Thioflavin T-Silent Denaturation Intermediates Support the Main-Chain

Article pubs.acs.org/biochemistry

Thioflavin T‑Silent Denaturation Intermediates Support the MainChain-Dominated Architecture of Amyloid Fibrils Sayaka Noda,† Masatomo So,† Masayuki Adachi,† József Kardos,‡ Yoko Akazawa-Ogawa,§ Yoshihisa Hagihara,§ and Yuji Goto*,† †

Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan Department of Biochemistry and MTA-ELTE NAP B Neuroimmunology Research Group, Eötvös Loránd University, Pázmány sétány 1/C, Budapest 1117, Hungary § National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan ‡

S Supporting Information *

ABSTRACT: Ultrasonication is considered one of the most effective agitations for inducing the spontaneous formation of amyloid fibrils. When we induced the ultrasonication-dependent fibrillation of β2-microglobulin and insulin monitored by amyloid-specific thioflavin T (ThT) fluorescence, both proteins showed a significant decrease in ThT fluorescence after the burst-phase increase. The decrease in ThT fluorescence was accelerated when the ultrasonic power was stronger, suggesting that this decrease was caused by the partial denaturation of preformed fibrils. The possible intermediates of denaturation retained amyloid-like morphologies, secondary structures, and seeding potentials. Similar denaturation intermediates were also observed when fibrils were denatured by guanidine hydrochloride or sodium dodecyl sulfate. The presence of these denaturation intermediates is consistent with the main-chain-dominated architecture of amyloid fibrils. Moreover, in the three types of denaturation experiments conducted, insulin fibrils were more stable than β2-microglobulin fibrils, suggesting that the relative stability of various fibrils is independent of the method of denaturation. myloid fibrils are fibrillar aggregates of proteins with a width of 10 nm and a length of several micrometers.1−3 Although they have been associated with more than 30 types of amyloidoses, including dialysis-related amyloidosis caused by the amyloid fibrils of β2-microglobulin (β2m), various proteins not associated with diseases have also been shown to form amyloid fibrils, indicating that amyloid fibrillation is a generic property of denatured proteins. There are several structural models of amyloid fibrils.1,2,4 The dominant secondary structure is a cross-β-structure stabilized by an ordered hydrogen bond network. It has been argued that while globular native forms have a side-chain-dominated compact structure that evolves by pursuing a unique fold with the optimal packing of amino acid residues,5,6 amyloid fibrils have a main-chaindominated structure with an extensive hydrogen bond network.7−10 On the other hand, the tight packing of side chains, as observed for the microcrystals of some short amyloidogenic peptides, created a steric zipper model of amyloid fibrils that involves interdigitation of side chains to form a tight interface.1,11 A recent cryo-electron microscopy (cryo-EM) study of light-chain-derived amyloid fibrils supported the steric zipper structure.12 It is noted that the view of a main-chain-dominated amyloid architecture does not necessarily require optimal packing of the overall side chains. Our previous findings support this view contrasting side-chain-

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dominated protein folding and main-chain-dominated amyloid misfolding.9,10,13 Amyloid fibrils are formed in the supersaturated solutions of precursor proteins by a nucleation−growth mechanism, similar to the crystallization of solutes from solution.14−17 We revisited “supersaturation” and argued its critical involvement in amyloid fibrillation.18−22 The role of supersaturation at the proteome level in neurodegenerative diseases has recently been reported.23,24 Among the various kinds of agitations used to break supersaturation,25,26 ultrasonic irradiation18,27−30 has been shown to be highly effective in forcing amyloid fibrillation. In the case of ultrasonication-forced fibrillation, we suggested that interactions with the hydrophobic surfaces of cavitation bubbles condensed proteins, leading to the breakdown of supersaturation followed by fibrillation.18,31 Ultrasonication is now recognized as one of the important approaches in elucidating the mechanisms underlying amyloid fibrillation.31,32 When we monitored the ultrasonication-dependent fibrillation of proteins by thioflavin T (ThT), we often observed a significant decrease in ThT fluorescence after the burst-phase increase.29,32−34 This decrease in ThT fluorescence was Received: March 13, 2016 Revised: June 22, 2016 Published: June 25, 2016 3937

DOI: 10.1021/acs.biochem.6b00231 Biochemistry 2016, 55, 3937−3948

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binding to amyloid fibrils was employed to quantify fibril formation. We previously confirmed that the ultrasonic irradiation used did not decompose the chemical structure of β2m34 or insulin.21 Under ultrasonic conditions with cycles of irradiation for 4 min and quiescence for 1 min at 100 mM NaCl and pH 2.0, monomeric β2m transformed to amyloid fibrils in 2 h,29,32 which were used as seed fibrils. When seed fibrils at 5% (v/v) were added to the monomeric solution under quiescent conditions, ThT fluorescence increased without a lag time and reached saturation within 2 h. This solution was used as “preformed fibrils”. Seeding potentials in which 5% (v/v) of the seeds were added to the monomeric solution under quiescent conditions were examined. Seeding reactions were monitored by ThT fluorescence with an MTP-810 microplate reader or an SH9000 microplate reader (Corona Electric Co., Ibaraki, Japan). The fluorescence intensity of ThT at 490 nm was measured with excitation at 450 nm at 37 °C. Effects of Ultrasonication on Preformed Fibrils. The sample solution of preformed fibrils in a cuvette with a 1 cm light path was irradiated with ultrasonication pulses by tightly attaching an ultrasonic generator to a sidewall of the cuvette.18 The solution was stirred with a stirring magnet at 600 rpm. We applied four different conditions of ultrasonication: cycles of ultrasonication for 9 min and quiescence for 1 min with a high or low ultrasonic amplitude and cycles of ultrasonication for 4 min and quiescence for 1 min with a high or low amplitude. Experiments performed under the same conditions were repeated in triplicate, and one representative result was shown for the respective conditions. Chemical Denaturation. Various concentrations of GdnHCl (0−7.0 M) or sodium dodecyl sulfate (0−30 mM) were added to the preformed fibrils of β2m or insulin at 0.1 mg/mL fibrils, 100 mM NaCl, and 5 μM ThT at pH 2.0 and 37 °C. The extent of denaturation of fibrils was estimated by ThT fluorescence, circular dichroism, and transmission electron microscopy. Tryptophan and Tyrosine Fluorescence. Tryptophan fluorescence was measured by the F7000 Hitachi fluorescence spectrophotometer with excitation at 295 nm and emission at 300−400 nm. Tyrosine fluorescence was measured by the same fluorometer with excitation at 270 nm and emission at 270− 380 nm. Circular Dichroism (CD) and Transmission Electron Microscopy (TEM). Far-UV CD measurements were performed using a Jasco J820 or J720 spectropolarimeter using a quartz cuvette with a 1 mm path length at a protein concentration of 0.1 mg/mL and 37 °C. Spectra were expressed as the mean residue ellipticity, [θ] (degrees square centimeters per decimole). Sample solutions (5 μL) for the TEM measurements were spotted onto a collodion-coated copper grid (Nisshin EM Co., Tokyo, Japan). After 1 min, the remaining solution was removed with filter paper and 5 μL of 2% (w/v) ammonium molybdate was spotted onto collodion-coated copper grids. After 1 min, the remaining solution was removed in the same manner. TEM (H-7650, Hitachi, Tokyo, Japan) images were obtained with a 80 kV voltage and a 15000× magnification. Secondary structure contents were estimated using BeStSel46 and/or CDNN47 algorithms (Tables S1−S6). However, the properties of the amyloid fibrils (e.g., the tendency to form precipitates) or the solution conditions (e.g., high absorption of

