Ferric Ions Inhibit the Amyloid Fibrillation of Î²-Lactoglobulin at High ...

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Ferric Ions Inhibit the Amyloid Fibrillation of β‑Lactoglobulin at High Temperature Rita Guzzi,*,†,‡ Bruno Rizzuti,§ Cristina Labate,† Bruno Zappone,§ and Maria P. De Santo† †

Department of Physics, University of Calabria, Ponte P. Bucci 31C, 87036 Rende (CS), Italy CNISM Unit, University of Calabria, Ponte P. Bucci, Cubo 31C, 87036 Rende (CS), Italy § CNR-NANOTEC, LICRYL-UOS Cosenza and CEMIF.Cal, c/o Department of Physics, University of Calabria, 87036 Rende, Italy ‡

ABSTRACT: The energetics of amyloid fibrillar aggregation of β-lactoglobulin (βLG) following incubation at high temperature and acid pH was studied by differential scanning calorimetry in the presence of Cu2+ or Fe3+ cations, and without any metal. Cu2+ and metal-free protein solutions showed a distinct exothermic response that disappeared almost completely when the Fe3+ molar concentration was ten times greater than the βLG concentration. Thioflavin T fluorescence studies in solution and atomic force microscopy analysis of the deposit left on flat mica substrates by heat-incubated βLG solutions correlated the absence of exothermic response of Fe3+-βLG solutions with a lack of fibril production. In contrast, abundant fibril deposits were observed for Cu2+-βLG solutions, with a rich polymorphism of multistrand fibrillar structures. Electron paramagnetic resonance revealed that Fe3+ permanently binds to βLG in the aggregate state whereas Cu2+ plays a catalytic role without binding to the protein. We propose that Fe3+ inhibits fibril production after binding to a key region of the protein sequence, possibly interfering with the nucleation step of the fibrillation process and opening a nonfibrillar aggregation pathway. These findings suggest that transition metal ions can be utilized to effectively modulate protein self-assembly into a variety of structures with distinct morphologies at the nanoscale level.

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stability of globular proteins is well established,8,9 only a few works have been concerned with the stability of protein aggregates.1,10,11 The study is particularly complex because the disruption of the amyloid structure inevitably generates a heterogeneous distribution of fragments, often showing a wide polymorphism. Spectroscopic and calorimetric approaches have been employed10,12,13 to characterize the stability of aggregated proteins and elucidate the complex mechanism of fibril polymerization and depolymerization. A number of experimental parameters (protein concentration, temperature, pH, ionic strength, presence of additives, etc.) can be varied to modulate the protein aggregation process and diversify the morphology, such as worm-like or rigid fibrils and amorphous aggregates.14,15 Interactions with metal ions can deeply affect protein aggregation, causing rapid precipitation, increased fibrillogenesis, and morphology alterations.16−18 Transition metals such Cu2+, Fe3+, and Zn2+ are particularly interesting as they were found in high concentrations (∼400, ∼950, and ∼1100 μM, respectively) in the core and periphery of senile amyloid plaque deposits.19,20 They were also used to modulate the in vitro formation of protein nano-objects for creating functional materials.5,6 Interestingly, Fe3+ ions were found to affect aggregation in opposite ways depending on the protein:

INTRODUCTION Self-assembly of proteins into ordered fibrillar aggregates is a general phenomenon intrinsically related to the molecular structure of polypeptide chains.1,2 First recognized in amyloid pathologies such as Alzheimer and Creutzfeldt-Jacobs diseases, amyloid fibrillation has also been reproduced in vitro using a variety of nonpathogenic proteins and polypeptides.3,4 Despite differences in sequences and secondary structures of proteins, amyloid fibrils share a common structural organization characterized by a highly ordered β-sheet conformation stabilized by an extensive network of intermolecular hydrogen bonds. Sequence-specific features, such as the presence of buried hydrophobic surfaces or favorable packing of side chains, further contribute to the stability of the amyloid structure. The high degree of structural organization makes amyloid fibrils very resistant to degradation and depolymerization. Recently, amyloid fibrillation has become the subject of an intense research effort in the soft matter and material science communities, as a promising route toward the bottom-up fabrication of functional materials via the self-assembly of nanostructured fibrils with controlled morphology and properties.5,6 Amyloid fibrillation is a multistep process starting from nonnative states of isolated proteins, followed by protein aggregation into oligomeric nuclei and elongation of fibrillar structures, possibly with a secondary nucleation stage related to fibril fragmentation.1,7 While the energetics and conformational © 2015 American Chemical Society

