Different propensity to form amyloid fibrils by two homologous proteins-Human stefins A and B: searching for an explanation

By using ThT fluorescence, X-ray diffraction, and atomic force microscopy (AFM), it has been shown that human stefins A and B (subfamily A of cystatins) form amyloid fibrils. Both protein fibrils show the 4.7 A and 10 A reflections characteristic for cross beta-structure. Similar height of approximately 3 nm and longitudinal repeat of 25-27 nm were observed by AFM for both protein fibrils. Fibrils with a double height of 5.6 nm were only observed with stefin A. The fibril's width for stefin A fibrils, as observed by transmission electron microscopy (TEM), was in the same range as previously reported for stefin B (Zerovnik et al., Biochem Biophys Acta 2002;1594:1-5). The conditions needed to undergo fibrillation differ, though. The amyloid fibrils start to form at pH 5 for stefin B, whereas in stefin A, preheated sample has to be acidified to pH < 2.5. In both cases, adding TFE, seeding, and alignment in a strong magnetic field accelerate the fibril growth. Visual analysis of the three-dimensional structures of monomers and domain-swapped dimers suggests that major differences in stability of both homologues stem from arrangement of specific salt bridges, which fix alpha-helix (and the alpha-loop) to beta-sheet in stefin A monomeric and dimeric forms.     

Amyloid fibril formation by human stefin B in vitro: immunogold labelling and comparison to stefin A

The mechanism by which proteins form amyloid fibrils is of high interest to the scientific community as its understanding could resolve questions relevant to conformational diseases. The structural and energetic basis of the process is still largely unknown. The main controversial issue is the co-existence of several protein conformations. Three models for the mechanism of protein fibrillogenesis have been proposed which need to be tested by experiments. In this report, amyloid fibrils grown from human stefin B (type I cystatin) are described. This physiologically relevant protein readily forms fibrils in vitro, in contrast to the homologue--human stefin A--which forms fibrils under extreme conditions only. In order to specifically label stefin B fibrils in vitro, rabbit polyclonal antibody and mouse monoclonal antibody A6/2 against human stefin B were used for immunogold labelling. Samples were examined by transmission electron microscopy. Fibrils of stefin B were strongly labelled using polyclonal antibody and Protein A gold, whereas no positive reaction was observed with monoclonal antibody A6/2.     

Differences in aggregation properties of three site-specific mutants of recombinant human stefin B

We describe expression, purification, and characterization of three site-specific mutants of recombinant human stefin B: H75W, P36G, and P79S. The far- and near-UV CD spectra have shown that they have similar secondary and tertiary structures to the parent protein. The elution on gel-filtration suggests that recombinant human stefin B and the P36G variant are predominantly monomers, whereas the P79S variant is a dimer. ANS dye binding, reflecting exposed hydrophobic patches, is highest for the P36G variant, both at pH 5 and 3. ANS dye binding also is increased for stefin B and the other two variants at pH 3. Under the chosen conditions the highest tendency to form amyloid fibrils has been shown for the recombinant human stefin B. The P79S variant demonstrates a longer lag phase and a lower rate of fibril formation, while the P36G variant is most prone to amorphous aggregation. This was demonstrated by ThT fluorescence as a function of time and by transmission electron microscopy.     

Amyloid fibril formation by human stefins: Structure,mechanism & putative functions

Many questions in the field of protein aggregation to amyloid fibrils remain open. In this review we describe predominantly in vitro studies of oligomerization and amyloid fibril formation by human stefins A and B. In human stefin B amyloidogenesis in vitro we have observed some general and many specific properties of its prefibrillar oligomers and amyloid fibrils. One characteristic feature in common to stefins and cystatins (and possibly some other amyloid proteins) is domain-swapping. In addition to solution structure of the domain-swapped dimer of stefin A, we recently have determined 3D structure of stefin B tetramer, which proved to be composed from two domain-swapped dimers, whose interaction occurs by a proline switch in the loop surrounding the conserved Pro 74. Studying the mechanism of fibril formation by stefin B, we found that the nucleation and fibril elongation reactions have energies of activation (E_a’s) in the range of proline isomerisation, strongly indicating importance of the Pro at site 74 and/or other prolines in the sequence. Correlation between toxicity of the prefibrillar oligomers and their interaction with acidic phospholipids was demonstrated. Stefin B was shown to interact with amyloid-beta peptide of Alzheimer’s disease in an oligomer specific manner, both in vitro and in the cells. It also has been shown that endogenous stefin B (with E at site 31) but especially the EPM1 mutant R68X and Y31-stefin B variant, and to a lesser extent EPM1 mutant G4R, are prone to form aggregates in cells.     

