Spectroscopic and Microscopic Studies of Aggregation and Fibrillation

Article pubs.acs.org/JPCB

Spectroscopic and Microscopic Studies of Aggregation and Fibrillation of Lysozyme in Water/Ethanol Solutions Alessandra Giugliarelli,† Luigi Tarpani,‡ Loredana Latterini,‡ Assunta Morresi,† Marco Paolantoni,† and Paola Sassi*,† †

Dipartimento di Chimica, Biologia e Biotecnologie and ‡Dipartimento di Chimica, Biologia e Biotecnologie and Centro di Eccellenza Materiali Innovativi Nanostrutturati CEMIN, Università di Perugia, Via Elce di Sotto 8, 06123 Perugia, Italy S Supporting Information *

ABSTRACT: The thermal aggregation of lysozyme has been analyzed in water/ethanol solutions at low pH to induce the specific protein aggregation pathway which leads to fibrillar structures in a few hours. In this solvating medium, the protein undergoes a conformational rearrangement promoting the formation of fibrils that are structurally similar to amyloid ones. As the process evolves with different steps, a multitechnique approach has been used by means of analytical probes that can be selectively sensitive in the detection of the different stages of protein association. Fourier transform infrared spectroscopy, intrinsic fluorescence, stationary fluorescence anisotropy, transmission electron microscopy (TEM), and atomic force microscopy (AFM) measurements have been carried out at different times to access and characterize the whole aggregation pathway. The data recorded with different experimental setups revealed different sensitivity to different stages of protein assembling. The whole set of data together with the direct visualization of different aggregate structures by use of TEM and AFM imaging enable to discuss a possible mechanism of fibrillation.

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final fibrils.20−23 Therefore, the characterization of the mechanism of production and growing of clusters is crucial for the comprehension of the aggregation process and hence to set up strategies devoted to its inhibition. To this extent, it is particularly important to know how the intermediates are onor off-pathway during the amyloid fibril formation.15−17,24−26 Among globular proteins, lysozyme is a good model to investigate conformational changes and the formation pathway of aggregate species due to its ability to form intermolecular βsheets structures at different temperatures and solvent compositions.7,27−31 Moreover, human lysozyme (similar to hen lysozyme) is involved in diseases related to amyloid formation.5,30 The ordered aggregated species of lysozyme are also interesting in biomaterial sciences since they form hydrogels whose properties can be modulated by changing the aggregating conditions.32 In this study, the experimental conditions (solvent composition, pH, and temperature) were chosen in order to induce a cost-effective thermal aggregation process that selectively leads to the production of amyloid-like structures in a few hours. Recently, some of us analyzed the thermodynamics and kinetics of lysozyme aggregation after dissolution of the protein in water/ethanol solutions at low pH values.12−14 By operating at pH = 3.0 and in the presence of

INTRODUCTION Protein unfolding and refolding are critical processes that can lead to the formation of particular structures where the protein has lost its native conformation and function. In general, protein systems rearrange to minimize the interaction between hydrophobic residues and the polar solvent.1 Depending on solvating conditions, the stabilization driving force can favor the folding of the protein system toward its native structure or can lead to misfolding and/or aggregation of protein molecules.2−4 Such an aggregation process is characterized by different steps during which the protein undergoes conformational rearrangements and intermolecular association to form stable structures of increasing complexity, from small clusters to amyloid fibrils, possibly passing through intermediate species. These species are involved in many neurodegenerative diseases.3,5,6It has been reported that amyloid fibrils produced from different native states show a common feature, an intermolecular β-sheet core,7−9 whose formation can be strongly influenced by the solvent properties.10−14 In fact, as the aggregation is usually observed from partially unfolded species, the protein’s environment strongly affects the onset and the development of the aggregation process. Despite the great amount of literature on the formation of amyloid fibrils, there are still open questions regarding the whole mechanism of protein aggregation and fibrillation and the key role of the early aggregated species.15−19 In fact, it has been suggested that the small protein clusters could be toxic to the cells more than the © XXXX American Chemical Society