Received: March 19, 2015 Revised: May 15, 2015 Published: May 19, 2015 1794

DOI: 10.1021/acs.biomac.5b00371 Biomacromolecules 2015, 16, 1794−1801

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Biomacromolecules they promoted fibril nucleation for α-synuclein, but inhibited fibrillation for the β-amyloid 1−42 peptide.16,21 In a previous study22 we showed that copper has a catalytic activity on the aggregation of β-lactoglobulin (βLG), increasing the rate of fibril nucleation without permanently binding to the protein or altering the fibril morphology. βLG is a small globular protein (molecular weight 18.3 kDa) that constitutes the major protein component of bovine milk whey, and is widely studied due to its availability and relevance to food industry. βLG self-assembles in a variety of different morphologies as a function of the experimental conditions and is an ideal choice for studying protein self-assembly into the context of nanostructured functional materials.5 In this work, we extended our previous study and examined the effect of Fe3+ cations on the βLG fibrillation by using differential scanning calorimetry (DSC), atomic force microscopy (AFM), and electron paramagnetic spectroscopy (EPR). In particular, we investigated the thermal response of βLG aggregates formed without metal ions and in solutions containing CuCl2 and FeCl3 at protein−ion molar ratios of 1:1 (equimolar) and 1:10. Exothermic effects in the DSC thermograms revealed the energetics of fibril formation as a function of ion concentration. The DSC profiles obtained for metal-free solution and in the presence of Cu2+ were found to be consistent with the formation of mature fibrils, whereas Fe3+ ions almost completely inhibited fibrillation when the concentration was increased to a 1:10 molar ratio.

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subtracted from the sample DSC scans to determine the apparent heat capacity, Cp,app. Fluorescence. The presence of βLG fibrils in solution was quantified with a Thioflavin T (ThT) fluorescence assay. Aliquots of 0.1 mL of protein samples before and after DSC experiments were diluted in 0.9 mL of 10 μM ThT solution in 50 mM glycine−NaOH buffer (pH 8.5) following the protocol in ref 13. The spectra were recorded at room temperature on a LS 50B spectrofluorimeter (PerkinElmer), using excitation and emission wavelength of 442 and 482 nm, respectively. Electron Paramagnetic Resonance. Conventional EPR spectra were recorded for 0.12 mM solutions of βLG at pH 2 containing FeCl3 at 1:10 molar ratio, and for protein-free reference solution of FeCl3 at the same concentrations and pH. βLG aggregation was induced by incubating the EPR-grade quartz tubes containing samples at 80 °C for 4 h, and then rapidly cooling them in an ice−water bath. The EPR spectra of the reference solution, incubated samples and nonincubated samples were recorded by plunging the tube into a finger dewar containing liquid nitrogen. Finally, the samples were inserted into a standard rectangular cavity (Bruker ER4201 TE102) for EPR measurements. The measurements were performed on a ESP 300 X band spectrometer (Bruker, Karlsruhe, Germany) equipped with an ESP 1600 data acquisition system. Ferric ions show a wide variety of EPR spectra depending on the metal−ligand coordination geometry. For high-spin Fe3+ ion (S = 5/2) in a crystal lattice, the 2S + 1 spin levels are splitted even in the absence of an external magnetic field, due to the local electric field at the paramagnetic center produced by charges in the coordination atoms. The overall effects of the local electric and static magnetic field are reflected on the spin energy levels separation24,25 given by the spin Hamiltonian:

⎡ ⎤ 1 / = gβB · S + D⎢Sz 2 − S(S + 1)⎥ + E[Sx 2 − Sy 2] ⎣ ⎦ 3

MATERIALS AND METHODS

Protein Solutions. Bovine βLG (from Sigma-Aldrich) was dissolved in purified water (Millipore Q, resistivity 18.2 MΩ·cm at 25 °C) at pH 2, obtained by adding HCl. The protein was solubilized by stirring for 1.5 h at 4 °C with a Teflon-coated magnetic stir-bar on a stir-plate set to 200 rpm. The protein solution was then dialyzed against the same solvent for 3 days at 4 °C by using a Spectra-Por membrane with a molecular weight cutoff of 3.5 kDa, prepared as in ref 23. The solvent volume outside the membrane was about 100 times the volume inside the membrane. After dialysis, the protein concentration was in the mM range, as measured by UV−vis spectrophotometry (JASCO mod. 7850) using an extinction coefficient ε278 = 17600 M−1 cm−1. Either CuCl2 or FeCl3 was added with the same molar concentration as the βLG protein (βLG/ metal = 1:1, equimolar solution) or 10× this concentration (βLG/ metal = 1:10). Differential Scanning Calorimetry. DSC measurements were performed on a VP-DSC MicroCalorimeter (MicroCal, Inc.) with a cell volume of 0.52 mL and a temperature resolution of 0.1 °C. Before loading, samples and reference were properly degassed by using the ThermoVac MicroCal station. To determine the baseline, we carried out at least four scans with pure solvent (pH 2) in both the sample cell and reference cell. Protein samples with concentration 0.12 mM were left equilibrating in the calorimeter cell for 60 min at 10 °C before starting any measurement. DSC scans were obtained by heating the samples from 10 to 100 °C at a rate of 90 °C/h. The data were analyzed using the Origin software package (MicroCal). The background solvent baseline was subtracted from the protein/solvent scans and the data were normalized by the protein concentration to obtain the molar heat capacity, Cp. We also studied aggregation of βLG during consecutive DSC scans in the temperature range from 10 to 120 °C, heating at a rate of 90 °C/h. The first scan was stopped at 80 °C and the protein solution was left incubating at this temperature for 4 h to induce fibril formation. After cooling the sample back to 10 °C, three further scans were recorded upon heating up to 120 °C. Experiments were not recorded on cooling. Only the solvent−solvent background was

(1)

where the first term describes the Zeeman splitting and the second and third terms represent the axial and rhombic component of the zerofield splitting. The latter contribution is responsible for the splitting of the 6-fold ground state of iron ions into three Kramers doublets. The E/D ratio in eq 125 determines the g-value dependence of the EPR spectrum and is related to the symmetry of the local ion environment. Atomic Force Microscopy. After completing the calorimetric scans, the βLG solutions with and without metals were deposited on freshly cleaved mica surfaces. βLG molecules were left adsorbing for 5−7 min before abundantly rinsing with purified water. After drying the samples in air overnight, the topography of the dried deposit was imaged using an Atomic Force Microscope (Nanoscope IIIa from Veeco-Bruker) operating in tapping mode with silicon cantilevers (RTESPA by Bruker), bearing a conical tip with a final radius of curvature of about 10 nm. AFM images were analyzed using the freely distributed analysis software WSXM 5.0.26 We selected isolated fibrils and fibril portions that did not show branching, splitting and sudden variations of height, and did not overlap (cross) or run closely side by side with other fibrils. The height of the substrate, z = 0, was chosen to be the average height measured in the randomly rough regions between fibrils. For each fibril we determined the height and, when the fibril showed a periodic height modulation, we calculated the fast Fourier transform (FFT) of the modulation along the fibril length to determine the period p. In this case, h was the maximum height.

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RESULTS βLG Unfolding and Fibrillation in the Absence of Metal Ions. Figure 1a shows the DSC run from 40 to 100 °C, recorded at the scan rate of 90 °C/h, for metal-free βLG solution at pH 2. The heat capacity profile showed a maximum at Tmax = 77 °C, corresponding to the transition between the native folded state and the denatured unfolded state.27 1795