Chemical dissection and reassembly of amyloid fibrils formed by a peptide fragment of transthyretin

We have examined the chemical dissection and subsequent reassembly of fibrils formed by a ten-residue peptide to probe the forces that drive the formation of amyloid. The peptide, TTR(10-19), encompasses the A strand of the inner beta-sheet structure that lines the thyroid hormone binding site of the human plasma protein transthyretin. When dissolved in water under low pH conditions the peptide readily forms amyloid fibrils. Electron microscopy of these fibrils indicates the presence of long (>1000 nm) rigid structures of uniform diameter (approximately 14 nm). Addition of urea (3 M) to preformed fibrils disrupts these rigid structures. The partially disrupted fibrils form flexible ribbon-like arrays, which are composed of a number of clearly visible protofilaments (3-4 nm diameter). These protofilaments are highly stable, and resist denaturation in 6 M urea at 75 degrees C over a period of hours. High concentrations (>50%, v/v) of 2,2,2-trifluoroethanol also dissociate TTR(10-19) fibrils to the constituent protofilaments, but these slowly dissociate to monomeric, soluble peptides with extensive alpha-helical structure. Dilution of the denaturant or co-solvent at the stage when dissociation to protofilaments has occurred results in the efficient reassembly of fibrils. These results indicate that assembly of fibrils from protofilaments involves relatively weak and predominantly hydrophobic interactions, whereas assembly of peptides into protofilaments involves both electrostatic and hydrophobic forces, resulting in a highly stable and compact structures.     

Amyloid fibril formation by human stefin B: influence of the initial pH-induced intermediate state

The amyloid fibril field is briefly described, with some stress put on differences between various proteins and possible role for domain swapping. In the main body of the text, first, a short review is given of the folding properties of both human stefins, alpha/beta-type globular proteins of 53% identity with a known three-dimensional fold. Second, in vitro study of amyloid fibril formation by human stefin B (type I cystatin) is described. Solvents of pH 4.8 and pH 3.3 with and without 2,2,2-trifluoroethanol (TFE) were probed, as it has been shown previously that stefin B forms acid intermediates, a native-like and molten globule intermediate, respectively. The kinetics of fibrillation were measured by thioflavin T fluorescence and CD. At pH 3.3, the protein is initially in the molten globule state. The fibrillation is faster than at pH 4.8; however, there is more aggregation observed. On adding TFE at each pH, the fibril formation is further accelerated.     

The cross-road between the mechanisms of protein folding and aggregation; study of human stefin B and its H75W mutant

The role of the aromatic residue at site 75 to protein stability, the mechanism of folding and the mechanism of amyloid-fibril formation were investigated for the human stefin B variant (bearing Y at site 31) and its point mutation H75W. With an aim to reveal the conformation at the cross-road between folding and aggregation, first, the kinetics of folding and oligomer formation by human stefin B(Y31) variant were studied. It was found to fold in three kinetic phases at pH 4.8 and 10% TFE; the pH and solvent conditions that transform the protein into amyloid fibrils at longer times. The same pH leads to the formation of native-like intermediate (known from previous studies of this variant), meaning that the process of folding and amyloid-fibril formation share the same structural intermediate, which is in this case native-like and dimeric. At pH 5.8 and 7.0 stefin B folded to the native state in four kinetic phases over two intermediates. In distinction, the mutant H75W did not fold to completion, ending in intermediate states at all pH values studied: 4.8, 5.8 and 7.0. At pH 4.8 and 5.8, the mutant folded in one kinetic phase to the intermediate of the “molten globule” type, which leads to the conclusion that its mechanism of folding differs from the one of the parent stefin B at the same pH. At pH 7.0 the mutant H75W folded in three kinetic phases to a native-like intermediate, analogous to folding of stefin B at pH 4.8.     

