Homotypic fibrillization of TMEM106B across diverse neurodegenerative diseases.

Misfolding and aggregation of disease-specific proteins, resulting in the formation of filamentous cellular inclusions, is a hallmark of neurodegenerative disease with characteristic filament structures, or conformers, defining each proteinopathy. Here we show that a previously unsolved amyloid fibril composed of a 135 amino acid C-terminal fragment of TMEM106B is a common finding in distinct human neurodegenerative diseases, including cases characterized by abnormal aggregation of TDP-43, tau, or α-synuclein protein. A combination of cryoelectron microscopy and mass spectrometry was used to solve the structures of TMEM106B fibrils at a resolution of 2.7 Å from postmortem human brain tissue afflicted with frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP, n = 8), progressive supranuclear palsy (PSP, n = 2), or dementia with Lewy bodies (DLB, n = 1). The commonality of abundant amyloid fibrils composed of TMEM106B, a lysosomal/endosomal protein, to a broad range of debilitating human disorders indicates a shared fibrillization pathway that may initiate or accelerate neurodegeneration.

Magic angle spinning NMR of proteins: high-frequency dynamic nuclear polarization and (1)H detection.

Magic angle spinning (MAS) NMR studies of amyloid and membrane proteins and large macromolecular complexes are an important new approach to structural biology. However, the applicability of these experiments, which are based on (13)C- and (15)N-detected spectra, would be enhanced if the sensitivity were improved. Here we discuss two advances that address this problem: high-frequency dynamic nuclear polarization (DNP) and (1)H-detected MAS techniques. DNP is a sensitivity enhancement technique that transfers the high polarization of exogenous unpaired electrons to nuclear spins via microwave irradiation of electron-nuclear transitions. DNP boosts NMR signal intensities by factors of 10(2) to 10(3), thereby overcoming NMR's inherent low sensitivity. Alternatively, it permits structural investigations at the nanomolar scale. In addition, (1)H detection is feasible primarily because of the development of MAS rotors that spin at frequencies of 40 to 60 kHz or higher and the preparation of extensively (2)H-labeled proteins.

Cryo-EM structures of functional and pathological amyloid ribonucleoprotein assemblies.

Amyloids are implicated in neurodegenerative and systemic diseases, yet they serve important functional roles in numerous organisms. Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins (RBPs) that control central events of RNA biogenesis in normal and diseased cellular conditions. Many of these proteins contain prion-like sequences of low complexity, which not only assemble into functional fibrils in response to cellular cues but can also lead to disease when missense mutations arise in their sequences. Recent advances in cryo-electron microscopy (cryo-EM) have provided unprecedented high-resolution structural insights into diverse amyloid assemblies formed by hnRNPs and structurally related RBPs, including TAR DNA-binding protein 43 (TDP-43), Fused in Sarcoma (FUS), Orb2, hnRNPA1, hnRNPA2, and hnRNPDL-2. This review provides a comprehensive overview of these structures and explores their functional and pathological implications.

Spatiotemporal formation of a single liquid-like condensate and amyloid fibrils of α-synuclein by optical trapping at solution surface.

Liquid-like protein condensates have recently attracted much attention due to their critical roles in biological phenomena. They typically show high fluidity and reversibility for exhibiting biological functions, while occasionally serving as sites for the formation of amyloid fibrils. To comprehend the properties of protein condensates that underlie biological function and pathogenesis, it is crucial to study them at the single-condensate level; however, this is currently challenging due to a lack of applicable methods. Here, we demonstrate that optical trapping is capable of inducing the formation of a single liquid-like condensate of α-synuclein in a spatiotemporally controlled manner. The irradiation of tightly focused near-infrared laser at an air/solution interface formed a condensate under conditions coexisting with polyethylene glycol. The fluorescent dye-labeled imaging showed that the optically induced condensate has a gradient of protein concentration from the center to the edge, suggesting that it is fabricated through optical pumping-up of the α-synuclein clusters and the expansion along the interface. Furthermore, Raman spectroscopy and thioflavin T fluorescence analysis revealed that continuous laser irradiation induces structural transition of protein molecules inside the condensate to ß-sheet rich structure, ultimately leading to the condensate deformation and furthermore, the formation of amyloid fibrils. These observations indicate that optical trapping is a powerful technique for examining the microscopic mechanisms of condensate appearance and growth, and furthermore, subsequent aging leading to amyloid fibril formation.

