Supersaturation-Limited and Unlimited Phase Spaces Compete to

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Supersaturation-limited and unlimited phase spaces compete to produce maximal amyloid fibrillation near the critical micelle concentration of sodium dodecyl sulfate Masatomo So, Akira Ishii, Yasuko Hata, Hisashi Yagi, Hironobu Naiki, and Yuji Goto Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b02186 • Publication Date (Web): 20 Aug 2015 Downloaded from http://pubs.acs.org on August 23, 2015

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Supersaturation-limited and unlimited phase spaces compete to produce maximal amyloid fibrillation near the critical micelle concentration of sodium dodecyl sulfate Masatomo So1, Akira Ishii1, Yasuko Hata1, Hisashi Yagi1†, Hironobu Naiki2, and Yuji Goto1* 1

Institute for Protein Research, Osaka University, Osaka 565-0871, Japan

2

Faculty of Medical Science, University of Fukui, Fukui 910-1193, Japan

KEYWORDS: Amyloid fibrillation; β2-microglobulin; critical micelle concentration; protein aggregation; solubility; ultrasonication.

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ABSTRACT: Although various natural and synthetic compounds have been shown to accelerate or inhibit the formation of amyloid fibrils, the mechanisms by which they achieve these adverse effects in a concentration-dependent manner currently remain unclear. SDS, one of the compounds that has adverse effects on fibrillation, is the most intensively studied. We here examined the effects of a series of detergents including SDS on the amyloid fibrillation of β2-microglobulin at pH 7.0, a protein responsible for dialysis-related amyloidosis. In all the detergents examined (i.e., SDS, sodium decyl sulfate, sodium octyl sulfate, and sodium deoxycholate), amyloid fibrillation was accelerated and inhibited at concentrations near the critical micelle concentration (CMC) and higher than CMC, respectively. The most stable conformation changed from monomers with a β-structure to amyloid fibrils with a β-structure and then to α-helical complexes with micelles with an increase in detergent concentrations. These results suggest that competition between supersaturation-limited fibrillation and unlimited mixed micelle formation between proteins and micelles underlies the detergent concentration-dependent complexity of amyloid fibrillation.


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INTRODUCION Amyloid fibrils are highly ordered assemblies of misfolded proteins and that have been implicated in more than 30 degenerative diseases including Alzheimer’s and Parkinson’s diseases.1-4 Considering the presence of non-pathogenic amyloids called “functional amyloids”, which participate in normal biological functions such as the storage of peptide hormones in the secretory granules of the endocrine system and bacteria biofilms, amyloid fibrillation is one of the fundamental properties of proteins.5-9 Therefore, clarifying the mechanisms of fibrillation may contribute not only to the treatment of amyloidoses, but also to obtaining a deeper understanding of the physicochemical properties of proteins. Amyloid fibrils form in supersaturated solutions of precursor proteins by a nucleation and growth mechanism that is characterized by a lag phase or by seed-dependent growth without a lag phase.10-14 In supersaturated solutions, precursor proteins remain soluble although their concentrations are higher than solubility (or critical concentration). Thus, supersaturation is one of the most important factors for understanding the development of degenerative diseases.15 Actin fiber formation,16-18 microtubule polymerization,19 and protein crystallization20-21 also occur under supersaturation, suggesting that the various biological and physicochemical processes that produce highly ordered structures are controlled by supersaturation. Although the many studies have attempted to predict amyloidogenicity from amino acid sequences,22-25 difficulties have 3 ACS Paragon Plus Environment

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been associated with fitting all the data under physiological conditions because fibrillation is strongly concentration-dependent and modified by surrounding factors such as solvent conditions.26-27 Various small compounds exhibit the ability to inhibit or accelerate amyloid fibrillation.28-30 The abilities of detergents to accelerate or inhibit the fibrillation of proteins or peptides have been studied extensively.31-34 The effects of detergents on the amorphous aggregation of proteins are often adverse and depend on the concentrations of detergents.35-38 These findings suggest that chemical compounds interacting with proteins or peptides have the potential to accelerate and inhibit fibrillation. We herein examined the effects of various detergents on the fibrillation of β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis.39-40 We selected physiological conditions at pH 7.0, at which patients deposit fibrils, and additives such as sodium dodecyl sulfate (SDS) or 2,2,2-trifluoroethanol (TFE) were useful for spontaneous fibrillation in test tubes.36,

