model. Finally, an expression is found for the mean time of the first appearance of an n-sized fibril in the protein solution. The results obtained are applicable to the formation of other aggregates corresponding to the OK fibrils, such as the one-dimensional Kossel-Stranski crystals and Ising ferromagnets.

Self-Assembly of Protein Fibrils in Microgravity

Abstract Deposits of insoluble protein fibrils in human tissue are associated with amyloidosis and neurodegenerative diseases. Different proteins are involved in each disease; all are soluble in their native conformation in vivo, but by molecular self-assembly, they all form insoluble protein fibril deposits with a similar cross β-sheet structure. This paper reports the results of an experiment in molecular self-assembly carried out in microgravity on the International Space Station (ISS). The Self-Assembly in Biology and the Origin of Life (SABOL) experiment was designed to study the growth of lysozyme fibrils in microgravity. Lysozyme is a model protein that has been shown to replicate the aggregation processes of other amyloid proteins. Here the design and performance of the experimental hardware is described in detail. The flight experiment was carried to the ISS in the Dragon capsule of the SpaceX CRS-5 mission and returned to Earth after 32 days. The lysozyme fibrils formed in microgravity aboard the ISS show a distinctly different morphology compared to fibrils formed in the ground-control (G-C) experiment. The fibrils formed in microgravity are shorter, straighter, and thicker than those formed in the laboratory G-C experiment. For two incubation periods, (2) about 8.5 days and (3) about 14.5 days, the average ISS and G-C fibril diameters are respectively: Period 2 D ISS = 7.5 nm ± 31 % , and D G ‐ C = 3.4 nm ± 31 % Period 3 D ISS = 6.2 nm ± 33 % , and D G ‐ C = 3.6 nm ± 33 % . \matrix{{Period\,2} \hfill & {} \hfill & {{D_{ISS}} = 7.5{\rm{nm}} \pm 31\% ,} \hfill \cr {} \hfill & {\rm and} \hfill & {{D_{G - C}} = 3.4{\rm{nm}} \pm 31\%} \hfill \cr {Period\,3} \hfill & {} \hfill & {{D_{ISS}} = 6.2{\rm{nm}} \pm 33\% ,} \hfill \cr {} \hfill & {\rm and} \hfill & {{D_{G - C}} = 3.6{\rm{nm}} \pm 33\% .}}

TRANSLATIONAL QUANTITATIVE SYSTEMS PHARMACOLOGY MODEL OF TAUOPATHIES

Quantitative systems pharmacology modelling can be valuable tool for understanding such a multifaceted process as tau protein accumulation. For prediction of the results of tau targeted therapy we propose a mechanistic model describing such tauopathies as Alzheimer's disease (AD), and frontotemporal degeneration (FTD) in human and tau accumulation in preclinical tauopathy model, mice carrying P301S(L) tau mutation. The model describes tau production, tau-microtubules interaction, tau modification, aggregation and propagation through different brain compartments. Tau oligomers serve as the key intermediates for fibril growth and as mediators of tau pathology connectivity-driven propagation mediators from entorhinal cortex through limbic system to neocortex. The driver of londitudinal disease progression in AD model is the decrease of autophagic degradation of tau fibrils. For FTD case we use assumption about tau mutation influence on polymerization and microtubule binding confirmed by in vitro data. The human model versions were calibrated across published biochemical data for soluble, insoluble tau, CSF tau and PET (18F-AV-1451 SUVR) data. The mouse model was calibrated across biochemical data for soluble (RAB, RIPA soluble fractions), insoluble tau (FA soluble fraction) in several brain structures, interstitial fluid and CSF of mouse. Model satisfactorily describes consecutive appearance of insoluble tau in different regions corresponding to Braak stages of AD. FTD data verification revealed that tau propagation parameters differ from AD situation. Mouse model version satisfactorily describes reduction of soluble tau in brain and accumulation of insoluble tau in the mouse P301S(L). Tau pathology propagation and gradual appearance of polymerized tau starting from entorhinal cortex through the limbic system to neocortex is also predicted correctly and follows the experimentally observed trend. Data on immunotherapy by antibodies HJ8.5 in mouse are described satisfactorily only if significant (10 times) increase of insoluble tau degradation is assumed, while blocking tau propagation only does not allow for describing the mouse data. Analogous degradation activation in humans, would lead to complete disappearance of tau in AD, but cause moderate effect in FTD according to the model. The proposed QSP model could be considered as platform for investigation of therapeutic impact on tau pathology and translational analysis.