accelerated when the lag time was shorter, suggesting that this reduction was caused by the ultrasonication-dependent transformation of preformed fibrils into distinct conformational states.29,32−34 Our previous findings suggested that this decrease in ThT fluorescence represented the formation of a “denaturation” intermediate of fibrils in which the secondary structures were retained but some specific side-chain structures responsible for ThT binding were disrupted.34 We here use “denaturation” to describe the destruction or depolymerization of amyloid fibrils into monomers assuming that amyloid fibrils are in a misfolded yet ordered conformational state. We investigated whether such intermediates resembled the molten globule intermediate observed for globular proteins, in which the secondary structures and compactness of the native states were largely retained, whereas the side chains were significantly disordered.35−38 If amyloid fibrils are formed by the mainchain-dominated interactions, the accumulation of such denaturation intermediates is more likely to occur than during the unfolding of the native states of globular proteins. In the case of native proteins, protein unfolding is one of the most important approaches to addressing the structure, dynamics, and mechanism of protein folding. In the same way, we believe that denaturation of amyloid fibrils is an important approach to achieving a comprehensive understanding of amyloid fibrils.9,10,39−42 Recent studies43,44 showed that non-native and toxic oligomers were produced during fibril disassembly in slightly acidic pH regions, indicating the biological relevance of denaturation studies of amyloid fibrils. To address the possibility of the molten globule-like denaturation intermediates of amyloid fibrils, we herein examined the denaturation of β2m and insulin fibrils induced by extensive ultrasonication, guanidine hydrochloride (GdnHCl), or sodium dodecyl sulfate (SDS). β2m and insulin are responsible for dialysis-related amyloidosis and insulinoma, respectively, and are considered to be highly amyloidogenic model proteins that are useful for elucidating the mechanism underlying amyloid fibrillation. The results obtained suggested that the denaturation intermediates with perturbed side chains were common to the amyloid fibrils of β2m and insulin, although their accumulation depended on the stability of fibrils.

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EXPERIMENTAL PROCEDURES Proteins and Chemicals. Recombinant human β2m with an additional Met residue at the N-terminus was expressed by Escherichia coli BL21 and purified as described previously.45 Recombinant human insulin (Roche Diagnostic GmbH) was purchased from Nacalai Tesque (Kyoto, Japan) and used without further purification. ThT was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents were obtained from Nacalai Tesque (Kyoto, Japan). Amyloid Fibrillation. Lyophilized β2m was dissolved at a concentration of 0.3 mg/mL in 10 mM HCl (pH 2.0) containing 100 mM NaCl and 5 μM ThT. Insulin was also dissolved at a concentration of 0.4 mg/mL in the same solution that was used for β2m. We used a water bath-type ultrasonic transmitter with a temperature controller (ELESTEIN, Elekon Science Co.) to induce fibrillation.27,29 A cycle involving ultrasonication for 4 min and quiescence for 1 min was repeated. The temperature of the water bath was set to 37 °C. Fibrils were detected by monitoring the fluorescence of ThT using the Hitachi fluorescence spectrophotometer (F7000). The excitation and emission wavelengths were 445 and 485 nm, respectively. The increase observed in ThT fluorescence upon 3938


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by seeding. We first monitored the decrease in the ThT fluorescence of β2m fibrils under various ultrasonic conditions by varying the ultrasonic power and irradiation period (Figure 1A). It is noted that ThT fluorescence before fibrillation was practically 0. We applied β2m fibrils to four different conditions of ultrasonication: cycles of ultrasonication for 9 min with quiescence for 1 min with a high or low ultrasonic amplitude