Received: August 3, 2015 Revised: September 22, 2015

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DOI: 10.1021/acs.jpcb.5b07487 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

Figure 1. (a) IR spectra in the amide I region of lysozyme 120 mg/mL in xEtOD = 0.15, pH = 3.0, at room temperature (dashed line), and at 50 °C as a function of time. Evolution of absorbance intensity at (b) 1650 and (c) 1618 cm−1 monitored at 50 °C for ca. 8 h. The sample was heated inside the IR cell.

hierarchical mechanism of fibrillation have been obtained by this combined spectroscopic and microscopic investigation.

ethanol (molar fraction of ethanol of 0.15), the aggregation process can be directed toward the formation of ordered aggregates (intermolecular β-sheet rich) instead of inhomogeneous species.26,33 Under these conditions, the melting temperature of lysozyme, which is correlated to the characteristic temperature of protein assembling, is lower than that observed in aqueous solution;12−14 this allowed evidencing that the population of ordered aggregates reduce at T > 65 °C. Thus, in these solutions, ordered oligomers are found to exist in the 45−80 °C range.13 The presence of ethanol also changes the kinetics of self-assembling since at 120 mg/mL protein concentration the aggregation is so fast to cause a rapid gelation of the sample.12 In vitro studies can help explain the complex mechanism of clustering and fibrillation by the use of different techniques, which are sensitive to different types of structuring.34−36 In the present investigation, spectroscopic and microscopic techniques have been employed in order to access the aggregates’ evolution from both molecular and microscopic points of view. Lysozyme aggregation has been induced by heating a protein solution at 50 °C and pH = 3.0 in water/ethanol solvent (xEtOH = 0.15)and samples have been analyzed at different incubation time, following the aggregation process up to ca. 40 h. IR spectroscopy has been used to probe the rearrangement of the secondary structure and the nucleation of ordered aggregates made up of intermolecular antiparallel β-sheets; these species can possibly evolve under different conditions into fibrillar structures.13,37,38 Intrinsic fluorescence from tryptophan (Trp) residues is a sensitive internal probe of protein tertiary structure due to the high sensitivity of Trp residues to changes in their microenvironment.39−45 In the present work, the excitation and emission spectra were monitored during the induced aggregation process with the aim of investigating the occurrence of structural modifications on the protein. AFM (atomic force microscopy) and TEM (transmission electron microscopy) techniques enable characterizing the morphology of micro- and nanodimensional protein structures.46 Their use has allowed us to obtain information about the dimension and morphology of assembled species produced in the prefibrillar and fibrillar state.24,36 New insights on the key steps of the complex

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EXPERIMENTAL SECTION Materials. Hen egg white lysozyme, deuterated water (D2O), and partially deuterated and nondeuterated ethanol were purchased from Sigma-Aldrich and used without further purification. A 120 mg/mL lysozyme solution was prepared by dissolving the protein in EtOH/H2O at xEtOH = 0.15 and pH= 3.0 at room temperature. A diluted (30 mg/mL) protein solution was also prepared and maintained at 50 °C; small aliquots were taken at different times and analyzed at room temperature with different techniques including UV−vis absorption, fluorescence emission, and FT-IR−ATR (Fourier transform infrared spectroscopy−attenuated total reflectance). For UV−vis absorption and emission spectra the solution was further diluted to have a final 1 mg/mL protein concentration. IR Experiments. Lysozyme solution (30 μL) at 120 mg/mL was placed between CaF2 windows and maintained at 50 °C in a homemade cell described in a previous work.42 FT-IR spectra in the 5000−400 cm−1 frequency range were acquired with a Bruker Tensor 27 performing measurements as a function of time up to 8 h. Each spectrum was the average of 30 scans with a 2 cm−1 resolution. IR spectra were normalized with respect to the integrated area of the amide I profile. Deuterated solvents (D2O and EtOD) were used for FTIR experiments in order to eliminate the overlapping of the H2O bending vibration (1640 cm−1) to amide I band (1600−1700 cm−1). As a consequence, the buffer had no contribution in the investigated region of the IR spectrum. For the ATR experiments, lysozyme 10 mg/mL was dissolved in H2O/EtOH xEtOH = 0.15 and heated in a water bath at 50 °C for 40 h. A drop of the sample at different heating times was dried on a ZnSe crystal (SpecacGateway ATR), and the corresponding FT-IR spectra were acquired with the same resolution of transmission measurements. Fluorescence Measurements. Tryptophan fluorescence emission and excitation spectra, corrected for the instrumental response, were acquired by use of a Fluorolog-2 (Spex, F112AI) fluorimeter: a right-angle detection geometry was set.41,43 Using polarized light at 280 nm to excite the sample solutions, steadystate fluorescence anisotropy measurements were performed. B