41

The results obtained showed a clear dependence of fibrillation on the detergent

concentration; concentrations near the critical micelle concentration (CMC) were the most effective at accelerating fibrillation. We previously proposed that a phase diagram of the conformational states of β2m dependent on the solvent and protein concentrations was useful for understanding the supersaturation-limited amyloid fibrillation of proteins.13, 26-27 Moreover, we 4 ACS Paragon Plus Environment

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recently proposed that a competitive mechanism of amyloid fibrillation and amorphous aggregation reproduced the observed aggregation kinetics of β2m.42 We here showed that a competitive mechanism between the detergent-assisted fibrillation and detergent-assisted formation of micelle-protein complexes (i.e. mixed micelles) explains the complexity of detergent-dependent amyloid fibrillation.

EXPERIMENTAL SECTION Materials. Recombinant human β2m protein with an additional methionine residue at the N terminus was expressed in Escherichia coli and purified as previously reported.43 The concentration of β2m was determined by measuring absorbance using a molar extinction coefficient of 19,300 M−1 cm−1 at 280 nm.43 Sodium decyl sulfate (C10) and sodium octyl sulfate (C8) were purchased from Sigma-Aldrich. Thioflavin T (ThT) was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). SDS (C12) and all other reagents were obtained from Nacalai Tesque (Kyoto, Japan). Amyloid Fibrillation. Lyophilized β2m was dissolved in 3.2 mM HCl (pH 2.5) or 50 mM phosphate buffer (pH 7.0) at 0.3 mg/mL containing 100 mM NaCl and 5 µM ThT, and 0.2-mL samples were distributed to the wells of a 96-well microplate (675076, Greiner Bio-one Co., Ltd., Frickenhausen, Germany). The microplate was set on a water bath-type ultrasonicator 5 ACS Paragon Plus Environment

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(ELESTEIN SP070-PG-M, Elekon Science Co., Ltd., Chiba, Japan) on which the plate received maximal ultrasonication. Ultrasonic pulses were applied to the microplate from three directions for cycles of 1 min, followed by a quiescent period of 5 min at 37 ºC. The frequency and power of the ultrasonic pulses were set to approximately 19 kHz and 700 watts, respectively. Amyloid fibrils were detected using ThT fluorescence with a microplate reader (MTP-810, Corona Electric Co., Ltd., Tokyo, Japan) with excitation and emission wavelengths of 445 nm and 485 nm, respectively. A multiple data collection mode with 9 data points was employed. In seeding experiments, seed fibrils (5% (v/v)) formed under ultrasonic conditions were added to monomeric β2m solutions at a concentration of 0.3 mg/mL containing 5 µM ThT. Fibrillation was measured using a microplate reader at 37 ºC without agitation. In order to decrease variations in the ultrasonic amplitude irradiated to a microplate, the water in the bath of the ultrasonicator was degassed using a degassing pump (WRS-40006A, Kaijo Co., Ltd., Tokyo, Japan). The levels of degasification were estimated by the concentration of dissolved oxygen checked by a dissolved oxygen meter (OM-51, Horiba Ltd., Kyoto, Japan). Since variations in the lag time of fibrillation on a microplate were significantly decreased under degassing, unless otherwise stated, we performed experiments under the condition of maximal degassing (Fig. S1). Measurements of Critical Micelle Concentrations. The CMC values of detergents were 6 ACS Paragon Plus Environment

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determined by 1-anilino-8-naphthalene sulfonate (ANS) fluorescence.44-45 Fluorescence titrations were performed with 50 µM of ANS containing 100 mM NaCl and 50 mM sodium phosphate buffer (pH 7.0) at 37 ºC. Fluorescence measurements were performed with a Hitachi F-4500 fluorescence spectrophotometer with excitation and emission wavelengths of 350 nm and 485 nm, respectively. The change in ANS fluorescence was fit with two straight lines and the point of intersection of these lines was used to define the CMC. Transmission Electron Microscopy (TEM). A sample solution (5 µL) was 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/w) uranyl acetate or 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 (Hitachi H-7650, Tokyo, Japan) images were obtained at 20 °C with a voltage of 80 kV and magnification of 15,000. Circular Dichroism (CD) Measurements. β2m solutions were diluted to 0.1 mg/mL and far-UV CD spectra (198-250 nm) were obtained with a Jasco J-720 spectropolarimeter (Jasco Co., Ltd., Tokyo, Japan) using a quartz cell with a 1-mm path length at 37 ºC. CD data were expressed by the mean residue ellipticity.