Gdn-HCl) have limited the applicability or reliability of the structure estimation. The results are summarized in Tables S1− S6. Fourier Transform Infrared (FTIR) Spectroscopy. FTIR measurements were taken on a Bruker Equinox 55 instrument equipped with an MCT detector in CaF2 cells with 100 μm Teflon spacers at 1 cm−1 resolution as described previously.21,34 The native insulin solution at low pH was prepared by dissolving the lyophilized insulin powder in D2O solutions containing 10 mM DCl and 0.1 M NaCl. Fibrillar samples were concentrated by centrifugation for 30 min at 40000 rpm with a Beckman TL-100 ultracentrifuge using a TLA100.3 rotor. The supernatant was used as a background reference. The spectra shown were baseline-subtracted, corrected for vapor and HDO contamination, and normalized to an area that is approximately equal to that of a 5 mg/mL protein solution. The second derivative of the spectra was used to determine the positions of the components.48 The fitting was performed as described previously.34 Cytotoxicity Measurements. PC12 cells were obtained from ACTT (CRL1721). The cells were maintained in RPMI1640 (Nacalai Tesque) supplemented with 5% (v/v) fetal bovine serum (FBS) (Gibco-Life Technologies), 10% (v/ v) horse serum (HS) (Nichimen America, Los Angeles, CA), and 1% (v/v) penicillin/streptomycin (Nacalai Tesque). The cells were kept in an incubator at 37 °C in a 5% CO2/95% air atmosphere. Twenty-four hours prior to experimentation, the PC12 cells were seeded onto a type I collagen-coated 96-well culture plate (nunc) at a density of 1.0 × 104 cells/well in RPMI1640 containing 10% FBS and 5% HS. For cell differentiation, the cells were treated with 50 ng/mL NGF in RPMI1640 containing 1% HS for 9 days. The WST-8 cell viability assay (Dojindo, Kumamoto, Japan) is based on the cleavage of the tetrazolium salt WST-8 to form a red formazan dye by viable cells and was performed according to the manufacturer’s instructions. Cells in microtiter plates were incubated for 24 h with various concentrations of insulin and β2m. The absorbance of the colored formazan was determined using an automated microplate reader at 450 and 620 nm (Multiskan Ascent Labsystems). The mean absorbance of control wells (cells without insulin and β2m) represented 100% cell viability. The viability of β2m- or insulin-treated cells was determined in at least triplicate and related to the absorbance of control cells. To detect direct Cecropin-induced cell lysis, we performed LDH release assays (Roche Molecular Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. The cells were incubated with various concentrations of β2m and insulin for 24 h. To determine the maximal LDH release, cells were treated with 2% Triton X-100 for 10 min before the assay was run. Untreated cells served as controls for spontaneous LDH release. The percentage of cytotoxicity was calculated using the equation LDH release (%) = (ODexp − ODmedium)/ (ODmaximum − ODmedium) × 100.

Figure 1. Stability of β2m fibrils under ultrasonication at pH 2.0. (A) Time-dependent changes in ThT fluorescence under various conditions of ultrasonication: cycles of ultrasonication for 9 min with quiescence for 1 min with a high (red) or low (blue) ultrasonic amplitude or cycles of ultrasonication for 4 min with quiescence for 1 min with a high (orange) or low (cyan) amplitude. The time courses of ThT fluorescence under stirring are also shown (black). (B) CD spectra of β2m fibrils under various conditions as described above. The spectra of monomers at pH 2 (---) and pH 7 (···) are also shown. (C) Tryptophan fluorescence spectra of β2m fibrils under high (red) and low (blue) ultrasonication with a cycle of irradiation for 1 min and quiescence for 1 min, and stirring (black). (D) TEM images of normal β2m fibrils (i), fibrils incubated under cycles of ultrasonication for 9 min and quiescence for 1 min with a high amplitude (ii), and fibrils incubated under cycles of ultrasonication for 4 min and quiescence for 1 min with a low amplitude (iii). The scale bar indicates 200 nm. (E) Seeding reactions of β2m monitored by ThT fluorescence with preformed fibrils incubated under various ultrasonic conditions as described above. The dashed line indicates the seeded fibrillation using the seeds formed under quiescent conditions. (F and G) CD spectra and TEM images, respectively, of fibrils elongated by seeding under stirring [black, (i), or cycles of ultrasonication for 9 min and quiescence for 1 min with a high (red) (ii) and low (blue) (iii) amplitude]. CD spectra (B and F), fluorescence spectra (C), and TEM images (D and G) were obtained at the end of reactions as shown in panels A and E.

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RESULTS Effects of Ultrasonication on Preformed Amyloid Fibrils. We previously reported ultrasonication-dependent decreases in ThT fluorescence for the fibrils of β2m,29,32,34 Aβ,49 and α-synuclein.33 To examine the ultrasonicationdependent decrease in ThT fluorescence in more detail, we herein used β2m and insulin. The amyloid fibrils of β2m were prepared by ultrasonication-forced fibrillation and propagation 3939


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seeding experiments may have been due to the fragmentation of long fibrils and the increase in the active ends of fibrils. Although the decreased ThT fluorescence intensity after seeding suggests that the reproduced fibrils were not exactly the same as those of preformed fibrils (Figure 1E), the validity of this remains unclear. Taken together, under extensive ultrasonic conditions, β2m fibrils were fragmented and transformed to distinct fibrillar species with decreased ThT fluorescence while retaining their β-structure and seeding potential. The effects of extensive ultrasonication on amyloid fibrils were also examined using human insulin (Figure 2A). The amyloid fibrils of insulin were prepared as described above for β2m fibrils at 0.4 mg/mL insulin, 100 mM NaCl, and pH 2.0. The ultrasonic conditions used were the same as those used for β2m. Upon ultrasonic irradiation with a high amplitude, insulin fibrils showed a decrease in ThT fluorescence irrespective of the irradiation cycles, whereas insulin fibrils under ultrasonic cycles with a low amplitude showed no change in ThT fluorescence (Figure 2A). Thus, a high amplitude of ultrasonic irradiation was required to decrease the ThT fluorescence of insulin fibrils. The secondary structures of insulin fibrils before and after ultrasonic irradiation were examined by measuring far-UV CD spectra (Figure 2B and Table S2). Although the CD spectra of insulin fibrils varied depending on the ultrasonic conditions, all of them showed β-sheet-dominated structures even after the significant decrease in ThT fluorescence (see ref 21) for the various CD spectra of insulin fibrils under distinct solvent conditions. Insulin has four tyrosine residues. We also performed tyrosine fluorescence measurements. Insulin fibrils showed a fluorescence spectrum with a maximum at 310 nm (Figure 2C). Although the intensity of Tyr fluorescence of fibrils under strong ultrasonication slightly decreased compared with those under stirring and weak ultrasonication, the shapes of the spectra were almost the same. TEM images showed that insulin fibrils incubated under ultrasonication were fragmented into short fibrils [Figure 2D (ii) and (iii)] compared with preformed fibrils [Figure 2D (i)]. However, the extent of fragmentation was smaller than those of β2m fibrils (Figure 1D). We also examined the seeding abilities of ultrasonicationtreated insulin fibrils (Figure 2E−G). The elongation kinetics, final ThT fluorescence values, and CD spectra of insulin fibrils prepared by seeding with ultrasonication-treated seed fibrils were identical to those of preformed fibrils. TEM images of elongated fibrils were indistinguishable from those of preformed fibrils. These results showed that the seeding abilities of insulin fibrils were unaffected by extensive ultrasonication. In other words, cross-seeding with ThTnegative seeds produced ThT-positive amyloid fibrils. Gdn-HCl Denaturation of Amyloid Fibrils. We investigated the stability of amyloid fibrils against Gdn-HCl, one of the most common denaturants of protein structures.39,50,51 Various concentrations of Gdn-HCl (0−7.0 M) were added to the preformed fibrils of β2m and insulin at 0.1 mg/mL fibrils, 100 mM NaCl, 5 μM ThT, pH 2.0, and 37 °C. The extent of denaturation was estimated by ThT fluorescence, CD, tryptophan fluorescence, and TEM. The ThT fluorescence of β2m fibrils decreased with increases in the concentration of Gdn-HCl (Figure 3A). The CD spectra of β2m fibrils also changed with increases in the concentration of Gdn-HCl (Figure 3B and Table S3). The tryptophan