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following 10−20 min, the Amide I band-shape changed since the shoulders at 1618 and 1690 cm−1 appeared and grew in intensity. As reported in the literature,28,29 these signals were assigned to the antiparallel intermolecular β-sheets characteristic of ordered aggregates, and their intensity was associated with the amount of interchain contacts produced on selfassembling. Figure 1b,c shows the intensity evolution at 1650 and 1618 cm−1, indicating the decrease of α-helix structures and the corresponding increase of intermolecular β-sheet contacts, respectively. These intensity variations are correlated since the protein unfolding activates the formation of ordered aggregates. According to Figure 1c, a rapid formation of antiparallel intermolecular β-sheets is realized in the first hour; between the first and the third hour a slight increase of the relative intensity at 1618 cm−1 is observed; after about 6 h, no major secondary structure changes are detected with IR spectroscopy. To explore possible changes on the tertiary structure under the same thermal treatment, the lysozyme intrinsic fluorescence in H2O/EtOH solutions was measured. Six tryptophan (Trp) and two tyrosine (Tyr) residues are present in this protein; however, the intrinsic fluorescence of the native lysozyme is dominated by the emission of two Trp residues (Trp 62 and Trp 108) whose spectral contributions have been resolved since Trp62 emits at longer wavelengths due to the localization in a more polar environment.48 Interestingly, Trp 62 and 63 are located in the amyloidogenic region of HEWL (approximately residues 49−101), which is involved in the formation of fibrils.31 Thus, the fluorescence behavior of these residues can give insight on the mechanism of fibril formation. Figure 2 shows both the emission and excitation spectra of a lysozyme solution (30 mg/mL) acquired at different times while the sample was maintained at 50 °C.

Anisotropy values (r) were calculated according to the following equation r=

I − I⊥ I + 2I⊥

where I|| and I⊥ correspond, respectively, to the fluorescence intensity parallel and perpendicularly polarized respect to the incident polarized light. TEM. Protein solution (10 μL) at 30 mg/mL concentration was diluted with 1 mL of distilled water, placed on a copper grid (200 mesh), and allowed to dry overnight. Before each measurement, the grid was stained with 2% uranyl acetate for 5 s and then washed with distilled water. TEM images were acquired with a Philips model 208. AFM. In order to follow the different steps of the aggregation process, we acquired AFM images of the protein deposited on mica surface.47 Lysozyme (10 mg) was dissolved in 1 mL of H2O. A drop of the sample was placed on a freshly cleaved mica support and allowed to dry overnight, and then the AFM images were acquired. Afterward, this sample was put on a Petri dish and covered with H2O/EtOH solution (xEtOH = 0.15, pH = 3). The Petri dish was heated at ca. 50 °C (the temperature was checked on the top of the dish), and the same sample was analyzed after different incubation times equal to 0.5, 6, and 28 h. Before each AFM analysis, the mica support was washed with distilled water to eliminate the excess solvent and dried overnight. AFM images were acquired under the same conditions used for the blank. With this procedure, aggregation of lysozyme was induced directly on the mica surface and the different aggregation steps were monitored by analyzing the same sample. A NT-MDT SolverPro microscope operating in tapping mode and equipped with a cantilever oscillating in the frequency range 140−390 kHz was used to acquire the images.