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(apolar) hydrophobic protein residues that became exposed to the (polar) water solvent during protein unfolding. The heat capacity of the native state (pretransition baseline, Cp,N) showed a slight linear increase as a function of the temperature, whereas that of the denatured state (post-transition baseline, Cp,U) had a small curvature. From the plot we can estimate the difference ΔCp = Cp,U − Cp,N = 10.8 ± 0.8 kJ mol−1 °C1−, which agrees with previous DSC studies on βLG.27,28 Figure 1b shows the thermograms recorded for metal-free βLG during four consecutive heating DSC scans. In the second DSC scan, after incubation at 80 °C for 4 h, a marked exothermic behavior was observed, with Cp,app rapidly decreasing as a function of the temperature. Cp,app showed a broad minimum at a temperature Tmin of about 90 °C, followed by a gradual increase up to 120 °C, where Cp,app was close to the value measured at 30 °C. The second scan also presented a small endothermic peak at the same Tmax of the first scan, superposed to the broad exothermic peak, due to the unfolding of a residual component of monomeric folded proteins. This indicated that some proteins refolded to the native state during cooling or did not unfold during the first scan and incubation. The Cp,app traces of the following two runs showed a substantial similarity with each other, the exothermic effect being maintained and the minimum shifted to 80 °C, whereas the endothermic peak disappeared. Therefore, refolding of the native protein structure was completely abolished, while the process associated with the exothermic peak remained quite effective. βLG samples taken from the DSC after a complete set of scans were deposited on a freshly cleaved mica surface for AFM imaging (Figure 2). The images showed the coexistence on the same substrate of fibrils with different morphology, height and periodicity, in agreement with previous AFM studies of βLG incubated at high temperature.29 As reported by Mezzenga and co-workers,30 fibrils are formed by the self-assembly of aperiodic protofilaments with height h1 = 2 nm. Multistrand fibrils have a twisted ribbon morphology (Figure 2b) and can be classified according to their maximum height hn = nh1 and periodicity, where n is the number of protofilaments in the fibril.22,30 In the absence of metals, the most abundant type of fibrils were aperiodic protofilaments (Figure 2a). A few filaments of

Figure 1. (a) DSC profile of metal-free βLG at pH 2. The thermogram is corrected for the instrumental baseline and normalized for protein concentration. The molar heat capacity of the native state, Cp,N, and of the unfolded state, Cp,U, are shown together with their difference, ΔCp. (b) Effect of repeated heating runs from 10 to 120 °C for metal-free βLG at pH 2. The first run was stopped at 80 °C and the protein was incubated at this temperature for 4 h before cooling and reheating. Tmax and Tmin indicate, respectively, the maximum position of the endothermic peak and the minimum position of the exothermic peak.

The heat capacity of the unfolded state, Cp,U, was larger than the value for the folded state, Cp,N, as usually observed for small globular proteins.8 The increase of Cp was due to hydration of

Figure 2. AFM images of βLG fibrils created in metal-free solution after completing the DSC scans. (a) 1st order fibril. The height scale is 5 nm (color scale from black to white). (b) 2nd order fibril. The inset below the figure shows the twisted ribbon assembly. The height scale is 6 nm. (c) αtype protofilament. The height scale is 3 nm. In all figures, h is the maximum height of the fibril and p the distance (period) between two consecutive maxima along the fibril axis. The errors on h and p are, respectively, 0.5 and 1 nm. 1796


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Biomacromolecules the 2nd type, that is, containing n = 2 protofilaments, with an average height of 4.0 ± 0.5 nm and a period of 36 nm, were also observed (Figure 2b). Other structural details were also evident in these samples, such as 2nd type fibrils splitting or thinning, with the height of the thicker portions being almost twice as large as the thinner parts (data not shown). In addition, few filaments were found with an average height of 1 nm and no distinct periodicity (Figure 2c), formerly identified as α-type protofilaments.22 βLG Unfolding and Fibrillation in the Presence of Metal Ions. Figure 3 compares the DSC scans for metal-free

Figure 4. Comparison of the fourth calorimetric run of βLG in water at pH 2 with and without Cu2+ and Fe3+ metal ions at equimolar and 10-fold excess.