or cycles of ultrasonication for 4 min with quiescence for 1 min with a high or low amplitude. At the high ultrasonic amplitude, a decrease in ThT fluorescence was observed regardless of the irradiation period. There was no degradation of β2m upon ultrasonic irradiation as described previously.34 At the low ultrasonic amplitude, ThT fluorescence slightly increased and then decreased, implying that ultrasonication with a low amplitude accelerated the dispersion and fragmentation of stacked fibrils and, thus, exposure of buried ThT binding sites, followed by the slight degradation of fibrils. A slight decrease in ThT fluorescence was observed under stirring without ultrasonication, indicating that the fibrils under these conditions maintained a stable conformation. These results suggested that ultrasonication induced a certain degree of degradation in amyloid structures. We performed CD measurements to examine the secondary structures of ultrasonicated fibrils under the respective conditions (Figure 1B and Table S1). Preformed β2m fibrils showed a far-UV spectrum typical for predominantly βstructures. The CD spectra of ultrasonication-treated fibrils under various ultrasonic conditions were essentially the same as those of nonirradiated fibrils even though these fibrils showed low ThT fluorescence, indicating that extensive ultrasonication did not change the secondary structures of β2m fibrils. The results implied the accumulation of the molten globule-like intermediate observed for globular proteins, in which the secondary structures and compactness of the native states were largely retained, whereas the side chains were significantly disordered.35−38 β2m has two tryptophan residues at positions 60 and 95. In both the native structure at neutral pH and amyloid fibrils at pH 2.0, Trp95 is buried while Trp60 is exposed.50,51 We performed tryptophan fluorescence measurements of ultrasonicated fibrils (Figure 1C). The fibrils treated with weak ultrasonication under stirring retained a high fluorescence intensity with a maximum at 338 nm, whereas the fibrils treated with strong ultrasonication showed a decrease in fluorescence intensity with a maximum at 330 nm. This suggested that strong ultrasonication induced the changes in the surface conformation of fibrils. We examined the morphologies of ultrasonication-treated fibrils using TEM (Figure 1D). Preformed β2m fibrils produced by seeding exhibited long and straight morphologies [Figure 1D (i)]. On the other hand, irrespective of the ultrasonic conditions, TEM images showed that predominantly short fibrils coexisted with a small amount of globular aggregates [Figure 1D (ii) and (iii)], suggesting that ultrasonication induced the fragmentation of long fibrils as well as a small amount of amorphous aggregates. We then examined the seeding potentials of ultrasonicated fibrils with decreased ThT fluorescence (Figure 1E). After the addition of 5% (v/v) of the fibrils formed under various ultrasonic periods to β2m monomers, the β2m solutions were incubated under quiescence without being stirred. Although fibrillation kinetics became faster than those with nonultrasonicated fibrils, the final ThT fluorescence intensities were smaller than those of the control experiment without the ultrasonic treatment. Nevertheless, the CD spectra were essentially the same as those of the mature fibrils (Figure 1F), and long fibril morphologies were observed by TEM (Figure 1G). These results suggested that fibrils with decreased ThT fluorescence retained the ability to be seeds for the production of ThT-positive fibrils. The higher growth rate in 3940


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Figure 2. Stability of insulin fibrils under ultrasonication at pH 2.0. (A) Time-dependent changes in ThT fluorescence under various conditions of ultrasonication: cycles of ultrasonication for 9 min with quiescence for 1 min with a high (red) or low (blue) ultrasonic amplitude or cycles of ultrasonication for 4 min with quiescence for 1 min with a high (orange) or low (cyan) amplitude. (B) CD spectra of insulin fibrils under various conditions as described above. The spectrum of monomers at pH 2 is also shown (---). (C) Tyrosine fluorescence spectra of insulin fibrils under high (red) or low (blue) ultrasonication amplitude with a cycle of irradiation for 1 min and quiescence for 1 min, and stirring (black). (D) TEM images of control insulin fibrils (i), fibrils incubated under cycles of ultrasonication for 9 min and quiescence for 1 min with a high amplitude (ii), and those incubated under cycles of ultrasonication for 4 min and quiescence for 1 min with a low amplitude (iii). The scale bar indicates 200 nm. (E− G) Seeding reactions of ultrasonication-treated insulin fibrils. (E) Elongation kinetics with seed fibrils monitored by ThT fluorescence under various conditions described above. (F and G) CD spectra and TEM images, respectively, of fibrils elongated by seeding under stirring [black, (i), or cycles of ultrasonication for 9 min and quiescence for 1 min with a high (red) (ii) and low (blue) (iii) amplitude]. CD spectra except that of monomers (B and F), fluorescence spectra (C), and TEM images (D and G) were obtained at the end of reactions shown in panels A and E.