In vitro study of stability and amyloid-fibril formation of two mutants of human stefin B (cystatin B) occurring in patients with EPM1

Myoclonus epilepsy of type 1 (EPM1) is a rare monogenic progressive and degenerative epilepsy, also known under the name Unverricht-Lundborg disease. With the aim of comparing their behavior in vitro, wild-type (wt) human stefin B (cystatin B) and the G4R and the R68X mutants observed in EPM1 were expressed and isolated from the Escherichia coli lysate. The R68X mutant (Arg68Stop) is a peptide of 67 amino acids from the N terminus of stefin B. CD spectra have shown that the R68X peptide is not folded, in contrast to the G4R mutant, which folds like wild type. The wild type and the G4R mutant were unfolded by urea and by trifluoroethanol (TFE). It has been shown that both proteins have closely similar stability and that at pH 4.8, where a native-like intermediate was demonstrated, TFE induces unfolding intermediates prior to the major transition to the all-alpha-helical state. Kinetics of fibril formation were followed by Thioflavin T fluorescence while the accompanying changes of morphology were followed by the transmission electron microscopy (TEM). For the two folded proteins the optimal concentration of TFE producing extensive lag phases and high fibril yields was predenaturational, 9% (v/v). The unfolded R68X peptide, which is highly prone to aggregate, formed amyloid fibrils in aqueous solution and in predenaturing 3% TFE. The G4R mutant exhibited a much longer lag phase than the wild type, with the accumulation of prefibrillar aggregates. Implications for pathology in view of the higher toxicity of prefibrillar aggregates to cells are discussed.     

Association of thin filaments into thick filaments revealing the structural hierarchy of amyloid fibrils

Beta2-Microglobulin (beta2-m) is a major structural component of dialysis-related amyloid fibrils. Kozhukh et al. [J. Biol. Chem. 277 (2002) 1310] prepared a series of peptide fragments of beta2-m by the protease digestion and examined their ability to form amyloid fibrils in citrate buffer at pH 2.5. Among various peptides, a 22-residue K3 peptide corresponding to Ser20-Lys41 spontaneously formed amyloid fibrils in aqueous solution. This peptide also formed amyloid protofibrils in 20% (v/v) 2,2,2-trifluoroethanol (TFE). To investigate the influence of solvent conditions on fibril formation, we studied their structures by atomic force microscopy. In aqueous solution, fibrils had a diameter of 4 or 8 nm and tended to cluster each other. On the other hand, protofibrils in 20% (v/v) TFE had a diameter of 2 nm with no tendency of clustering. Intriguingly, when the K3 protofibrils were transferred from 20% (v/v) TFE to aqueous solution, some of them associated to form thicker fibrils with a diameter of 4-15 nm and a left-handed helical twist. TFE is a hydrophobic solvent, so that hydrophobic interactions between molecules may be weakened. The results suggest that the fibrils in aqueous conditions are formed by the cooperative association of protofibrils at the growing ends of the fibrils, in which hydrophobic interactions play a major role.     

Exclusion of the native alpha-helix from the amyloid fibrils of a mixed alpha/beta protein

Members of the cystatin superfamily are involved in an inherited form of cerebral amyloid angiopathy and readily form amyloid fibrils in vitro. We have determined the structured core of human stefin B (cystatin B) amyloid fibrils using quenched hydrogen exchange and NMR. The core contains residues from four of the five strands of the native β-sheet, delimited by unprotected loop regions analogous to those of the native monomeric structure. However, non-native features are also apparent, the most striking of which is the exclusion of the native α-helix. Before forming amyloid in vitro, cystatins dimerise via 3D domain swapping, and assemble into tetramers with trans to cis isomerism of a conserved proline. In the fibril, the hinge loop that forms an extended β-structure in the dimer remains protected, consistent with the domain-swapping interface being maintained. However, the fibril data are not compatible with a simple 3D domain-swapping model for amyloid formation, and the displacement of the helix points to alternative packing arrangements of native-like β-structure, in which proline isomerism is important in preventing steric clashing.     

Differences in the effects of TFE on the folding pathways of human stefins A and B.