RESULTS AND DISCUSSION 7 ACS Paragon Plus Environment

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Effects of Degassing. Fibrillation experiments of β2m were performed at 100 mM NaCl, pH 2.5, and 37 °C with a water bath-type ultrasonicator and microplate reader.46 When a microplate was used, the lag time varied depending on the location of the wells because of fluctuations in the ultrasonic power irradiated to the microplate (Fig. S1a). We previously proposed that rotation or movement of the microplate on the water bath of the ultrasonicator was effective for averaging the ultrasonic power.46 We here employed a different approach to improve the synchronization of amyloid fibrillation. Ultrasonic irradiation to an aqueous solution has been shown to generate small cavitation bubbles.47 The growth of bubbles occurs by coalescence of these bubbles and rectified diffusion.48 The bubbles that reach the resonance size collapse, inducing sonochemical reactions. However, when the size of bubbles exceeds the resonance size, the bubbles remain at the pressure antinode of a standing ultrasonic wave, thereby preventing transmission of the ultrasonic wave. In order to decrease variations in the ultrasonic amplitude on a microplate, the water in the bath of the ultrasonicator was degassed by a degassing pump and the effects were monitored at various levels of degasification. As expected, well-dependent variations in the lag time of fibrillation decreased significantly under degassed conditions (Fig. S1b-d). Thus, we herein performed the experiments under the conditions of maximal degassing. Effects of Various Detergents. We investigated the effects of various concentrations of 8 ACS Paragon Plus Environment

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SDS on the spontaneous fibrillation of β2m under ultrasonic conditions at 100 mM NaCl, pH 7.0, and 37 °C. To achieve uniform ultrasonic irradiation under degassing, 40-60 wells at the center of a microplate were used. We used at least 6 wells for each of the conditions in order to estimate variations in fibrillation kinetics. We employed standard ultrasonic cycles of 1 min with a quiescence of 4 min. Yamamoto et al.36 performed similar experiments without ultrasonication, but with seed fibrils. They reported that fibrillation was most effective near the CMC value of SDS. The CMC of SDS varied depending on the environment such as the temperature and concentration of salts. Although the CMC of SDS in the water at 25 °C was previously reported to be 8 mM,49 the value determined by Yamamoto et al.36 using ANS fluorescence was 0.5 mM under the conditions of 100 mM NaCl, 50 mM sodium phosphate, pH 7.0, and 37 °C. We obtained the same value under the same solvent conditions, except for the presence of ultrasonic irradiation (Fig. 1a). Fibrillation was not detected at SDS concentrations lower than 0.3 mM. At 0.4-1.8 mM SDS, significant increases in ThT fluorescence were observed after a lag time (Fig. 1b). The maximum intensity of ThT fluorescence (Fig. 1c) and the lag time for increases in ThT fluorescence (Fig. 1d) plotted against the SDS concentration showed that the rate and extent of fibrillation varied depending on the concentration of SDS, with an optimum at approximately 0.5 mM, which is close to the CMC of SDS. These results were consistent with the findings of Yamamoto et al.36 The 9 ACS Paragon Plus Environment

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formation of fibrils was confirmed on electron micrographs, which revealed the existence of ultrasonicated short fibrils (Fig. 1e). These fibrils had the ability to promote the subsequent elongation of fibrils as seeds (Fig. 1f).

Figure 1. Effects of SDS on amyloid fibrillation of β2m at pH 7.0 and 37 ºC. (a) Determination of CMC for SDS using ANS fluorescence. The CMC value of SDS was determined to be 0.57 mM. The chemical structure of SDS is also shown. (b) Fibrillation kinetics at various SDS concentrations as defined by a color scale bar. (c, d) Dependencies of the maximum ThT intensity 10 ACS Paragon Plus Environment

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(c) and lag time (d) on SDS concentration. Lines are fittings with a degree-4 polynomial function. (e) TEM images of fibrils formed at 0, 0.5, and 2.0 mM SDS. A scale bar represents 200 nm. (f) Seed-dependent fibrillation at 0.5 mM SDS. Fibrils formed under ultrasonication at 0.5 mM SDS were used as seeds. Different traces represent reactions in different wells.