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RESULTS AND DISCUSSION Figure 1a shows the IR spectra of a 120 mg/mL sample of lysozyme in D2O/EtOD (xEtOD = 0.15, pH = 3.0) recorded at different times at the constant temperature of 50 °C (incubation time up to 8 h). The FT-IR spectrum of lysozyme registered at room temperature (dashed gray line in Figure 1a) before the heating process began showed the Amide I band profile centered at 1650 cm−1, which is characteristic of the native protein having α + β structure.28,44,45 At the ethanol molar fraction used in this experiment, the immediate aggregation of lysozyme at room temperature due to an “ethanol shock” does not occur. This shock effect was recently discussed by Nemzer and coworkers,25 whose experiments led to the production of nonfibrous spherical clusters with a stochastic process that depends on pH and ethanol concentration. The experimental conditions selected in the present work (xEtOD = 0.15 and pH = 3.0) determined that the aggregation occurred at about 50 °C, the produced aggregates were stable in a wide temperature range, and the study of fibrils formation was completely assessed in vitro.7,27,31 The first spectrum acquired after 1 min of incubation at 50 °C (cyan line) was sensitively blue-shifted with respect to the native spectral profile. This is related to the increase of the fraction of unfolded species;11−14 in fact, under the same experimental conditions a 30% unfolded protein was previously estimated on a diluted lysozyme solution.14 Then, in the

Figure 2. Emission (right; λexcitation = 280 nm) and excitation (left; λemission = 335 nm) spectra of lysozyme in H2O/EtOH (xEtOH = 0, 15, pH = 3) recorded at different times during thermal treatment at 50 °C. Spectra were corrected for the fraction of absorbed light. Inset: full width at half height (fwhh) of emission band.

The emission spectrum acquired at the beginning of the thermal treatment at 50 °C (t = 0) is centered at 330 nm and thus slightly blue-shifted compared to what observed in water; this is in agreement with what previously obtained for lysozyme in the presence of alcohols, which change the solvation properties of the exposed Trp.41,43,49,50 The excitation spectrum presents differences with the UV absorption spectrum of the protein in the same medium (see Figure S1, Supporting C


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wavelengths revealed a progressive lengthening of the average decay times at early stages, further supporting the hypothesis that important changes in Trp microenvironment are occurring in this timespan.54 At longer incubation times, the mean decay time is almost constant, similar to what observed is for the anisotropy values (Figure 3) indicating that no major variations are taking place in the environment of Trp units. On the other hand, the bandwidth of emission spectra, acquired after 24 h, still presents important modifications (inset of Figure 2); the occurrence of structural changes is also supported by the comparison of the fluorescence spectra recorded at different excitation wavelength (Figure 4). In

Information), suggesting that in the present experimental conditions energy-transfer processes are taking place. At an early stage of the heating treatment (first 6 h), the emission spectra display a gradual red-shift with the maximum moving from 330 to 340 nm (Figure 2); the red-shift comes together with a remarkable intensity increase (the spectra are corrected for the fraction of absorbed light). The spectral shift is due to modifications in the protein conformation leading to an increased exposure of the Trp units to a more polar environment;16,44,51these modifications in protein conformation could be associated with the unfolding process, in agreement with IR data of diluted protein solutions.14 However, the intensity increase is not justified by the degree of exposure of Trp fluorescent residues since, generally, the fluorescence quantum yield of Trp is lower in a hydrophilic environment than in a hydrophobic or aprotic one. The intensity increase during the unfolding process could be due to an enhanced contribution from Trp residues whose fluorescence is generally quenched in the native protein. This effect is also supported by the change of spectral width, measured as the full width at half-maximum (fwhm, inset Figure 2) of the emission spectra, which increases in the first 6 h incubation. These observations indicate that at early stages of the thermal treatment important structural changes are occurring in lysozyme which likely involves Trp residues normally located in hydrophobic environment (Trp108) or quenched (Trp63) in the native protein. This analysis is also supported by fluorescence anisotropy and the time-resolved fluorescence measurements (Figure 3),