Figure 3. Stability of β2m fibrils at various concentrations of Gdn-HCl at pH 2.0. (A) Time courses of changes in the ThT fluorescence of β2m fibrils at 485 nm at various concentrations of Gdn-HCl as indicated. (B) CD spectra of β2m fibrils at various concentrations of Gdn-HCl. (C) Tryptophan fluorescence spectra of β2m fibrils at various concentrations of Gdn-HCl. (D) Dependence of ThT fluorescence (○), CD intensities at 220 nm (●), and the ratio of tryptophan fluorescence at 330 and 350 nm (□). (E) TEM images of β2m fibrils at 2.0, 4.0, and 6.0 M Gdn-HCl. Scale bars indicate 500 nm. (F) Seeding reactions with β2m fibrils incubated at various concentrations of Gdn-HCl monitored by ThT fluorescence. (G) CD spectra of elongated fibrils with seeds as described for panel F. The CD spectra with decreased intensity with seeds prepared at 6 and 7 M Gdn-HCl were more likely to be caused by the precipitation of products at final Gdn-HCl concentrations of 0.3 and 0.35 M, respectively. (H) TEM images of seeded fibrils. Different colors in panels A−C, F, and G indicate different concentrations of Gdn-HCl as shown in panel A. CD spectra except that of monomers (B and G), fluorescence spectra (C), and TEM images (E and H) were obtained at the end of reactions shown in panels A and F.

TEM images obtained from samples at various concentrations of Gdn-HCl indicated the persistence of amyloid fibrils at 2.0 and 4.0 M Gdn-HCl, whereas no fibril was detected at 6.0 M Gdn-HCl, suggesting that β2m fibrils were dissolved in the presence of 6.0 M Gdn-HCl (Figure 3E). These results were consistent with our previous findings for the denaturation of β2m fibrils by Gdn-HCl.39,50,51 Seeding reactions using β2m fibrils formed at various concentrations of Gdn-HCl were also examined via ThT fluorescence, in which the concentration of Gdn-HCl was diluted 20-fold from the condition of denaturation (Figure 3F). Although the lag times of seeding reactions were longer than that of β2m fibrils formed at 0 M Gdn-HCl, fibrils incubated at various Gdn-HCl concentrations were effective as seeds.

fluorescence of β2m fibrils changed in a manner that was dependent on the concentration of Gdn-HCl (Figure 3C). The emission maximum shifted from 330 to 350 nm with an increase in the concentration of Gdn-HCl, indicating the exposure of buried tryptophan residues upon the depolymerization of fibrils. 50,51 The decrease observed in ThT fluorescence occurred before changes in CD intensity at 220 nm or the ratio of the emission maximum of tryptophan fluorescence at 330 and 350 nm (Figure 3D). 3941


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Biochemistry Considering the fact that the spontaneous fibrillation of β2m at pH 2 takes ∼20 h under shaking (data not shown), the ability as seeds was retained even after treatment with 7 M Gdn-HCl. TEM images of seeded fibrils showed the existence of fibrils at all Gdn-HCl concentrations (Figure 3H), although the fibrils formed at 6 M Gdn-HCl seemed to be more dispersed and shorter than those at lower concentrations. The CD spectra of seeded fibrils recorded after the seeding experiments (at ∼25 h) showed a typical β-structure, which was similar to that of fibrils formed at 0 M Gdn-HCl, except for those treated with seeds prepared at Gdn-HCl concentrations of >5.0 M (Figure 3G). Those CD spectra with seeds prepared at 6 or 7 M Gdn-HCl showed an unusual shape, suggesting that aggregated fibrils prevented accurate CD measurements. Taken together, β2m fibrils incubated at a high concentration of Gdn-HCl retained their seeding potential even after a decrease in ThT fluorescence. Similar denaturation experiments were performed with insulin fibrils prepared at pH 2.0 (Figure 4). The ThT fluorescence intensity started to decrease at approximately 2.0 M Gdn-HCl (Figure 4A,D). However, the ThT intensity at 1 M Gdn-HCl was reproducibly higher than that at 0 M, implying that the addition of a low concentration of Gdn-HCl loosened the stacked fibrils causing a slight increase in ThT intensity. The CD spectra obtained were typical for the β-structure at lower than 5 M Gdn-HCl, although the spectra showed slight variations depending on the Gdn-HCl concentration (Figure 4B and Table S4). Those at 6 and 7 M Gdn-HCls showed a low signal intensity, suggesting the denaturation of fibrils. However, TEM images (Figure 4E) and seeding experiments (Figure 4F) showed the existence of the remaining fibrils. A large number of precipitations were often found in a cuvette, implying the difficulty of the CD measurements in the presence of 6 or 7 M Gdn-HCl. Taken together, the conformation under these conditions was unclear because of the presence of high concentrations of Gdn-HCl and the presence of large aggregates. The tyrosine fluorescence spectrum of insulin fibrils increased monotonously in a manner that depended on the concentration of Gdn-HCl (Figure 4C,D), suggesting the absence of cooperative conformational changes with an increase in Gdn-HCl concentration. Moreover, TEM images at 2.0, 4.0, and 6.0 M Gdn-HCl (Figure 4E) confirmed that they retained a fibrillar morphology similar to those of preformed fibrils. Furthermore, the insulin fibrils formed at various concentrations of Gdn-HCl retained their seeding abilities (Figure 4F). Although it seemed that partly depolymerized fibrils at high concentrations of Gdn-HCl served as better seeds for fibrillation, their significance was unclear. The CD spectra of seeded fibrils were typical for the β-structure at all Gdn-HCl concentrations (Figure 4G). The fibril morphologies of seeded fibrils were confirmed to be long and straight by TEM (Figure 4H). In contrast, native insulin, which is helical and dimeric in the absence of Gdn-HCl at low pH, showed a spectral change at 5.0 M Gdn-HCl indicative of the denaturation of native helical structures (Figure 4B). Thus, insulin fibrils were more stable than the dimeric native state against denaturation by Gdn-HCl at pH 2.0. SDS Denaturation of Amyloid Fibrils. We also examined the denaturation of amyloid fibrils by SDS. Various concentrations of SDS were added to the preformed fibrils of β2m or insulin at 0.1 mg/mL, containing 100 mM NaCl and 5