containing a number n > 2 of protofilaments were more frequently observed because Cu2+ accelerated the rate of fibril self-assembly, allowing the formation of higher order aggregates during the incubation time of 4 h.22 For instance, 3rd order right-handed twisted ribbons were frequently observed with a maximum height h = 6.5 ± 0.5 nm and a period p = 90 nm (Figure 5a). The value of p was about 20% larger than the one reported for βLG in metal-free solution, possibly due to the increased ionic strength of the metal-containing solution.31 Moreover, in the presence of Cu2+ ions at 1:10 concentration other morphologies could also be observed such as helical ribbons (Figure 5b) and tubular structures (Figure 5c), previously identified for metal-free βLG solutions incubated for longer times, higher temperature and higher concentration.29 In addition, we observed a previously unreported ladder-like structure where two fibrils, one tubular in shape, running side by side appeared to be loosely woven together (Figure 5d). The periodicity of such structure was 170 nm, and the two parallel fibrils had a maximum height of 9.0 ± 0.5 and 1.5 ± 0.5 nm, respectively. Such rich polymorphism of βLG fibrillation has been already investigated and was attributed to the association of multiple filaments into transient structures.29 AFM images of βLG in the presence of Fe3+ ions at equimolar concentration showed fibrillar structures with the same morphology found for metal-free and copper solutions at the same concentration (Figure 6). However, the surface coverage of fibrils adsorbed from Fe3+ solution was much lower than the one obtained in Cu2+ solution at 1:10 molar ratio. At this molar ratio, a few isolated fibrils were found to cover an area of 10 μm × 10 μm area for Fe3+ (Figure 6b), to be compared to about 50 fibrils observed over the same area in the presence of Cu2+ (Figure 6a). To quantify the presence of βLG fibrils in solution, ThT fluorescence measurements were done on protein samples before and after completing DSC scans (Figure 6c). The results clearly show a reduction of ThT fluorescence emission in the presence of Fe3+, which correlates well with the AFM and DSC findings. EPR Study of Metal−βLG Binding. Cu2+ and Fe3+ are paramagnetic ions (external electronic shell 3d9 and 3d5, respectively) and EPR can be used to compare the metal environment under different experimental conditions. EPR experiments were recorded at liquid nitrogen temperature for protein-free FeCl3 solutions and βLG:Fe3+ 1:10 solutions, both before and after incubation at 80 °C for 4 h (Figure 7). EPR spectrum of high-spin ferric ions is

Figure 3. Calorimetric profiles of metal-free βLG and protein solution containing CuCl2 and FeCl3 at 1:1 or 1:10 molar ratio. The thermograms were corrected for the baseline and normalized for protein concentration to give the molar heat capacity Cp.

βLG with those obtained by adding either CuCl2 or FeCl3. The presence of Cu2+ ions, either at molar ratio 1:1 or 1:10, did not alter the transition temperature Tmax but slightly reduced the relative heat capacity of the unfolded state. The area under the Cp peak, representing the enthalpy of the transition, decreased from 351 to 339 kJ mol−1 (±1 kJ mol−1), as previously reported.22 Furthermore, ΔCp was reduced to about 7 kJ mol−1 °C−1 compared to metal-free solutions, suggesting a lower internal packing density of the βLG molecule. A significantly different behavior was observed in the DSC thermogram when FeCl3 was added to the βLG solution (Figure 3). At equimolar ratio, the molar heat capacity Cp was smaller after the unfolding transition than for the native protein state. Increasing the βLG/Fe3+ molar ratio to 1:10 also decreased the transition temperature to Tmax = 75 °C and produced a severe distortion of the Cp profile. Such distortions are usually assigned to irreversible aggregation of the unfolded polypeptide.9 The DSC thermograms obtained during the fourth heating scan are compared in Figure 4. The main result is that the exothermic effect was maintained when Cu2+ was present in solution, regardless of the molar ratio. In contrast, addition of Fe3+ ions at equimolar concentration strongly reduced the exothermic effect. The Cp,app curve for βLG/Fe3+ molar ratio 1:1 (Figure 4) showed a broad minimum centered at about 70 °C, whereas no exothermic effects could be detected for 1:10 molar ratio. AFM images were obtained for the dried deposit left on mica by copper-βLG solutions at both molar ratio. In Figure 5a is shown the basic twisted ribbon fibril morphology already found for the metal-free protein solution (Figure 2b). Twisted ribbons 1797


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Figure 5. AFM images of βLG fibrils obtained after completing the DSC scans in solutions containing CuCl2 at molar ratio 1:10. The horizontal scale bar is 200 nm. (a) 3rd order twisted ribbon fibril. The height scale is 8 nm. (b) Multistrand helical ribbon. The height scale is 12 nm. (c) Multistrand assembly with tubular morphology. Color scale is 8 nm. (d) Multistrand assembly with ladder morphology. The height scale is 10 nm. The errors on h and p are, respectively, 0.5 and 1 nm. The morphology of the fibril assembly is shown below each figure.