Figure 4. Emission spectra of lysozyme in H2O/EtOH (xEtOH = 0, 15, pH = 3) at t = 0 (a) and t = 40 h (b), obtained by exciting the samples at λexc = 260 nm (red triangles) and λexc = 295 nm (black circles).

particular, the spectrum obtained at t = 0 upon excitation at 260 nm (where both Trp and Tyr residues absorb) has a higher contribution on the red side compared to the spectrum acquired exciting the sample at 295 nm (Figure 4a); on the contrary, these differences are not observed after prolonged treatment times (Figure 4b). These data indicate that different populations of fluorescent amino acids are detectable during the entire process. Thus, if we consider the fluorescence data as a whole set, we evidence changes during the entire treatment (0 < t < 40 h); moreover, the different evolution of emission properties at short (below 6 h) and long (above 6 h) heating times suggests the occurrence of a multistage process. This can be connected to the unfolding process and the formation and growth of lysozyme aggregates. To reach a direct visualization of species involved in the aggregation process and have insights on the formation of complex supramolecular structures, the analysis has been completed by the TEM and AFM microscopic investigation. TEM images have been acquired for two protein samples deposited at different times during the aging at xEtOD = 0.15. Figure 5A presents the TEM image of the lysozyme sample deposited after 30 min incubation at 50 °C. The presence of particles with an average size smaller than 500 nm

Figure 3. Steady-state anisotropy (λexcitation = 280 nm, circles) and average decay times on exciting at 295 nm (λemission = 335 nm, triangles; λemission = 375 nm, squares) of lysozyme in H2O/EtOH (xEtOH = 0, 15, pH = 3.0) recorded at different times during thermal treatment at 50 °C.

although these data are generally quite complex to interpret for protein containing different fluorescent residues. In the early stage of the heating treatment (first 6 h) the steady-state anisotropy data display a 30% increase (see Figure 3), compatible with a rearrangement of the protein structure.52 The fluorescence decays recorded upon excitation at 295 nm and monitoring at 335 and 375 nm show a nonexponential behavior, as previously observed for other proteins containing different emitting units.53 The decays could be satisfactorily fitted with three-exponential functions (see Table 1of Supporting Information)and the average decay times (τmean) are reported in Figure 3. The decay curves collected at both D