We examined the dependence of fibrillation on the detergent concentration with sodium decyl sulfate (C10) and sodium octyl sulfate (C8), which are analogues to SDS, but with shorter alkyl chain lengths. The CMC values of the C10 and C8 compounds were determined to be 9.5 and 76.1 mM, respectively, by the ANS fluorescence method (Fig. 2a, b). The CMC values of the C10 and C8 compounds in aqueous solution at 25 °C were previously reported to be 33 and 110 mM, respectively,49 which were slightly larger than our values. We performed fibrillation experiments using various concentrations of the C10 and C8 detergents (Fig. 2c, d). The lag times and ThT fluorescence intensities obtained were the minimum and maximum, respectively, at detergent concentrations near the CMC values (Fig. 2e-h). TEM images showed the formation of similar fibrils to those induced by SDS (Fig. 3a, b). These ultrasonicated fibrils had the ability to be seeds for subsequent seed-dependent fibrillation, in which we added the optimal concentration of detergents (Fig. 3c, d).


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Figure 2. Effects of sodium decyl sulfate (C10) and sodium octyl sulfate (C8) on amyloid fibrillation of β2m at pH 7.0 and 37 ºC. (a, b) Determination of CMC values using ANS fluorescence. The CMC values of C10 (a) and C8 (b) were determined to be 9.5 mM and 76.1 12 ACS Paragon Plus Environment

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mM, respectively. (c, d) Fibrillation kinetics at various C10 (c) or C8 (d) concentrations as defined by a color scale bar. (e-h) Dependencies of the maximum ThT intensity (e, f) and lag time (g, h) on the concentrations of C10 (e, g) and C8 (f, h), respectively. Lines are fittings with a degree-4 polynomial function.

Figure 3. Morphologies and seed potentials of β2m fibrils formed in the presence of sodium decyl sulfate (C10) or sodium octyl sulfate (C8). (a, b) TEM images of fibrils formed at 6 and 10 mM C10 (a) or 60 and 100 mM C8 (b). Scale bars represent 200 nm. (c, d) Seed-dependent fibrillation at 6 mM C10 (c) or 60 mM C8 (d). Seeds were obtained from the fibrils formed under ultrasonication at the same detergent concentrations. Different traces represent the kinetics in 13 ACS Paragon Plus Environment

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different wells.

We also examined the effects of sodium deoxycholate, another type of detergent constituting bile acids. The CMC value of sodium deoxycholate estimated from the ANS fluorescence method under quiescence was 5.4 mM (Fig. 4a, inset). Sodium deoxycholate exhibited the ability to accelerate the fibrillation of β2m (Fig. 4b). However, the maximal concentration of sodium deoxycholate needed to accelerate fibrillation was approximately 25 mM and the weak or no ability to accelerate fibrillation was detected at concentrations lower and higher than 25 mM (Fig. 4c, d). The formation of amyloid fibrils was confirmed by TEM images (Fig. 4e). Since the apparent CMC value (5.4 mM) determined under quiescence was markedly lower than the optimal concentration for fibrillation (25 mM), we performed CMC measurements under ultrasonication. The CMC value of sodium deoxycholate under ultrasonication was 31.6 mM, which was consistent with the optimal concentration for fibrillation determined under ultrasonication (Fig. 4a). The CMC values of other detergents (SDS, C10, and C8) did not depend on the presence or absence of ultrasonication (Fig. S2). Although the exact mechanism underlying ultrasonication-dependent changes in the apparent CMC value remains unknown, strong agitation appeared to be important for dissolving sodium deoxycholate completely.


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Figure 4. Effects of sodium deoxycholate on amyloid fibrillation of β2m at pH 7.0 and 37 ºC. (a) The CMC of deoxycholate obtained from ANS fluorescence measurements. The CMC value under quiescent conditions was 5.4 mM (inset), whereas that under ultrasonic conditions was 31.6 mM. The chemical structure of deoxycholate is also shown. (b) Fibrillation kinetics at various deoxycholate concentrations as defined by a color scale bar. (c, d) Dependences of the maximum ThT intensity (c) and lag time (d) on the deoxycholate concentration. Lines are fittings with a degree-4 polynomial function. (e) TEM image of fibrils formed at 30 mM deoxycholate. A 15 ACS Paragon Plus Environment

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scale bar represents 200 nm. (f) Seed-dependent fibrillation under quiescent conditions at 30 mM (solid lines) or 5 mM (dashed lines) deoxycholate. Fibrils formed under ultrasonication at 30 mM deoxycholate were used as seeds. Different traces represent the kinetics in different wells.