Amyloid seeding of transthyretin by ex vivo cardiac fibrils and its inhibition

Each of the 30 human amyloid diseases is associated with the aggregation of a particular precursor protein into amyloid fibrils. In transthyretin amyloidosis (ATTR), mutant or wild-type forms of the serum carrier protein transthyretin (TTR), synthesized and secreted by the liver, convert to amyloid fibrils deposited in the heart and other organs. The current standard of care for hereditary ATTR is liver transplantation, which replaces the mutant TTR gene with the wild-type gene. However, the procedure is often followed by cardiac deposition of wild-type TTR secreted by the new liver. Here we find that amyloid fibrils extracted from autopsied and explanted hearts of ATTR patients robustly seed wild-type TTR into amyloid fibrils in vitro. Cardiac-derived ATTR seeds can accelerate fibril formation of wild-type and monomeric TTR at acidic pH and under physiological conditions, respectively. We show that this seeding is inhibited by peptides designed to complement structures of TTR fibrils. These inhibitors cap fibril growth, suggesting an approach for halting progression of ATTR.

Watching proteins' weight

Biophysics Careful measurements of light scattering can provide information on individual macromolecules and complexes. Young et al. used a light-scattering approach for accurate mass determination of proteins as small as 20 kDa (see the Perspective by Lee and Klenerman). Movies of protein complex association and dissociation were analyzed to extract biophysical parameters from single molecules and assemblies without labeling. Using this approach, the authors determined in vitro kinetics of fibril and aggregate growth and association constants for a complex protein-glycoprotein assembly. Science , this issue p. [423][1]; see also p. [378][2] [1]: /lookup/doi/10.1126/science.aar5839 [2]: /lookup/doi/10.1126/science.aat5851

Strikingly different effects of cholesterol and 7-ketocholesterol on lipid bilayer-mediated aggregation of amyloid beta (1-42)

Oxidized cholesterol has been widely reported to contribute to the pathogenesis of Alzheimer's disease (AD). However, the mechanism by which they affect the disease is not fully understood. Herein, we aimed to investigate the effect of 7-ketocholesterol (7keto) on membrane-mediated aggregation of amyloid beta (Aβ-42), one of the critical pathogenic events in AD. We have shown that when cholesterol is present in lipid vesicles, kinetics of Aβ nuclei formation is moderately hindered while that of fibril growth was considerably accelerated. The partial substitution of cholesterol with 7keto slightly enhanced the formation of Aβ-42 nuclei and remarkably decreased fibril elongation, thus maintaining the peptide in protofibrillar aggregates, which are reportedly the most toxic species. These findings add in understanding of how cholesterol and its oxidation can affect Aβ-induced cytotoxicity.

Inhibition of α‐Synuclein Amyloid Fibril Elongation by Blocking Fibril Ends

AbstractMisfolding of the protein α‐synuclein (αSyn) into amyloid fibrils plays a central role in the development of Parkinson's disease. Most approaches for the inhibition of αSyn fibril formation are based on stabilizing the native monomeric form of the protein or destabilizing the fibrillized misfolded form. They require high concentrations of inhibitor and therefore cannot be easily used for therapies. In this work, we designed an inhibitor (Inh‐β) that selectively binds the growing ends of αSyn fibrils and creates steric hindrance for the binding of monomeric αSyn. This approach permits the inhibition of fibril formation at Inh‐β concentrations (IC50=850 nm) much lower than the concentration of monomeric αSyn. We studied its kinetic mechanism in vitro and identified the reactions that limit inhibition efficiency. It is shown that blocking of αSyn fibril ends is an effective approach to inhibiting fibril growth and provides insights for the development of effective inhibitors of αSyn aggregation.