Alzheimer's disease seeded tau forms paired helical filaments yet lacks seeding potential.

Alzheimer's disease (AD) and many other neurodegenerative diseases are characterized by pathological aggregation of the protein tau. These tau aggregates spread in a stereotypical spatiotemporal pattern in the brain of each disease, suggesting that the misfolded tau can recruit soluble monomers to adopt the same pathological structure. To investigate whether recruited tau indeed adopts the same structure and properties as the original seed, here we template recombinant full-length 0N3R tau, 0N4R tau, and an equimolar mixture of the two using sarkosyl-insoluble tau extracted from AD brain and determine the structures of the resulting fibrils using cryoelectron microscopy. We show that these cell-free amplified tau fibrils adopt the same molecular structure as the AD paired-helical filament (PHF) tau but are unable to template additional monomers. Therefore, the PHF structure alone is insufficient for defining the pathological properties of AD tau, and other biochemical components such as tau posttranslational modifications, other proteins, polyanionic cofactors, and salt are required for the prion-like serial propagation of tauopathies.

Physiological and pathological functions of TMEM106B in neurodegenerative diseases.

As an integral lysosomal transmembrane protein, transmembrane protein 106B (TMEM106B) regulates several aspects of lysosomal function and is associated with neurodegenerative diseases. The TMEM106B gene mutations lead to lysosomal dysfunction and accelerate the pathological progression of Neurodegenerative diseases. Yet, the precise mechanism of TMEM106B in Neurodegenerative diseases remains unclear. Recently, different research teams discovered that TMEM106B is an amyloid protein and the C-terminal domain of TMEM106B forms amyloid fibrils in various Neurodegenerative diseases and normally elderly individuals. In this review, we discussed the physiological functions of TMEM106B. We also included TMEM106B gene mutations that cause neurodegenerative diseases. Finally, we summarized the identification and cryo-electronic microscopic structure of TMEM106B fibrils, and discussed the promising therapeutic strategies aimed at TMEM106B fibrils and the future directions for TMEM106B research in neurodegenerative diseases.

An allosteric HTRA1-calpain 2 complex with restricted activation profile.

SignificanceClassic serine proteases are synthesized as inactive precursors that are proteolytically processed, resulting in irreversible activation. We report an alternative and reversible mechanism of activation that is executed by an inactive protease. This mechanism involves a protein complex between the serine protease HTRA1 and the cysteine protease calpain 2. Surprisingly, activation is restricted as it improves the proteolysis of soluble tau protein but not the dissociation and degradation of its amyloid fibrils, a task that free HTRA1 is efficiently performing. These data exemplify a challenge for protein quality control proteases in the clearing of pathogenic fibrils and suggest a potential for unexpected side effects of chemical modulators targeting PDZ or other domains located at a distance to the active site.

ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification.

Prion-like self-perpetuating conformational conversion of proteins into amyloid aggregates is associated with both transmissible neurodegenerative diseases and non-Mendelian inheritance. The cellular energy currency ATP is known to indirectly regulate the formation, dissolution, or transmission of amyloid-like aggregates by providing energy to the molecular chaperones that maintain protein homeostasis. In this work, we demonstrate that ATP molecules, independent of any chaperones, modulate the formation and dissolution of amyloids from a yeast prion domain (NM domain of Saccharomyces cerevisiae Sup35) and restricts autocatalytic amplification by controlling the amount of fragmentable and seeding-competent aggregates. ATP, at (high) physiological concentrations in the presence of Mg2+, kinetically accelerates NM aggregation. Interestingly, ATP also promotes phase separation-mediated aggregation of a human protein harboring a yeast prion-like domain. We also show that ATP disaggregates preformed NM fibrils in a dose-independent manner. Our results indicate that ATP-mediated disaggregation, unlike the disaggregation by the disaggregase Hsp104, yields no oligomers that are considered one of the critical species for amyloid transmission. Furthermore, high concentrations of ATP delimited the number of seeds by giving rise to compact ATP-bound NM fibrils that exhibited nominal fragmentation by either free ATP or Hsp104 disaggregase to generate lower molecular weight amyloids. In addition, (low) pathologically relevant ATP concentrations restricted autocatalytic amplification by forming structurally distinct amyloids that are found seeding inefficient because of their reduced ß-content. Our results provide key mechanistic underpinnings of concentration-dependent chemical chaperoning by ATP against prion-like transmissions of amyloids.