Trifluoroethanol (TFE) has been used to probe differences in the stability of the native state and in the folding pathways of the homologous cysteine protein inhibitors, human stefin A and B. After complete unfolding in 4.5 mol/L GuHCl, stefin A refolded in 11% (vol/vol) TFE, 0.75 mol/L GuHCl, at pH 6.0 and 20 degrees C, with almost identical first-order rate constants of 4.1 s-1 and 5.5 s-1 for acquisition of the CD signal at 230 and 280 nm, respectively, rates that were markedly greater than the value of 0.11 s-1 observed by the same two probes when TFE was absent. The acceleration of the rates of refolding, monitored by tyrosine fluorescence, was maximal at 10% (vol/vol) TFE. Similar rates of refolding (6.2s-1 and 7.2 s-1 for ellipticity at 230 and 280 nm, respectively) were observed for stefin A denatured in 66% (vol/vol) TFE, pH 3.3, when refolding to the same final conditions. After complete unfolding in 3.45 mol/L GuHCl, stefin B refolded in 7% (vol/vol) TFE, 0.57 mol/L GuHCl, at pH 6.0 and 20 degrees C, with a rate constant for the change in ellipticity at 280 nm of 32.8 s-1; this rate was only twice that observed when TFE was absent. As a major point of distinction from stefin A, the refolding of stefin B in the presence of TFE showed an overshoot in the ellipticity at 230 nm to a value 10% greater than that in the native protein; this signal relaxed slowly (0.01 s-1) to the final native value, with little concomitant change in the near-ultraviolet CD signal; the majority of this changes in two faster phases. After denaturation in 42% (vol/vol) TFE, pH 3.3, the kinetics of refolding to the same final conditions exhibited the same rate-limiting step (0.01 s-1) but were faster initially. The results show that similarly to stefin A, stefin B forms its hydrophobic core and predominant part of the tertiary structure faster in the presence of TFE. The results imply that the alpha-helical intermediate of stefin B is highly structured. Proteins 1999;36:205-216.     

High affinity copper binding by stefin B (cystatin B) and its role in the inhibition of amyloid fibrillation

We show that human stefin B, a protease inhibitor from the family of cystatins, is a copper binding protein, unlike stefin A. We have used isothermal titration calorimetry to directly monitor the binding event at pH 7 and pH 5. At pH 7 stefin B shows a picomolar affinity for copper but at pH 5 the affinity is in the nanomolar range. There is no difference in the affinity of copper between the wildtype stefin B (E31 isoform) and a variant (Y31 isoform), whereas the mutant (P79S), which is tetrameric, does not bind copper. The conformation of stefin B remains unaltered by copper binding. It is known that below pH 5 stefin B undergoes a conformational change and amyloid fibril formation. We show that copper binding inhibits the amyloid fibril formation and, to a lesser degree, the initial aggregation. Similarities to and differences from other copper binding amyloidogenic proteins are discussed.     

Essential role of Pro 74 in stefin B amyloid-fibril formation: Dual action of cyclophilin A on the process

We report that Pro74 in human stefin B is critical for fibril formation and that proline isomerization plays an important role. The stefin B P74S mutant did not fibrillate over the time of observation at 25 °C, and it exhibited a prolonged lag phase at 30 °C and 37 °C. The peptidyl prolyl cis/trans isomerase cyclophilin A, when added to the wild-type protein, exerted two effects: it prolonged the lag phase and increased the yield and length of the fibrils. Addition of the inactive cyclophilin A R55A variant still resulted in a prolonged lag phase but did not mediate the increase of the final fibril yield. These results demonstrate that peptidyl prolyl cis/trans isomerism is rate-limiting in stefin B fibril formation.     

Nucleobindin 1 caps human islet amyloid polypeptide protofibrils to prevent amyloid fibril formation

Many human diseases are associated with amyloid fibril deposition, including type 2 diabetes mellitus where human islet amyloid polypeptide (hIAPP) forms fibrils in the pancreas. We report here that engineered, soluble forms of the human Ca(2+)-binding protein nucleobindin 1 (NUCB1) prevent hIAPP fibril formation and disaggregate preexisting hIAPP fibrils. Scanning transmission electron microscopy (STEM) and atomic force microscopy indicate that NUCB1 binds to and stabilizes heterogeneous prefibrillar hIAPP species. The NUCB1-stabilized prefibrillar species were isolated by size-exclusion chromatography and analyzed by STEM, dynamic light scattering, and multi-angle light scattering. The stabilized prefibrillar species show a size range of 2-6 million Da and have other similarities to hIAPP protofibrils, but they do not progress to become mature fibrils. The effects of NUCB1 are absent in the presence of Ca(2+). We postulate that the engineered forms of NUCB1 prevent hIAPP fibril formation by a mechanism where protofibril-like species are "capped" to prevent further fibril assembly and maturation. This mode of action appears to be different from other protein-based inhibitors, suggesting that NUCB1 may offer a new approach to inhibiting amyloid formation and disaggregating amyloid fibrils.     