Figure 6. (a, b) Large-area AFM images of βLG fibrils obtained after completing the DSC scans in solutions containing Cu2+ and Fe2+ ions at 1:10 molar ratio. The height scale from black to white is 6 nm. (c) Normalized fluorescence intensity of thioflavin T at wavelength 482 nm for protein-free solutions of Cu2+ and Fe3+ ions, native βLG solutions with and without metal ions, and the same βLG solutions after completing the DSC scans. The ion molar concentration was 10× the βLG concentration. The dashed line corresponds to native βLG.

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the energetics of fibrillar protein aggregates have been conducted so far.10,11,38 Interestingly, the features of the Cp,app profiles of βLG have a remarkable resemblance to the DSC thermograms of amyloid fibrils formed under comparable experimental condition (pH 2.5) by β2-microglobulin (β2m),39 a small globular protein with a β-sheet structural arrangement similar to βLG. The exothermic behavior observed in DSC scans of β2m has been identified as a signature of the thermal response of mature protein fibrils, with Tmin corresponding to the fibril melting temperature.39,40 Identifying such a similarity in the thermal response of βLG and β2m fibrils is particularly significant, because it helps clarify the thermodynamic properties and the stability of fibrillar aggregates. The origin of the exothermic behavior in the melting curves of amyloid fibrils was explained by Goto and co-workers,40 as induced by the association of hydrophobic surfaces exposed to water. Furthermore, the spatial approach among fibrils is favored by thermal agitation during heating, as it is evidenced by the decrease of Cp,app. In particular, exothermic effects are observed when fibrillation proceeds through a lag-phase or nucleation-dependent kinetic pathway, whereas an endothermic peak is observed for nucleation-independent aggregation processes. Our findings show that the character of the metal-βLG interaction is markedly different for Cu2+ and Fe3+ ions. The former does not permanently bind to βLG and plays a catalytic role in βLG fibrillation,22 increasing the nucleation rate and therefore accelerating the self-assembly of complex multistrand fibrillar structures such as high-order twisted ribbon fibrils, helical ribbons, tubular structures, and previously unreported “ladder-like” multistrand superstructures (Figure 5). The exothermic behavior and melting temperature of fibrilcontaining solutions (Tmin = 77−80 °C, Figure 4) are similar to those observed for metal-free solutions (Figure 1). At the opposite, Fe3+ binds to βLG in the aggregate state and inhibits the formation of fibrillar structures. This is evident from the reduction of Tmax observed in the DSC profile (Figure 3), the change in the line shape of the EPR spectra (Figure 7) and the absence of fibrils in AFM images (Figure 6). The depth of the exothermic peak of the Cp,app curve observed at Tmin for Cu2+ and Fe3+ solutions (Figure 4) can also be related to the different amount of fibrils in the solution (Figure 6c). Indeed, the thermal signature of fibrillar aggregation becomes weaker and completely disappear from the DSC scans as Fe3+ ions are added to the solution at 1:1 and 1:10 molar concentration (Figure 4), respectively, possibly activating a different aggregation pathway leading to less organized protein structures. Metals and other ligands that bind preferentially to the unfolded state contribute to the destabilization the native protein structure, stimulating denaturation.9 The EPR spectral features of the incubated protein-iron sample (Figure 7, line b) are very similar to that of Fe3+ complexes with phenolate- and pyridine/imidazole-containing ligands.41 In these complexes, used as models for Fe-tyrosinate proteins, the iron ion is coordinated to four nitrogen and two oxygen atoms. Furthermore, the EPR spectra of the βLG−Fe3+ complex are similar to those found for other nonheme ferric proteins such as transferrin, where the metal is in an octahedral configuration bound to five oxygen and one nitrogen atom,42 and neuromelanin.32 The preferential ligand residues of Fe3+ ions are Glu, Asp, Tyr, and Cys side chains.16 During prolonged high temperature incubation at low pH, βLG fragments are

Figure 7. EPR spectra of βLG interacting with FeCl3 at 1:10 molar ratio recorded at −196 °C (a) before and (b) after 4 h incubation at 80 °C. For comparison the spectrum of FeCl3 in water at pH 2 is also shown (c).