Figure 4. Stability of insulin fibrils at various concentrations of GdnHCl at pH 2.0. (A) Time courses of changes in the ThT fluorescence of insulin fibrils at 485 nm at various concentrations of Gdn-HCl. (B) CD spectra of insulin fibrils at various concentrations of Gdn-HCl at 40 min in panel A. Spectra of monomers before fibrillation at 0 (---) and 5 M Gdn-HCl () are also shown. (C) Tyrosine fluorescence spectra of insulin fibrils at various concentrations of Gdn-HCl at the end of reactions shown in panel A. (D) Dependencies of ThT fluorescence (○), CD intensity at 222 nm (●), and emission maximum of tyrosine fluorescence (□) on the Gdn-HCl concentration at the end of reactions shown in panel A. (E) TEM images of insulin fibrils incubated at 2.0 (i), 4.0 (ii), and 6.0 M (iii) Gdn-HCl. Scale bars indicate 200 nm. (F and G) Seeding abilities of insulin fibrils formed at various concentrations of Gdn-HCl. (F) Time courses of changes in ThT fluorescence. (G) CD spectra of elongated fibrils at 24 h. (H) TEM images of seeded fibrils at the end of reactions. Different colors in panels A−C, F, and G indicate different concentrations of Gdn-HCl as shown in panel B.

μM ThT at pH 2.0 and 37 °C. We varied the concentration of SDS between 0 and 30 mM. In the case of β2m, the intensities of ThT fluorescence increased and decreased with an increase in SDS concentration (Figure 5A). The dramatic increase in ThT induced by low concentrations of SDS was reproducible. This suggested that low concentrations of SDS changed the tertiary structure of fibrils so that more ThT binding sites were exposed. The higher concentrations of SDS caused the decrease in ThT intensity by the complete degradation of secondary structures of fibrils. The CD spectra also showed a conformational change that depended on SDS concentration (Figure 5B and Table S5). The intensities of ThT fluorescence and the absolute values of CD at 220 nm showed maxima at approximately 2.0 mM SDS (Figure 5D). The CD spectra at 3942


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Figure 5. Stability of β2m fibrils at various concentrations of SDS at pH 2.0. (A−C) Time courses of changes in ThT fluorescence at 485 nm, CD spectra, and tryptophan fluorescence spectra, respectively, of β2m fibrils at various SDS concentrations. Different colors indicate different concentrations of SDS shown in panel B. (D) Dependence of ThT fluorescence (○), CD intensity at 220 nm (●), and the ratio of the emission wavelength at 330 nm to that at 350 nm of tryptophan fluorescence (□) of β2m fibrils on SDS concentration. (E) TEM images of β2m fibrils at 0.5, 5.5, and 20 mM SDS. The image at 20 mM in right panel indicates the existence of a small amount of fibrils. Scale bars represent 500 nm. CD spectra (B), fluorescence spectra (C), and TEM images (E) were obtained at the end of reactions as shown in panel A.

Figure 6. Stability of insulin fibrils at various concentrations of SDS at pH 2.0. (A−C) Time courses of changes in ThT fluorescence at 485 nm, CD spectra, and tyrosine fluorescence spectra, respectively, of insulin fibrils at various concentrations of SDS. Different colors indicate different concentrations of SDS shown in panel B. (D) Dependence of ThT fluorescence (○), CD intensity at 220 nm (●), and emission maximum of tyrosine fluorescence (□) on SDS concentration. (E) TEM images of insulin fibrils at 0.5, 5.5, and 20 mM SDS. Scale bars represent 500 nm. CD spectra (B), fluorescence spectra (C), and TEM images (E) were obtained at the end of reactions as shown in panel A.

missing the 1621 cm−1 parallel β-component, which is the main component of mature fibrils, and similar to that of strongly sonicated fibrils. The 1618 cm−1 component was found in amorphous aggregates or in immature fibrils (∼1616−1618 cm−1).34,48 We also measured FTIR spectra of insulin fibrils under various conditions (Figure 8 and Table 7). All fibrils showed similar secondary structure contents. The spectra of the mature seeded fibrils and fibrils in the presence of SDS were almost overlapping, whereas the spectrum of sonicated fibrils showed a peak shift of the main component from 1629 to 1631 cm−1, which was assigned as a parallel β-component. These observations indicated that insulin fibrils were more resistant to SDS denaturation than those of β2m, but that both fibrils were degraded to a certain degree by ultrasonication. Although near-UV CD spectra have often been used to address the molten globule states of globular proteins, we could not obtain reliable spectra for insulin fibrils because of strong light scattering and a strong propensity to precipitate (data not shown). Although ANS fluorescence has been known to be a useful probe to specifically monitor the molten globule states,35,37,38 we assumed that ANS might not be a good probe for monitoring the denaturation of amyloid fibrils because both amyloid fibrils and amorphous aggregates show strong ANS fluorescence.34 Cytotoxicity of Ultrasonicated Fibrils. We performed cytotoxicity experiments to investigate the effects of ultrasonication on preformed fibrils. Many studies have reported