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Parts A and B of Figure 6 show the images of lysozyme in EtOH/water solution before thermal treatment. Slightly uniform and small particles are distributed on the surface. Profiles taken at two different cutoff lines (Figure 6C) show an average height of 3 nm. The small size of the detected particles and the absence of large clusters indicate that lysozyme is not aggregated under these conditions (the hydrodynamic radius of lysozyme is about 2 nm).15,47 This image can be considered as the blank of our experiment. After the protein was heated for 30 min at 50 °C in H2O/EtOH (Figure 6D,E), particles having a larger size appeared all over the surface with the characteristic elliptical shape of the protein aggregates, intermediates of fibrillation.15,24 As evidenced in Figure 6F, these species show an average diameter of a few hundred nanometers and 15 nm height as they are formed by association of many protein molecules. The microscopic image can be correlated with corresponding IR data (Figure 7) that evidence how these structures are βsheet rich. The position of the IR signal of the intermolecular βsheets has been recently discussed by Foley et al.55 as a structural fingerprint of oligomeric vs oligomer-free amyloid fibrils growth pathway: the intermolecular β-sheets IR signal occurs at lower wavenumbers (1618 ± 1 cm−1) if the aggregation proceeds through the formation of oligomers, compared to the case of the oligomer-free pathway (1623 ± 1 cm−1). Our data (Figure 7) show an average value of the aggregate IR peak at 1618 ± 2 cm−1, suggesting the occurrence of an oligomeric aggregation process.55 After 6 h of heating, AFM reveals a different arrangement of the protein aggregates (Figure 6G−I). These species are close to each other to form a more complex structure. They align on the surface creating structures a few micrometer long which are assigned to protofibrils.24,37,56It is interesting to note that AFM clearly displays the growth of protofibrils through the alignment of the same particles of Figure 6E, as confirmed by comparison of profiles in Figures 6F and 6I; it is noteworthy that small clusters are still visible on the mica surface together with protofibrils (Figure 6G,H). The corresponding ATR spectrum registered at t > 6 h shows only minor changes. The AFM data together with those obtained at the molecular level (IR and fluorescence) suggests that proto-fibrils grow as assemblies of elliptical particles without significant changes in the secondary structure of the single protein unit, but with a continuous modification of the higher packing degree. AFM images recorded after 28 h heating (Figures 6J−L), show the growth of proto-fibrils to form long and thin fibril-like structures. The profile was taken on the widest point of the proto-fibril (Figure 6L) which is 30 nm high and a few microns long, indicating that at this stage, the smaller species are fused into the fibrillar structure. This observation supports one of the proposed mechanism for fibrils formation: they grow through oligomers addition and fusion,15 as also seen for fibrils formed from A-β peptide at longer incubation time.24The A-β peptide is commonly used as a model for fibrillation in the human body; thus, our experimental conditions allow producing fibrillar species in a few hours with a mechanism similar to the one observed for clinical amyloid studies. The comparison of TEM and AFM images evidence the analogies between experiments conducted in solution (TEM) and on a surface (AFM). Figures 5A and 6E show the small aggregates produced in solution and on the mica disk after 30 min at 50 °C. In Figures 5B and 6H, identical protofibrils a few microns long are clearly evidenced, but in the TEM image (Figure 5B) oligomers

Figure 5. TEM images of lysozyme in xEtOH = 0.15 at 50 °C for 30 min (A) and 6 h (B). Reference bar corresponds to 1 μm. Image A shows the small particles that correspond to proteinaggregates; image B shows the formation of protofibril structures.

demonstrates the formation of large aggregates, produced according to the secondary and tertiary structure modifications described above. After 6 h at 50 °C, elongated assemblies can be observed (Figure 5B) which are assigned to the formation of protofibrils. They consist of thick structures having few microns length, which are formed by aggregates and are the first structural arrangement of the mature fibrils. Under our experimental conditions, the formation of mature fibrils has not been evidenced because this process usually takes many days to complete.30 Thus, the assembling process of lysozyme was monitored by the use of AFM microscopy to have deeper morphological information. For the AFM experiments, the aggregation process was followed directly on the mica surface; we observed that the same qualitative information on the morphology and size of assembled species can be obtained by forming aggregates in solution and then depositing the sample on the mica substrate, as usually reported in literature.15,24 The choice to apply the thermal treatment and follow the process directly on the mica surface was aimed at reducing the reproducibility problems due to the deposition. In fact, intermediate aggregates could modify their structure when transferred from solution to a substrate,15 and this could be particularly favored in the experimental conditions of our experiment causing a very fast aggregation kinetics (pH = 3.0 and ethanol as cosolvent). To monitor the secondary structure of lysozyme under the same conditions of AFM measurements, ATR spectra (Figure 7) were registered for the sample at the same concentration as described in the Experimental Section. E


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Figure 6. AFM images of lysozyme dissolved in water and dried on mica support (A, B) and in xEtOH = 0.15, pH = 3.0 incubated at 50 °C for 30 min (D, E), 6 h (G, H), and 28 h (J, K). The corresponding profiles are taken along the yellow lines.