characterized by a broad resonance line (peak to peak line width ΔHpp = 12.4 mT) at about 150 mT, corresponding to the Landé factor g = 4.3 (Figure 7, line c; see also eq 1). This EPR line is usually found for Fe3+ complexes with low rhombic symmetry in an octahedral or tetrahedral surrounding.32,33 The resonance is assigned to the middle Kramers doublet transitions of a rhombic distorted high-spin Fe3+ complexes with 6S ground state. The zero-field splitting parameter expected for this transition is E/D ≈ 0.33.34 EPR studies have shown that Cu2+ does not bind to βLG in both the native and denaturated protein state.22 Likewise, the EPR spectrum of freshly prepared βLG/Fe3+ sample was very similar to the corresponding protein-free FeCl3 solution (compare lines a and c in Figure 7), indicating that iron cations did not bind to βLG in the native state. In contrast, the resonance line became sharper after incubation of the sample, (ΔHpp reduces to 5.7 mT), clearly indicating that iron ions did bind to βLG in the aggregate state. For comparison, we also checked that incubation of a protein-free FeCl3 solution (data not shown) did not produce any modification of the spectrum obtained before incubation (Figure 7, line c).

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DISCUSSION Fibrillation of βLG and other model proteins after prolonged incubation at high temperature and low pH is the topic of intense research.31,35−37 By combining AFM and calorimetric results obtained for metal-free βLG we observed that high temperature incubation of protein solution produces mature fibrils characterized by an exothermic response. Structured protein aggregates show a definite thermal response when heated to high temperature, with a melting temperature identified by either a maximum or a minimum value in the Cp profiles, corresponding respectively to an endothermic or exothermic peak.11 When βLG fibrils were melted at high temperature, the reassociation of the resulting depolymerized unfolded proteins and protein fragments back into fibrils during cooling was quite effective. This was evidenced by the persistence of a clear exothermic peak in consecutive thermal scans (Figure 1b), indicating the presence of organized supramolecular structures, combined with direct AFM imaging of fibrils with defined morphology (Figure 2). In spite of the enormous importance of protein fibrillation in the molecular biology research, only a few studies concerning 1799


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produced by hydrolysis. The fragments that occur more frequently in fibrils belong to the first portion of the amino acid sequence, Nseq ≤ 64, and are characterized by low charge and high number of hydrophobic residues.43 Interestingly, five Asp residues are located in this protein region at positions 11, 28, 33, 53, and 64, and may be available for Fe3+ binding, possibly interfering in the fibrillation process. The resulting effect is the almost complete inhibition of the βLG fibrillation by Fe3+ (Figure 6), which corresponds to a flat Cp,app trace at high metal concentration. It is interesting to note that ferric ion hindered ordered fibrillation of the β-amyloid 1−42 peptide,44 which contains three Asp residues. Polymorphism in protein amyloid fibrillation has also been found in other model proteins such as bovine serum albumin,45 insulin,46 and lysozyme.37 Nevertheless, the abundance and variety of structures that can be obtained for βLG is particularly noteworthy, and the possibility of using Cu2+ to assist such modulation and speed up the process is intriguing. Unfortunately, although the DSC experiments are able to reveal the formation of amyloid fibrils, the polymorphic nature of the protein samples complicates any further interpretation about the stability of such specific structures. The results of the present work may open new perspectives, including the potential use of iron to control the growth of tailored nanostructures (of βLG, as well as other proteins) in a number of applications, and the possibility to find a more comprehensive description for the energetics of formation of supramolecular protein structures.

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CONCLUSION In this article we demonstrate that transition metal ions, commonly found in senile amyloid plaques, can be used to modulate the in vitro self-assembly of model proteins such as βLG into fibrillar nanostructures. Namely, Fe3+ ions affect the protein response to denaturing conditions of high temperature and low pH, and is permanently bound to aggregated state, whereas Cu2+ ions stimulate fibrillation without binding. Our findings show that calorimetry is a valuable tool for investigating supramolecular protein aggregation, providing insights into the energetics of aggregate formation.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: rita.guzzi@fis.unical.it. Fax: +39.0984.494401. Notes

The authors declare no competing financial interest.

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REFERENCES

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