Modulation of aggregation and structural polymorphisms of ß-amyloid fibrils in cellular environments by pyroglutamate-3 variant cross-seeding.

Amyloidogenic deposition of ß-amyloid (Aß) peptides in human brain involves not only the wild-type Aß (wt-Aß) sequences, but also posttranslationally modified Aß (PTM-Aß) variants. Recent studies hypothesizes that the PTM-Aß variants may trigger the deposition of wt-Aß, which underlies the pathology of Sporadic Alzheimer's disease. Among PTM-Aß variants, the pyroglutamate-3-Aß (pyroE3-Aß) has attracted much attention because of their significant abundances and broad distributions in senile plaques and dispersible and soluble oligomers. pyroE3-specific antibodies are being tested as potential anti-Aß drugs in clinical trials. However, evidence that support the triggering effect of pyroE3-Aß on wt-Aß in cells remain lacking, which diminishes its pathological relevance. We show here that cross-seeding with pyroE3-Aß40 leads to accelerated extracellular and intracellular aggregation of wt-Aß40 in different neuronal cells. Cytotoxicity levels are elevated through the cross-seeded aggregation, comparing with the self-seeded aggregation of wt-Aß40 or the static presence of pyroE3-Aß40 seeds. For the extracellular deposition in mouse neuroblastoma Neuro2a (N2a) cells, the cytotoxicity elevation correlates positively with the seeding efficiency. Besides aggregation rates, cross-seeding with pyroE3-Aß40 also modulates the molecular level structural polymorphisms of the resultant wt-Aß40 fibrils. Using solid-state nuclear magnetic resonance (ssNMR) spectroscopy, we identified key structural differences between the parent pyroE3/ΔE3 and wt-Aß40 fibrils within their fibrillar cores. Structural propagation from seeds to daughter fibrils is demonstrated to be more pronounced in the extracellular seeding in N2a cells by comparing the ssNMR spectra from different seeded wt-Aß40 fibrils, but less significant in the intracellular seeding process in human neuroblastoma SH-SY5Y cells.

Secondary structure and toxicity of lysozyme fibrils are determined by the length and unsaturation of phosphatidic acid.

A progressive aggregation of misfolded proteins is a hallmark of numerous pathologies including diabetes Type 2, Alzheimer's disease, and Parkinson's disease. As a result, highly toxic protein aggregates, which are known as amyloid fibrils, are formed. A growing body of evidence suggests that phospholipids can uniquely alter the secondary structure and toxicity of amyloid aggregates. However, the role of phosphatidic acid (PA), a unique lipid that is responsible for cell signaling and activation of lipid-gated ion channels, in the aggregation of amyloidogenic proteins remains unclear. In this study, we investigate the role of the length and degree of unsaturation of fatty acids (FAs) in PA in the structure and toxicity of lysozyme fibrils formed in the presence of this lipid. We found that both the length and saturation of FAs in PA uniquely altered the secondary structure of lysozyme fibrils. However, these structural differences in PA caused very little if any changes in the morphology of lysozyme fibrils. We also utilized cell toxicity assays to determine the extent to which the length and degree of unsaturation of FAs in PA altered the toxicity of lysozyme fibrils. We found that amyloid fibrils formed in the presence of PA with C18:0 FAs exerted significantly higher cell toxicity compared to the aggregates formed in the presence of PA with C16:0 and C18:1 FAs. These results demonstrated that PA can be an important player in the onset and spread of amyloidogenic diseases.

Post-translational modification sites are present in hydrophilic cavities of alpha-synuclein, tau, FUS, and TDP-43 fibrils: A molecular dynamics study.