We performed seeding experiments without ultrasonication to investigate whether fibrils formed under ultrasonication in the presence of sodium deoxycholate at pH 7.0 had the ability to accelerate fibrillation. When seed fibrils (5% (v/v)) formed under ultrasonic conditions were added to monomeric β2m solutions at a concentration of 0.3 mg/mL containing 5 or 30 mM of sodium deoxycholate, fibrillation without a lag time was observed with a faster rate at 30 mM sodium deoxycholate (Fig. 4f). These results suggested that the ultrasonication-induced fibrils formed in the presence of deoxycholate had the ability to be seeds for fibrillation and that destabilization of the native conformation below the CMC value increased the elongation rate. We then measured CD spectra before and after fibrillation in order to obtain structural information on detergent-induced fibrils. We could not measure the CD spectra of sodium deoxycholate due to the high viscosity of the solution. The CD spectra of β2m monomers in the presence of the three types of detergents before fibrillation are shown in Figure 5a, c, and e. β2m has a native structure with dominantly β-sheets at a neutral pH in the absence of detergents, whereas, for the three types of detergents, low concentrations of detergents below CMC induced 16 ACS Paragon Plus Environment

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changes in CD spectra, suggesting a slight increase in the α-helical content. At the end point of fibrillation, at which ThT fluorescence showed a maximum, the CD spectra typical for β-sheet structures were observed for the three types of detergents, thereby indicating that fibrils formed by different detergents had similar β-structures independent of the detergent types (Fig. 5b, d, and f).

Figure 5. Far-UV CD spectra of β2m before (a, c, e) and after (b, d, f) detergent-induced amyloid fibrillation at pH 7.0 and 37 ºC. (a, b) SDS-induced fibrillation. (c, d) Sodium decyl sulfate (C10)-induced fibrillation. (e, f) Sodium octyl sulfate (C8)-induced fibrillation. 17 ACS Paragon Plus Environment

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CMC and Amyloid Fibrillation. Previous studies reported using various proteins and peptides, such as Aβ, α-synuclein, and β2m31, 33-34, 36, 45, 50 that detergents, particularly SDS, accelerated or inhibited amyloid fibrillation in a manner that depended on their concentrations with a maximum near the CMC value. In the case of the 22-residue peptide, K3, corresponding to the Ser20 to Lys41 of β2m, the addition of SDS induced fibrils at pH 2 with a maximum at slightly below the CMC value.45 A similar maximum for fibrillation was also observed for K3 with highly fluorinated alcohols (i.e., TFE and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) in 10 mM HCl (pH ~2)): TFE- and HFIP-induced fibrillation exhibited a maximum at ~20 and ~10% (v/v), respectively. TFE or HFIP has been shown to form micelle-like dynamic clusters under these conditions.51 We recently showed using hen egg white lysozyme26 and human insulin27 that these proteins maximally formed amyloid fibrils at the TFE or HFIP concentrations of maximal alcohol clustering. These findings suggested that amyloid fibrillation by detergents or fluorinated alcohols was caused by a common mechanism, in which hydrophobic interactions between proteins and clusters of co-solvents (i.e., detergents or alcohols) play important roles.45 Detergents and alcohols are considered to mimic biological membranes or some amphiphilic compounds32, 52-55 therefore the underlying mechanism for maximal fibrillation near the CMC values of detergents needs to be clarified. 18 ACS Paragon Plus Environment

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In the presence of SDS near the CMC, hydrophobic interactions between proteins may be strengthened by bound hydrophobic SDS molecules, which contribute to intermolecular associations between proteins. Polar interactions between proteins may be simultaneously strengthened by bound SDS molecules, thereby contributing to the intermolecular hydrogen bonds substantiating the cross β-structures. The concerted actions of intermolecular hydrophobic and polar interactions assisted by bound SDS lead to amyloid fibrillation near the CMC. However, this kind of interaction may be very unstable and has often led to the formation of amorphous aggregates.35 The high propensity of K3 to form non-fibrillar aggregates has also been reported previously.45 On the other hand, proteins are solubilized into detergent micelles above the CMC because micelle-protein complexes (i.e. mixed micelles), in which proteins often assume an α-helical conformation, are energetically more stable than amyloid fibrils.45 These mixed micelles inhibit amyloid fibrillation and moreover induce the depolymerization of preformed fibrils. These consecutive accelerating and inhibitory effects may produce an optimum in detergent-assisted fibrillation near the CMC value. However, the energetics and kinetics underlying the presence of the optimum were largely unknown. We herein demonstrated that the relationship between CMC values and optimal concentrations for fibrillation was valid for the four types of detergents examined (SDS (C12), 19 ACS Paragon Plus Environment

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sodium decyl sulfate (C10), sodium octyl sulfate (C8), and sodium deoxycholate) (Fig. 6a), the CMC values of which varied from submillimolar to hundred millimolar levels. Moreover, based on previous findings, this relationship can be extended to various kinds of proteins and detergents with CMC values ranging from 0.1 – 500 mM (Fig. 6a).35, 38, 56 This correlation establishes that, combined with moderate denaturation effects on proteins, various detergents close to the CMC concentration accelerate fibrillation.