Figure 8). At 50 °C, the thermal unfolding of lysozyme is rapidly obtained (step b); protein chains self-assemble and rearrange into intermolecular β-sheets aggregates (step c). This is the nucleation step evidenced by the appearance of signals at 1618 and 1690 cm−1 in the FTIR spectrum. From previous experiments, we know that unfolding and aggregation are, respectively, reversible and irreversible denaturation processes.13,14 In the first hour of reaction, aggregates grow to form the elliptical particles shown by TEM and AFM images of Figures 5A and 6E, respectively. Their condensation (step d) and alignment (step e) lead to the production of elongated structures as observed in Figures 6H and 5B. Then, these segments fuse into the long and thin protofibrils (step f) shown in Figure 6K. The presence of a multistep process was

are not present: this evidence can be explained by considering that during aggregation the protein clusters are adherent on the surface and the mobility of these species is diminished; thus, part of the aggregates cannot align to form the fibrils. Moreover, the washing of the sample before TEM experiment isolates only large structures while smaller ones are washed away. By combining the molecular insights from IR and fluorescence experiments with the microscopic images acquired with TEM and AFM techniques, the whole pathway of amyloid fibrils production at 50 °C for lysozyme in water/ethanol solution at pH = 3 is reconstructed in Figure 8. Native lysozyme is maintained in water/ethanol solution xEtOH = 0, 15 at room temperature as confirmed by IR absorption (step a in F


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analysis of fwhm and intrinsic fluorescence decays at long heating times suggests that Trp residues experience the effect of different environments during the production of protein clusters and prefibrillar species. This is confirmed by TEM and AFM images acquired at different aging times. At first, TEM and AFM images show the formation of nanometric structures. AFM analysis indicates that these species grow through a mechanism of addition and fusion of clusters. Thus, they align to form the proto-fibril structures, following the same pathway previously proposed for amyloid fibrils production in vivo.24 Our data support the idea that the process of addition and fusion of intermediate protein clusters plays a key role in the formation of amyloid-like aggregates; thus, the study of aggregation inhibitors could be a target for these species characterized by an intermolecular β-sheet secondary structure.

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ASSOCIATED CONTENT

S Supporting Information *

Figure 7. Time evolution of IR spectra in the amide I region of lysozyme solution (30 mg/mL in xEtOH = 0.15, pH = 3.0) at 50 °C isothermal heating.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b07487. Fitting parameters of fluorescence decays (Table S1); absorption and excitation spectra of lysozyme H2O/ EtOH solution (Figure S1) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS L.L. acknowledges financial support from the University of Perugia and the Ministero per l’Università e la Ricerca Scientifica e Tecnologica (Rome, Italy). A.G. acknowledges the financial support of the 2007-2013 ESF “Competitiveness and Employment objective” Umbrian Regional Operational Programme (ROP), Avviso pubblico aiuti individuali per la realizzazione di progetti di ricerca.

Figure 8. Possible pathway for lysozyme aggregation under our experimental conditions: (a) native lysozyme, (b) unfolded lysozyme produced with thermal treatment, (c) formation of intermolecular βsheets structures (nucleation step of polymerization), (d) addition of protein aggregates, (e) alignment of assembled structures, (f) formation of protofibrils.

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confirmed by the differences in the fluorescence profile, decay times and steady-state anisotropy of Trp residues. This aggregation model is consistent with the one proposed for lysozyme aggregation at different ethanol concentration50 and also supports the “nucleated conformational conversion” model first evidenced for prion protein.57

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CONCLUSIONS A multitechnique approach has been applied to the different aggregation stages of lysozyme in water/ethanol at xEtOH = 0.15 at pH = 3.0; in these solvating conditions, the formation of fibril-like structures is favored both from thermodynamic and kinetic points of view. Thermal aggregation has been monitored for ca. 40 h with different spectroscopic and microscopic techniques; this has allowed characterizing the steps involved in the formation of ordered amyloid-like fibrils from the native lysozyme. The molecular changes (secondary and tertiary structures) of the protein system have been evaluated through intrinsic fluorescence of Trp residues and IR spectroscopy. Aggregates are characterized by the presence of intermolecular β-sheets that grow to give the nuclei of prefibrillar structures; after that their secondary structure remains stable. The careful G


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Spectroscopic and Microscopic Studies of Aggregation and Fibrillation

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