Hydration plays a crucial role in the refolding of intrinsically disordered proteins into amyloid fibrils; however, the specific interactions between water and protein that may contribute to this process are still unknown. In our previous studies of alpha-synuclein (aSyn), we have shown that waters confined in fibril cavities are stabilizing features of this pathological fold; and that amino acids that hydrogen bond with these confined waters modulate primary and seeded aggregation. Here, we extend our aSyn molecular dynamics (MD) simulations with three new polymorphs and correlate MD trajectory information with known post-translational modifications (PTMs) and experimental data. We show that cavity residues are more evolutionarily conserved than non-cavity residues and are enriched with PTM sites. As expected, the confinement within hydrophilic cavities results in more stably hydrated amino acids. Interestingly, cavity PTM sites display the longest protein-water hydrogen bond lifetimes, three-fold greater than non-PTM cavity sites. Utilizing the deep mutational screen dataset by Newberry et al. and the Thioflavin T aggregation review by Pancoe et al. parsed using a fibril cavity/non-cavity definition, we show that hydrophobic changes to amino acids in cavities have a larger effect on fitness and aggregation rate than residues outside cavities, supporting our hypothesis that these sites are involved in the inhibition of aSyn toxic fibrillization. Finally, we expand our study to include analysis of fibril structures of tau, FUS, TDP-43, prion, and hnRNPA1; all of which contained hydrated cavities, with tau, FUS, and TDP-43 recapitulating our PTM results in aSyn fibril cavities.

Von-Hippel Lindau protein amyloid formation. The role of GST-tag.

In the last decade, much attention was given to the study of physiological amyloid fibrils. These structures include A-bodies, which are the nucleolar fibrillar formations that appear in the response to acidosis and heat shock, and disassemble after the end of stress. One of the proteins involved in the biogenesis of A-bodies, regardless of the type of stress, is Von-Hippel Lindau protein (VHL). Known also as a tumor suppressor, VHL is capable to form amyloid fibrils both in vitro and in vivo in response to the environment acidification. As with most amyloidogenic proteins fusion with various tags is used to increase the solubility of VHL. Here, we first performed AFM-study of fibrils formed by VHL protein and by VHL fused with GST-tag (GST-VHL) at acidic conditions. It was shown that formed by full-length VHL fibrils are short heterogenic structures with persistent length of 2400 nm and average contour length of 409 nm. GST-tag catalyzes VHL amyloid fibril formation, superimpose chirality, increases length and level of hierarchy, but decreases rigidity of amyloid fibrils. The obtained data indicate that tagging can significantly affect the fibrillogenesis of the target protein.

Cholesteric Tactoids with Tunable Helical Pitch Assembled by Lysozyme Amyloid Fibrils.

Amyloid fibrils are biological rod-like particles showing liquid-liquid crystalline phase separation into cholesteric phases through a complex behavior of nucleation, growth, and order-order transitions. Yet, controlling the self-assembly of amyloids into liquid crystals, and particularly the resulting helical periodicity, remains challenging. Here, a novel cholesteric system is introduced and characterized based on hen egg white lysozyme (HEWL) amyloid fibrils and the results rationalized via a combination of experiments and theoretical scaling arguments. Specifically, the transition behaviors are elucidated from homogenous nematic, bipolar nematic to cholesteric tactoids following the classic Onsager model and the free energy functional model from Frank-Oseen elasticity theory. Additionally, the critical effects of pH and ionic strength on these order-order-transitions, as well as on the shape and helical pitch of the cholesteric tactoids are demonstrated. It is found that a small increase in pH from 2.0 to 2.8 results in a 34% decrease in pitch, while, on the contrary, increasing ionic strength from 0 to 10 mm leads to a 39% increase in pitch. The present study provides an approach to obtain controllable chiral nematic structures from HEWL amyloid fibrils, and may contribute further to the application of protein-based liquid crystals in pitch-sensitive biosensors or biomimetic architectures.

Influence of divalent metal cations on α-lactalbumin fibril formation.

The effect of binding of divalent metal cations (Ca2+, Cu2+, Mg2+, Mn2+, Zn2+) on the kinetics of fibril formation of bovine α-lactalbumin at acidic conditions is considered. The kinetic parameters of the process were determined using a thioflavin T fluorescence assay. The DSC thermograms of bovine α-lactalbumin in the presence and absence of cations were recorded. The duration of the lag period correlates with the changes in the thermal stability of the molten globule of the protein in the presence of cations. The final thioflavin T fluorescence intensity after formation of the mature fibrils decreases under the influence of calcium ions which strongly bind to the monomeric protein, and increases in solutions containing copper and especially zinc. These ions seem to accelerate secondary nucleation processes and change the fibril morphology, which was confirmed by atomic force microscopy imaging.

A Peptide Strategy for Inhibiting Different Protein Aggregation Pathways.