SDS concentrations of 5.0 mM showed the appearance of an α-helical component. Tryptophan fluorescence also showed a change depending on SDS concentration (Figure 5C); however, the ratio of the emission maximum at 330 nm to that at 350 nm seemed to be saturated at ∼2 mM SDS (Figure 5D). TEM images of β2m fibrils at 0.5 or 5.5 mM SDS showed fibrillar morphologies similar to those of preformed fibrils, whereas few fibrils were observed at 20 mM SDS (Figure 5E). In the case of insulin, a significant increase and a subsequent decrease in ThT fluorescence were observed with an increase in SDS concentration; however, practically no change was observed in the CD spectrum or tyrosine fluorescence spectrum at SDS concentrations between 0 and 30 mM (Figure 6A−D and Table S6). TEM images confirmed that they retained typical fibrillar morphologies (Figure 6E). These results indicated that insulin fibrils retained their typical fibrillar structures at various concentrations of SDS and that they were more stable than β2m fibrils. Structural Features of Amyloid Fibrils Detected by FTIR. We previously measured FTIR spectra of mature fibrils and amorphous aggregates of β2m.34 We further measured FTIR of amyloid fibrils dissolved in 20 mM SDS (Figure 7 and Table S7). Interestingly, the spectrum in the presence of 20 mM SDS was different from that of mature fibrils in terms of 3943


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that oligomeric intermediates showed more cytotoxicity than mature fibrils.52,53 Radford and co-workers reported that pHinduced fragmented fibrils of β2m showed distinct cytotoxicity and localization in cells.43,44,54,55 We also reported that strongly sonicated fibrils of α-synuclein showed cytotoxicity greater than that of ThT-positive fibrils.33 Therefore, we expected similar results for ultrasonicated fibrils of β2m and insulin. Cell viability (WST-8) and cell membrane defects (LDH) in the presence of monomers, preformed fibrils, or ultrasonicated fibrils are shown in Figure 9. β2m and insulin monomers showed no or little

Figure 7. FTIR spectra of various conformational states of β2m: (A) native monomers, (B) acid-denatured monomers, (C) fibrils formed by stirring, (D) ultrasonicated fibrils, and (E) SDS-denatured fibrils. The dotted, solid, and broken lines represent the experimental, fitted, and deconvoluted spectra, respectively, where the experimental and fitted spectra overlapped. (F) Comparison of experimental spectra shown in panels A−E. All spectra except for those of SDS were taken from ref 34.

Figure 9. Cytotoxicities of various conformational states of β2m and insulin. The (A and B) WST-1 cell viability assays and (C and D) LDH release assays were performed at various concentrations of (A and C) β2m and (B and D) insulin.

cytotoxicity. As expected, ultrasonicated fibrils of β2m were more toxic than preformed fibrils. On the other hand, insulin fibrils showed the same level of toxicity except in the LDH assay at 2.5 mg/mL. These results were consistent with the changes in ThT and FTIR spectra, suggesting that packing defects and surface exposure of side chains as expected for the molten globule-like conformation are associated with the increased cytotoxicity.

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DISCUSSION Accumulation of Denaturation Intermediates of Amyloid Fibrils. Although extensive ultrasonication decreased the ThT fluorescence of the preformed fibrils of β2m and insulin, both fibrils with decreased ThT fluorescence retained the β-sheet structures typical for amyloid fibrils as well as their seeding potentials to reproduce similar fibrils. However, the precise structures of these fibrils detected by FTIR were different from those of preformed fibrils, suggesting a slight distortion of secondary structures. ThT binding has been shown to occur against the surface-exposed grooves lined with aromatic amino acids or formed by hydrophobic side-chain ladders.56,57 Our previous study indicated a large variation in ThT fluorescence among fibrils of various mutants of β2m.45 Recently, a mutant of amyloid β-peptide has been shown to rapidly form fibrils with low ThT fluorescence.58 Amyloid fibrils without ThT fluorescence were also reported for fish IAPP.59 Thus, although the marked ThT fluorescence is a common feature of various amyloid fibrils, it does not seem to be a necessary condition for amyloid fibrils.56,57

Figure 8. FTIR spectra of various conformational states of insulin: (A) native dimers, (B) fibrils formed by seeding, (C) ultrasonicated fibrils, and (D) SDS-treated fibrils. The dotted, solid, and broken lines represent the experimental, fitted, and deconvoluted spectra, respectively, where the experimental and fitted spectra overlapped. (E) Comparison of experimental data shown in panels A−D.

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Main-Chain-Dominated Amyloid Fibrils versus SideChain-Dominated Globular Proteins. ThT fluorescence decreased slightly prior to changes in other probes, which was common to the three types of methods used to induce the denaturation of preformed fibrils (i.e, ultrasonication, GdnHCl, and SDS), whereas the difference between the transitions monitored by tryptophan fluorescence and far-UV CD was negligible. This result suggested the accumulation of the intermediates of fibril denaturation in which the fine tertiary structures responsible for ThT fluorescence were broken while amyloid β-structures were retained. These denaturation intermediates had seeding potentials, thereby producing mature fibrils with ThT binding. Amyloid fibrils are considered as main-chain-dominated structures,7−10,13 while the native states of globular proteins are side-chain-dominated structures in which side-chain packing is critically important as well as the secondary structures.5,6 On the other hand, molten globule states with nativelike secondary structures but largely disordered side-chain structures have been reported to accumulate under some extreme conditions such as an acidic pH in the presence of salt or during the process of kinetic folding.35−38 We consider that ThT-negative aggregates that accumulated under strong ultrasonication were molten globule-like even though perturbation of the side chains of amyloid fibrils was not very strong. Although the generality and significance of molten globule states in protein folding currently remain unclear, such intermediates may accumulate when the cooperativity of the main-chain and side-chain contributions is broken (Figure 10). The cooperative organization of the secondary and tertiary structures of globular proteins commonly prevents the accumulation of such intermediate structures, except under extreme solvent conditions or during kinetic refolding. In contrast, provided that main-chain interactions are more dominant for the formation of amyloid fibrils, the denaturation intermediates of amyloid fibrils with disordered fine side-chain structures but rigid β-structures are more likely to accumulate under various conditions than those of globular proteins. According to a steric zipper model of amyloid fibrils, the side chains are interdigitated to form tightly packed amyloid fibrils.1,4,11,12,57 Collapses of these packing of side chains are likely to be easily induced by physical or chemical denaturation compared to those of strong hydrogen bonds between strands, resulting in a decrease in only ThT fluorescence. However, it is still possible that the ability of amyloid fibrils to form intermediate of denaturation more often than globular proteins is just a stability issue. For example, molten globule states are often observed for large proteins, but generally not for small single-domain proteins of modest stability. Thus, it is possible that partially folded intermediates are more common to amyloid fibrils because amyloid fibrils are more stable than small globular proteins. To clarify the origin of the observed molten globule-like intermediate of amyloid denaturation, it will be important to examine the intermediate conformational states of amyloid fibrils under various conditions with various proteins. It has been shown using β2m that fibril fragmentation enhances amyloid cytotoxicity.43 More recently, Tipping et al.44 studied the pH-dependent fibril disassembly and the effects on cytotoxicity. Fibril disassembly at pH 6.4 resulted in the formation of nonnative spherical oligomers that disrupt synthetic membranes. By contrast, fibril dissociation at pH 7.4 results in the formation of nontoxic, native monomers. The