Figure 6. Mechanism underlying detergent-dependent amyloid fibrillation of proteins. (a) The linear relationship between the maximal detergent concentration of fibrillation and CMC. The values for benzalkonium chloride (BAC12, BAC14, and BAC16) with the keratoepithelin peptide 20 ACS Paragon Plus Environment

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were taken from Kato et al.,56 in which the number after BAC represented the length of alkyl chains. The values for lysophosphatidic acid (LPA) with β2m were from Pal-Gabor et al.38 (b) A phase diagram of conformational states that depended on SDS and protein concentrations at pH 2.5. (c) Dependencies of free energy changes in amyloid fibrillation (blue line), amorphous aggregation (blue dotted line), and micelle-protein complex formation (red line) on the NaCl concentration. (d) An illustrative model of the competition between amorphous aggregates, amyloid fibrils, and micelles of proteins and detergents.

Furthermore, disordered proteins or peptides with no significant conformation in the absence of detergents (e.g., keratoepithelin, Aβ, or K3) indicated that the interactions between disordered proteins with detergent molecules slightly below CMC were directly responsible for initiating a fibrillation pathway. Detergent-assisted nucleation can be explained by decreases in peptide solubility because of an increase in hydrophobicity upon detergent binding. On the other hand, a detergent concentration higher than the CMC inhibits fibrillation because stable micelles often capture proteins or peptides in an α-helical conformation, preventing intermolecular protein-protein interactions. However, as described above, no comprehensive mechanism has been proposed that considers equilibrium and kinetics.


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Phase Diagram of Amyloid Fibrillation. Amyloid fibrils form in supersaturated solutions of precursor proteins by a nucleation and growth mechanism.10-12,

13

On the other hand,

amorphous aggregates often prevail under certain solvent conditions and compete with amyloid fibrillation. We proposed that amyloid fibrils and amorphous aggregates were similar to the crystals and glasses of solutes, respectively, and that the conformational phase diagram was useful for obtaining a deeper understanding of the partition between amyloid fibrillation and amorphous aggregation.13 This phase diagram has been successfully used for fibrillation dependent on salt13, 42 or alcohol concentrations.26-27 We here propose that the phase diagram is also useful for understanding the detergent concentration-dependent complexity in amyloid fibrillation (Fig 6b, c). We assumed that protein concentration-dependent CMC values defined the phase boundary between micelles and other phases. The dependence of the lag time of fibrillation on SDS concentrations with a minimum near the CMC value (Fig. 1d) was reproduced by following a horizontal dotted line at 0.3 mg/ml β2m in the phase diagram. The same is true for the maximal ThT fluorescence representing the amount of amyloid fibrils (Fig. 1c). The major driving force of fibrillation in the presence of SDS is a decrease in the solubility of denatured proteins upon interactions with hydrophobic detergent molecules.32, 45 However, an alternative pathway may exist under decreased solubility, leading to amorphous aggregation.45 22 ACS Paragon Plus Environment

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Although detergent-assisted fibrillation is limited by supersaturation (i.e. a high free energy barrier of nucleation), the free energy barriers of amorphous aggregation are low so that amorphous aggregation occurs rapidly with saturating kinetics.13 This competitive mechanism resembles the effects of salts on amyloid fibrillation.42, 57 However, SDS brings an additional detergent effects by which mixed micelles of SDS and protein are formed and amyloid fibrillation was prevented (Fig. 6b). In other words, when the concentration of SDS exceeds that of the CMC, complexes of proteins and micelles become more stable both thermodynamically and kinetically. Based on the dynamic nature of the complexes, it is likely that the complex formation of proteins and micelles occurs without a lag phase. Three types of aggregates (i.e, amyloid fibrils, amorphous aggregates, and complexes with micelles) may compete in their populations depending on their relative stabilities, as observed for the salt-dependent competition between amyloid fibrils and amorphous aggregates (Fig 6d).13, 42 The dominant species is determined by the relative free energies of the respective phases, which we assumed to be proportional to the concentration of monomers (i.e., critical concentration) in equilibrium with the respective phases. In other words, the critical concentrations for the three types of aggregates change in distinct manners depending on the SDS concentration, causing a change in the dominant species.