Protein aggregation correlates with many human diseases. Protein aggregates differ in structure and shape. Strategies to develop effective aggregation inhibitors that reach the clinic failed so far. Here, we developed a family of peptides targeting early aggregation stages for both amorphous and fibrillar aggregates of proteins unrelated in sequence and structure. They act on dynamic precursors before mechanistic differentiation takes place. Using peptide arrays, we first identified peptides inhibiting the amorphous aggregation of a molten globular, aggregation-prone mutant of the Axin tumor suppressor. Optimization revealed that the peptides activity did not depend on their sequences but rather on their molecular determinants: a composition of 20-30 % flexible, 30-40 % aliphatic and 20-30 % aromatic residues, a hydrophobicity/hydrophilicity ratio close to 1, and an even distribution of residues of different nature throughout the sequence. The peptides also suppressed fibrillation of Tau, a disordered protein that forms amyloids in Alzheimer's disease, and slowed down that of Huntingtin Exon1, an amyloidogenic protein in Huntington's disease, both entirely unrelated to Axin. Our compounds thus target early stages of different aggregation mechanisms, inhibiting both amorphous and amyloid aggregation. Such cross-mechanistic, multi-targeting aggregation inhibitors may be lead compounds for developing drug candidates against various protein aggregation diseases.

Structure-Activity Relationship of Fluorinated Benzenesulfonamides as Inhibitors of Amyloid-ß Aggregation.

Amyloid-beta aggregation is considered one of the factors influencing the onset of the Alzheimer's disease. Early prevention of such aggregation should alleviate disease condition by applying small molecule compounds that shift the aggregation equilibrium toward the soluble form of the peptide or slow down the process. We have discovered that fluorinated benzenesulfonamides of particular structure slowed the amyloid-beta peptide aggregation process by more than three-fold. We synthesized a series of ortho-para and meta-para double-substituted fluorinated benzenesulfonamides that inhibited the aggregation process to a variable extent yielding a detailed picture of the structure-activity relationship. Analysis of compound chemical structure effect on aggregation in artificial cerebrospinal fluid showed the necessity to arrange the benzenesulfonamide, hydrophobic substituent, and benzoic acid in a particular way. The amyloid beta peptide aggregate fibril structures varied in cross-sectional height depending on the applied inhibitor indicating the formation of a complex with the compound. Application of selected inhibitors increased the survivability of cells affected by the amyloid beta peptide. Such compounds may be developed as drugs against Alzheimer's disease.

Investigation of the Effect of Pyrogallol on the Formation of Amylin Amyloid Fibrils as a Strategy for the Treatment of Type 2 Diabetic Patients: A Theoretical and Experimental Study.

In more than 50 to 90% of type 2 diabetic patients, under the influence of various factors, the production of islet amyloid polypeptide or amylin in pancreatic beta cells increases. Spontaneous accumulation of amylin peptide in the form of insoluble amyloid fibrils and soluble oligomers is one of the main causes of beta cell death in diabetic patients. The objective of the present study was to evaluate the effect of pyrogallol, as a phenolic compound, on inhibiting the formation of amylin protein amyloid fibrils. In this study, different techniques such as the thioflavin T (ThT) and 1-Anilino-8-naphthalene sulfonate (ANS) fluorescence intensity and the circular dichroism (CD) spectrum, will be used to investigate the effects of this compound on inhibiting the formation of amyloid fibrils. To investigate the interaction sites of pyrogallol with amylin, docking studies were performed. Our results that pyrogallol in a dose-dependent manner (0.5:1, 1:1, and 5:1, Pyr to Amylin) inhibits the amylin amyloid fibrils formation. Docking analysis revealed that pyrogallol forms hydrogen bonds with valine 17 and asparagine 21. In addition, this compound forms 2 more hydrogen bonds with asparagine 22. This compound also forms hydrophobic bonds with histidine 18. Considering this data and the direct relationship between oxidative stress and the formation of amylin amyloid accumulations in diabetes, the use of compounds with both antioxidant and anti-amyloid properties can be considered an important therapeutic strategy for type 2 diabetes.

Unfolding Mechanism and Fibril Formation Propensity of Human Prion Protein in the Presence of Molecular Crowding Agents.