Considering the mechanism of binding of ThT to amyloid fibrils, our results suggested the presence of denaturation intermediates of fibrils with their side chains perturbed, which may be common to various amyloid fibrils (Figure 10).

Figure 10. Schematic models for denaturation of side-chaindominated native states of globular proteins and main-chaindominated amyloid fibrils with a “molten globule-like” intermediate. Although the side-chain-perturbed intermediates of denaturation can accumulate in both globular proteins and amyloid fibrils under certain conditions in which the cooperativity of structural formation is broken, these intermediates are more likely to occur for amyloid fibrils because of the dominant roles of main-chain interactions. The effects of ultrasonication or chemical denaturants on the free energy landscape are shown in the bottom panel.

However, it was possible that the decreased level of ThT binding is a phenomenon specific to extensively ultrasonicated fibrils. To address the generality of denaturation intermediates, we examined the effects of Gdn-HCl and SDS on the two types of fibrils. The results obtained further suggested that the accumulation of denaturation intermediates is common to various amyloid fibrils and methodologies of denaturation. In the SDS-induced denaturation of β2m fibrils monitored by tryptophan fluorescence, the changes observed in tryptophan fluorescence agreed with those in far-UV CD. However, this does not contradict the side-chain-perturbed denaturation intermediates of amyloid fibrils because the extent of the exposure of tryptophan residues may not change upon such conformational changes. Although further studies are required to verify the structural changes responsible for the decrease in ThT fluorescence, it is intriguing that the results are consistent with the main-chain-dominated architecture of amyloid fibrils (see below). Furthermore, when fibrils were treated with extensive ultrasonication, we noted that insulin fibrils were more stable than β2m fibrils. The stability of insulin fibrils being higher than that of β2m fibrils was confirmed by TEM images. The higher stability of insulin fibrils was also valid for denaturation by GdnHCl or SDS. Taken together, insulin fibrils were more resistant than β2m fibrils to various forces of denaturation, and irrespective of the methodology, both fibrils exhibited denaturation intermediates with decreases in ThT fluorescence while retaining rigid β-structures. 3945


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results showed that a slight pH difference alters the depolymerization of preformed fibrils and thus the formation of fibril-derived toxic oligomers. We previously showed using αsynuclein that extensive ultrasonic irradiation transformed preformed fibrils into amorphous aggregates with higher cytotoxicity.60 Here, we showed that ultrasonicated fibrils of β2m or insulin were more toxic than preformed fibrils, suggesting that packing defects and surface exposure of side chains are associated with cytotoxicity. It will be important to further characterize the cytotoxicity of the molten globule-like denaturation intermediates of fibrils.

This work was supported by JSPS KAKENHI Grants 15H04362 and 15K14458. J.K. is supported by the Hungarian National Research, Development and Innovation Office (KTIA_NAP_13-2-2014-0017). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Kyoko Kigawa (Osaka University) for expression and purification of β2m. This work was performed under the Cooperative Research Program for Institute for Protein, Osaka University, ICR-15-02.

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CONCLUSIONS Starting with a decrease in ThT fluorescence upon the extensive ultrasonic treatment of β2m amyloid fibrils, we detected a denaturation intermediate in which ThT fluorescence decreased while the other properties of amyloid fibrils, including seeding potentials, were retained. Such intermediate states were also common to the ultrasonication-dependent denaturation of insulin fibrils; however, insulin fibrils exhibited a stability higher than that of β2m fibrils. The accumulation of such denaturation intermediates was also common to the denaturation induced by Gdn-HCl or SDS, suggesting its generality in various amyloid fibrils, although the significance of this suggestion should be further addressed using various amyloidogenic proteins under various conditions. The mainchain-dominated architecture of amyloid fibrils in contrast to the side-chain-dominated native states of globular proteins may underlie the accumulation of such denaturation intermediates. The high cooperativity of nucleation and growth of amyloid fibrillation may prevent the detection of such intermediates during the formation of amyloid fibrils. Alternatively, these intermediates formed through a new pathway out of the fibrillation from monomers. In conclusion, we first characterized the main-chain-retained denaturation intermediates of amyloid fibrils. The clarification of the structures and properties of such intermediates and their biological significance will be important for obtaining a deeper understanding of the formation and stability of amyloid fibrils and, moreover, for creating therapeutic strategies against various diseases associated with the deposition of amyloid fibrils.

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ABBREVIATIONS AFM, atomic force microscopy; β2m, β2-microglobulin; CD, circular dichroism; Gdn-HCl, guanidine hydrochloride; SDS, sodium dodecyl sulfate; TEM, transmission electron microscopy; ThT, thioflavin T.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00231. Tables containing the secondary structure assignments based on CD (Tables S1−S6) and FTIR (Table S7) spectra (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan. Telephone: +81-6-68798614. Fax: +81-6-6879-8616. E-mail: [email protected]. Author Contributions

S.N., M.S., and Y.G. designed the research. S.N., M.S., M.A., and J.K. performed the experiments. S.N., M.S., J.K., and Y.G. wrote the paper. S.N. and M.S. contributed equally to this work. 3946


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Thioflavin T-Silent Denaturation Intermediates Support the Main-Chain

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