Optimum amyloid fibril formation of a peptide fragment suggests the amyloidogenic preference of beta2-microglobulin under physiological conditions

Beta(2)-microglobulin (beta(2)m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Although full-length beta(2)m readily forms amyloid fibrils in vitro by seed-dependent extension with a maximum at pH 2.5, fibril formation under physiological conditions as detected in patients has been difficult to reproduce. A 22-residue K3 peptide of beta(2)m, Ser(20)-Lys(41), obtained by digestion with Acromobacter protease I, forms amyloid fibrils without seeding. To obtain further insight into the mechanism of fibril formation, we studied the pH dependence of fibril formation of the K3 peptide and its morphology using a ThT fluorescence assay and electron microscopy, respectively. K3 peptide formed amyloid fibrils over a wide range of pH values with an optimum around pH 7 and contrasted with the pH profile of the seed-dependent extension reaction of full-length beta(2)m. This suggests that once the rigid native-fold of beta(2)m is unfolded and additional factors triggering the nucleation process are provided, full-length beta(2)m discloses an intrinsic potential to form amyloid fibrils at neutral pH. The fibril formation was strongly promoted by dimerization of K3 through Cys(25). The morphology of the fibrils varied depending on the fibril formation conditions and the presence or absence of a disulfide bond. Various fibrils had the potential to seed fibril formation of full-length beta(2)m accompanied with a characteristic lag phase, suggesting that the internal structures are similar.     

Hexafluoroisopropanol induces amyloid fibrils of islet amyloid polypeptide by enhancing both hydrophobic and electrostatic interactions

Although amyloid fibrils deposit with various proteins, the comprehensive mechanism by which they form remains unclear. We studied the formation of fibrils of human islet amyloid polypeptide associated with type II diabetes in the presence of various concentrations of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) under acidic and neutral pH conditions using CD, amyloid-specific thioflavin T fluorescence, fluorescence imaging with thioflavin T, and atomic force microscopy. At low pH, the formation of fibrils was promoted by HFIP with an optimum at 5% (v/v). At neutral pH in the absence of HFIP, significant amounts of amorphous aggregates formed in addition to the fibrils. The addition of HFIP suppressed the formation of amorphous aggregates, leading to a predominance of fibrils with an optimum effect at 25% (v/v). Under both conditions, higher concentrations of HFIP dissolved the fibrils and stabilized the α-helical structure. The results indicate that fibrils and amorphous aggregates are different types of precipitates formed by exclusion from water-HFIP mixtures. The exclusion occurs through the combined effects of hydrophobic interactions and electrostatic interactions, both of which are strengthened by low concentrations of HFIP, and a subtle balance between the two types of interactions determines whether the fibrils or amorphous aggregates dominate. We suggest a general view of how the structure of precipitates varies dramatically from single crystals to amyloid fibrils and amorphous aggregates.     

Transthyretin fibrillogenesis entails the assembly of monomers: a molecular model for in vitro assembled transthyretin amyloid-like fibrils