The pathological process of prion diseases implicates that the normal physiological cellular prion protein (PrPC) converts into misfolded abnormal scrapie prion (PrPSc) through post-translational modifications that increase ß-sheet conformation. We recently demonstrated that HuPrP(90-231) thermal unfolding is partially irreversible and characterized by an intermediate state (ß-PrPI), which has been revealed to be involved in the initial stages of PrPC fibrillation, with a seeding activity comparable to that of human infectious prions. In this study, we report the thermal unfolding characterization, in cell-mimicking conditions, of the truncated (HuPrP(90-231)) and full-length (HuPrP(23-231)) human prion protein by means of CD and NMR spectroscopy, revealing that HuPrP(90-231) thermal unfolding is characterized by two successive transitions, as in buffer solution. The amyloidogenic propensity of HuPrP(90-231) under crowded conditions has also been investigated. Our findings show that although the prion intermediate, structurally very similar to ß-PrPI, forms at a lower temperature compared to when it is dissolved in buffer solution, in cell-mimicking conditions, the formation of prion fibrils requires a longer incubation time, outlining how molecular crowding influences both the equilibrium states of PrP and its kinetic pathways of folding and aggregation.

Inhibition of heat-induced protein aggregation by zirconium phthalocyanines.

Specific proteins found in food sources tend to aggregate into fibrils under heat treatment; studying these aggregation processes and developing tools to control protein heat-induced aggregation is an active area of research. Phthalocyanine complexes are known to exhibit antiprionic and anti-fibrillogenic activity. Thus, the anti-fibrillogenic effect of a series of Zr phthalocyanines with different out-of-plane coordinated ligands, namely positively charged (PcZrLys2 ), negatively charged (PcZrCitr2 ), and group able to form disulfide bridges (PcZrS2 ), on the heat-induced aggregation of such proteins as BLG, insulin, and lysozyme was studied. The inhibition of reaction activity up to about 90% was observed in the presence of these compounds for all proteins. The effective concentration of the inhibitor was calculated for the compound with the highest activity (PcZrS2 ) to be 10.6 ± 3.6 and 7.3 ± 1.2 µM/L, respectively. Fluorescence spectroscopy studies demonstrated similar binding constants of three phthalocyanines binding with BLG globule. This is consistent with the results of molecular dynamics simulation, which imply the interaction of the globule with the tetrapyrrole macrocycle of phthalocyanine, leading to the globule stabilization. At the same time, TEM shows that in the presence of phthalocyanine PcZrS2 , thinner and longer fibrils were formed compared to control in all three proteins (BLG, insulin, and lysozyme). Thus, we can conclude that phthalocyanine PcZrS2 affects the amyloid aggregation's general mechanism, which is typical for proteins of different structures. Therefore, the phthalocyanine PcZrS2 is proposed as an anti-amyloidogenic agent suppressing heat-induced aggregation of proteins of different structures, making it potentially suitable for application in the food industry.

Efficient 18.8 T MAS-DNP NMR reveals hidden side chains in amyloid fibrils.

Amyloid fibrils are large and insoluble protein assemblies composed of a rigid core associated with a cross-ß arrangement rich in ß-sheet structural elements. It has been widely observed in solid-state NMR experiments that semi-rigid protein segments or side chains do not yield easily observable NMR signals at room temperature. The reasons for the missing peaks may be due to the presence of unfavorable dynamics that interfere with NMR experiments, which result in very weak or unobservable NMR signals. Therefore, for amyloid fibrils, semi-rigid and dynamically disordered segments flanking the amyloid core are very challenging to study. Here, we show that high-field dynamic nuclear polarization (DNP), an NMR hyperpolarization technique typically performed at low temperatures, can circumvent this issue because (i) the low-temperature environment (~ 100 K) slows down the protein dynamics to escape unfavorable detection regime, (ii) DNP improves the overall NMR sensitivity including those of flexible side chains, and (iii) efficient cross-effect DNP biradicals (SNAPol-1) optimized for high-field DNP (≥ 18.8 T) are employed to offer high sensitivity and resolution suitable for biomolecular NMR applications. By combining these factors, we have successfully established an impressive enhancement factor of ε ~ 50 on amyloid fibrils using an 18.8 T/ 800 MHz magnet. We have compared the DNP efficiencies of M-TinyPol, NATriPol-3, and SNAPol-1 biradicals on amyloid fibrils. We found that SNAPol-1 (with ε ~ 50) outperformed the other two radicals. The MAS DNP experiments revealed signals of flexible side chains previously inaccessible at conventional room-temperature experiments. These results demonstrate the potential of MAS-DNP NMR as a valuable tool for structural investigations of amyloid fibrils, particularly for side chains and dynamically disordered segments otherwise hidden at room temperature.