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In the phase diagram, we assumed that the CMC was the SDS concentration at which the phase transition from fibrils or amorphous aggregates to mixed-micelles of proteins and detergents occurred. In other words, even preformed fibrils depolymerize upon the stabilization of SDS micelles. The CMC value of SDS has been shown to increase in the presence of proteins because additional amounts of detergents are required to make protein-SDS interactions below the CMC.32, 54 Moreover, it is likely that proteins form amorphous aggregates at higher protein concentrations before being dissolved by detergents.45 In the phase diagram, the region of amorphous aggregation can be located at higher protein concentrations before dissolution by micelles. Therefore, at low concentration of proteins, SDS-dependent phase transitions include monomers, fibrils, and micelles, whereas, at high concentration, monomers, fibrils, amorphous aggregates, and micelles are included. To explain these complexities in amyloid fibrillation in a manner that depends on the protein concentration, we assumed two lines representing the free energy of monomers relative to protein-micelle complexes at low and high protein concentrations. The exact phase boundaries of the conformational phases will depend on various factors. However, we propose that the phase diagram shown in Figure 6 will be common to various proteins and detergents, as observed in the case of salts.13, 42, 57

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Our results indicate that amyloid fibrillation in the presence of detergents is determined by competition between accelerating and decelerating factors. To form crystal-like amyloid fibrils, the concentrations of proteins and peptides have to be higher than their solubilities (i.e., protein critical concentration). Previous studies suggested that the aggregation of peptides or proteins was accelerated by a membrane.53-54, 58-60 Local biological environments such as the presence of amphiphilic compounds or membrane surfaces may significantly increase the local concentration, leading to the breakage of supersaturation. Glass-like amorphous aggregates under such concentrated conditions are likely to provide seed-competent conformations, explaining the SDS-induced acceleration of fibrillation. However, when the driving forces of amyloid fibrillation are too strong, protein molecules may form glass-like amorphous aggregates. In the case of detergents that form stable micelle structures, proteins are stabilized by forming stabile protein-micelle complexes, in which proteins often assume an α-helical conformation. Thus, the same detergents work as accelerators or inhibitors of amyloid fibrillation depending on their concentrations. These competitive pathways may also be controlled by kinetic factors because crystal-like amyloid fibrillation is rate-limited by supersaturation, while glass-like amorphous aggregation and the formation of micelle-protein complexes may independent of supersaturation.13 These will produce further kinetic complexity in amyloid fibrillation, in which transiently formed 25 ACS Paragon Plus Environment

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amorphous aggregates are slowly converted to amyloid fibrils. A conformational phase diagram combined with the underlying free energy profiles of respective species will be crucial for understanding the apparent complexity of amyloid fibrillation.


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AUTHOR INFORMATION

Corresponding Author

*Yuji Goto: [email protected]

Present Addresse †Department of Chemistry and Biotechnology, Graduate School of Engineering, and Center for Research on Green Sustainable Chemistry, Tottori University, Tottori 680-8552, Japan.

Funding Sources This work was supported by the Japanese Ministry of Education, Culture, Sports, Science and Technology and Japan Society of the Promotion of Science. This work was performed under the Cooperative Research Program of Institute for Protein Research, Osaka University.

ACKNOWLEDGMENT We thank Kyoko Kigawa (Osaka University) for expression and purification of β2m and Tetsuya Shimura (Kaijo Co. Ltd.) for technical support.

ABBREVIATIONS Aβ, amyloid β; ANS, 1-anilino-8-naphthalene sulfonate; β2m, β2-microglobulin; C8, sodium octyl sulfate; C10, sodium decyl sulfate; CD, circular dichroism; CMC, critical micelle 27 ACS Paragon Plus Environment

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concentration; HFIP, 1,1,1,3,3,3-hexafluoroisopropanol; SDS, sodium dodecyl sulfate; TEM, transmission electron microscopy; TFE, 2, 2, 2-trifluoroethanol; ThT, thioflavin T.