Extracellular accumulation of transthyretin (TTR) variants in the form of fibrillar amyloid deposits is the pathological hallmark of familial amyloidotic polyneuropathy (FAP). The TTR Leu55Pro variant occurs in the most aggressive forms of this disease. Inhibition of TTR wild-type (WT) and particularly TTR Leu55Pro fibril formation is of interest as a potential therapeutic strategy and requires a thorough understanding of the fibril assembly mechanism. To this end, we report on the in vitro assembly properties as observed by transmission electron microscopy (TEM), atomic force microscopy (AFM) and quantitative scanning transmission electron microscopy (STEM) for both TTR WT fibrils produced by acidification, and TTR Leu55Pro fibrils assembled at physiological pH. The morphological features and dimensions of TTR WT and TTR Leu55Pro fibrils were similar, with up to 300 nm long, 8 nm wide fibrils being the most prominent species in both cases. Other species were evident; 4-5 nm wide fibrils, 9-10 nm wide fibrils and oligomers of various sizes. STEM mass-per-length (MPL) measurements revealed discrete fibril types with masses of 9.5 and 14.0(+/-1.4) KDa/nm for TTR WT fibrils and 13.7, 18.5 and 23.2(+/-1.5) kDa/nm for TTR Leu55Pro fibrils. These MPL values are consistent with a model in which fibrillar TTR structures are composed of two, three, four or five elementary protofilaments, with each protofilament being a vertical stack of structurally modified TTR monomers assembled with the 2.9 nm axial monomer-monomer spacing indicated by X-ray fibre diffraction data. Ex vivo TTR amyloid fibrils were examined. From their morphological appearance compared to these, the in vitro assembled TTR WT and Leu55Pro fibrils examined may represent immature fibrillar species. The in vitro system operating at physiological pH for TTR Leu55Pro and the model presented for the molecular arrangement of TTR monomers within fibrils may, therefore, describe early fibril assembly events in vivo.     

Seeding-dependent propagation and maturation of amyloid fibril conformation

Recent studies of amyloid fibrils have focused on the presence of multiple amyloid forms even with one protein and their propagation by seeding, leading to conformational memory. To establish the structural basis of these critical features of amyloid fibrils, we used the amyloidogenic fragment Ser20-Lys41 (K3) of beta2-microglobulin, a protein responsible for dialysis-related amyloidosis. In 20% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl (pH approximately 2), K3 peptide formed two types of amyloid-like fibrils, f218 and f210, differing in the amount of beta-sheet as measured by circular dichroism spectroscopy and Fourier transform infrared spectroscopy. Atomic force microscopy showed that the fibril with a larger amount of beta-sheet (f210) is thinner and longer. Both fibrils were reproduced by seeding, showing the template-dependent propagation of a fibril's conformation. However, upon repeated self-seeding, f218 fibrils were gradually transformed into f210 fibrils, revealing the conformational maturation. The observed maturation can be explained fully by a competitive propagation of two fibrils. The maturation of amyloid fibrils might play a role during the development of amyloidosis.     

Quantitative morphological analysis reveals ultrastructural diversity of amyloid fibrils from alpha-synuclein mutants

High resolution atomic force microscopy is a powerful tool to characterize nanoscale morphological features of protein amyloid fibrils. Comparison of fibril morphological properties between studies has been hampered by differences in analysis procedures and measurement error determination used by various authors. We describe a fibril morphology analysis method that allows for quantitative comparison of features of amyloid fibrils of any amyloidogenic protein measured by atomic force microscopy. We have used tapping mode atomic force microscopy in liquid to measure the morphology of fibrillar aggregates of human wild-type alpha-synuclein and the disease-related mutants A30P, E46K, and A53T. Analysis of the images shows that fibrillar aggregates formed by E46K alpha-synuclein have a smaller diameter (9.0 +/- 0.8 nm) and periodicity (mode at 55 nm) than fibrils of wild-type alpha-synuclein (height 10.0 +/- 1.1 nm; periodicity has a mode at 65 nm). Fibrils of A30P have smaller diameter still (8.1 +/- 1.2 nm) and show a variety of periodicities. This quantitative analysis procedure enables comparison of the results with existing models for assembly of amyloid fibrils.     

Dissolution of beta2-microglobulin amyloid fibrils by dimethylsulfoxide

Increasing numbers of proteins have been found to aggregate into insoluble fibers, collectively referred to as amyloid fibrils. To address the conformational stability of amyloid fibrils, we studied the effects of dimethylsulfoxide (DMSO), 2,2,2-trifluoroethanol (TFE), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) on beta(2)-microglobulin amyloid fibrils by circular dichroism, thioflavin T fluorescence, light scattering, and electron microscopy. When measured by circular dichroism and thioflavin T fluorescence, HFIP, and TFE dissolved the fibrils, producing predominantly helical conformations. However, these alcohols did not dissolve the amyloid fibrils completely as monitored by light scattering and electron microscopy. On the other hand, DMSO completely dissolved the amyloid fibrils although a high concentration [i.e., 80% (v/v)] was required. These results are consistent with the important role of hydrogen bonds in stabilizing amyloid fibrils.