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Figure 1. Effects of SDS on amyloid fibrillation of β2m at pH 7.0 and 37 °C. (a) Determination of CMC for SDS using ANS fluorescence. The CMC value of SDS was determined to be 0.57 mM. The chemical structure of SDS is also shown. (b) Fibrillation kinetics at various SDS concentrations as defined by a color scale bar. (c, d) Dependencies of the maximum ThT intensity (c) and lag time (d) on SDS concentration. Lines are fittings with a degree-4 polynomial function. (e) TEM images of fibrils formed at 0, 0.5, and 2.0 mM SDS. A scale bar represents 200 nm. (f) Seed-dependent fibrillation at 0.5 mM SDS. Fibrils formed under ultrasonication at 0.5 mM SDS were used as seeds. Different traces represent reactions in different wells. 160x240mm (300 x 300 DPI)

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Figure 2. Effects of sodium decyl sulfate (C10) and sodium octyl sulfate (C8) on amyloid fibrillation of β2m at pH 7.0 and 37 °C. (a, b) Determination of CMC values using ANS fluorescence. The CMC values of C10 (a) and C8 (b) were determined to be 9.5 mM and 76.1 mM, respectively. (c, d) Fibrillation kinetics at various C10 (c) or C8 (d) concentrations as defined by a color scale bar. (e-h) Dependencies of the maximum ThT intensity (e, f) and lag time (g, h) on the concentrations of C10 (e, g) and C8 (f, h), respectively. Lines are fittings with a degree-4 polynomial function. 160x240mm (300 x 300 DPI)


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Figure 3. Morphologies and seed potentials of β2m fibrils formed in the presence of sodium decyl sulfate (C10) or sodium octyl sulfate (C8). (a, b) TEM images of fibrils formed at 6 and 10 mM C10 (a) or 60 and 100 mM C8 (b). Scale bars represent 200 nm. (c, d) Seed-dependent fibrillation at 6 mM C10 (c) or 60 mM C8 (d). Seeds were obtained from the fibrils formed under ultrasonication at the same detergent concentrations. Different traces represent the kinetics in different wells. 160x120mm (300 x 300 DPI)


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Figure 4. Effects of sodium deoxycholate on amyloid fibrillation of β2m at pH 7.0 and 37 °C. (a) The CMC of deoxycholate obtained from ANS fluorescence measurements. The CMC value under quiescent conditions was 5.4 mM (inset), whereas that under ultrasonic conditions was 31.6 mM. The chemical structure of deoxycholate is also shown. (b) Fibrillation kinetics at various deoxycholate concentrations as defined by a color scale bar. (c, d) Dependences of the maximum ThT intensity (c) and lag time (d) on the deoxycholate concentration. Lines are fittings with a degree-4 polynomial function. (e) TEM image of fibrils formed at 30 mM deoxycholate. A scale bar represents 200 nm. (f) Seed-dependent fibrillation under quiescent conditions at 30 mM (solid lines) or 5 mM (dashed lines) deoxycholate. Fibrils formed under ultrasonication at 30 mM deoxycholate were used as seeds. Different traces represent the kinetics in different wells. 160x180mm (300 x 300 DPI)


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Figure 5. Far-UV CD spectra of β2m before (a, c, e) and after (b, d, f) detergent-induced amyloid fibrillation at pH 7.0 and 37 °C. (a, b) SDS-induced fibrillation. (c, d) Sodium decyl sulfate (C10)-induced fibrillation. (e, f) Sodium octyl sulfate (C8)-induced fibrillation. 160x180mm (300 x 300 DPI)


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Figure 6. Mechanism underlying detergent-dependent amyloid fibrillation of proteins. (a) The linear relationship between the maximal detergent concentration of fibrillation and CMC. The values for benzalkonium chloride (BAC12, BAC14, and BAC16) with the keratoepithelin peptide were taken from Kato et al.,56 in which the number after BAC represented the length of alkyl chains. The values for lysophosphatidic acid (LPA) with β2m were from Pal-Gabor et al.38 (b) A phase diagram of conformational states that depended on SDS and protein concentrations at pH 2.5. (c) Dependencies of free energy changes in amyloid fibrillation (blue line), amorphous aggregation (blue dotted line), and micelle-protein complex formation (red line) on the NaCl concentration. (d) An illustrative model of the competition between amorphous aggregates, amyloid fibrils, and micelles of proteins and detergents. 160x140mm (300 x 300 DPI)


Supersaturation-Limited and Unlimited Phase Spaces Compete to

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