Soft Matter

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aCNR-IPCF, UOS di Cosenza, LICRYL and C

Cubo 33/B, Rende 87036, Italy. E-mail: brunbUniversita della Calabria, Dipartimento dicUnita CNISM, Universita della Calabria, C

Cite this: Soft Matter, 2013, 9, 2412

Received 18th October 2012Accepted 27th November 2012

DOI: 10.1039/c2sm27408f

www.rsc.org/softmatter

2412 | Soft Matter, 2013, 9, 2412–24

Catalytic activity of copper ions in the amyloidfibrillation of b-lactoglobulin

Bruno Zappone,*a Maria P. De Santo,b Cristina Labate,b Bruno Rizzutia

and Rita Guzzi*bc

The self-assembly of proteins and polypeptides in amyloid fibrillar aggregates is rapidly emerging as a

promising route towards the fabrication of nano-objects with controlled morphologies and properties.

Transition metal ions are known to play an important but elusive role in the amyloid fibrillation

associated with neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. We have

considered the effect of copper ions Cu2+ on the nanoscale morphology and fibrillation kinetics of

b-lactoglobulin (bLG), a model protein not related to amyloid diseases, which denatures and self-

assembles in nanofibrils upon heating at low pH and ionic strength. We found that an increasing level

of Cu2+ decreases the enthalpy of denaturation and significantly increases the rate of fibril nucleation,

also producing a small increase of the fibril elongation rate. Cu2+ acts as a catalytic agent during protein

denaturation and fibrillation, without binding to bLG before or after heating, and produces only minor

changes in fibril morphology. Beside possible implications for amyloid pathologies in vivo, our results

suggest that transition metal ions can be used to control the self-assembly of protein-based nano-

objects in vitro.

Introduction

Amyloid aggregation – the self-assembly of proteins in nano-brils stabilized by cross-b interactions1 – has long been asso-ciated with neurodegenerative disorders such as Alzheimer's,Parkinson's, Huntington's and Creutzfeldt–Jakob diseases.2

However, the discovery of (non-pathological) functional roles ofamyloid brils in biological processes3–5 has also opened a newperspective in materials science and nanotechnology: creatingfunctional nanostructured materials from the amyloid linearself-assembly of ‘design’ proteins and polypeptide units.6 Thenon-specic character of cross-b interactions, directly involvingthe polypeptide main chain common to all proteins rather thanthe specic sequence of amino acid side chains, makes this ideaappealing to scientists working in the broad eld of somatter,not necessarily restricted to biochemistry or medicine.

The key to develop amyloid-based nanostructures is to be ableto control the aggregation kinetics, stability and morphology ofthe brils by playing on a number of factors such as the aminoacid sequence, protein concentration, solvent properties (type,pH, and ionic strength), temperature and interactions with co-solutes and surfaces.7 There is strong evidence that transitionmetals, especially copper ions Cu2+, play a crucial role in the

emif.Cal, c/o Universita della Calabria,

o.zappone@cnr.it; rita.guzzi@s.unical.it

Fisica, Cubo 31/C, Rende 87036, Italy

ubo 31/C, Rende 87036 (CS), Italy

19

brillation of disease-related proteins in vivo.8High copper levelsare found in the brous plaques characteristic of Alzheimer'sand Parkinson's diseases9 and a high copper concentration inthe blood is associated with an increased risk of Creutzfeldt–Jakob disease.10 However, it is not clear whether metal ionsstimulate protein unfolding, promote bril aggregation orcombine with other biological molecules to increase oxidativestress and precipitation of the protein.8 Studies conducted invitro on disease-related proteins have not yet claried thescenario. For instance, copper is known to promote the aggre-gation of short oligomers and the brillation of a-synuclein(related to Parkinson's disease)11,12 but inhibits brillation ofamyloid-b (related to Alzheimer's disease)13–15 by favoringamorphous aggregation16 due to excessive ion bridging.17

In this work, we have considered the effect of copper ions onthe brillation of bovine b-lactoglobulin (bLG), a protein that isnot related to any amyloid disease and yet self-assembles intobrils with well-denedmorphology.18 bLG has been extensivelystudied owing to its abundance in cow milk whey, ease ofpurication and relevance to the production of diary food,making it an ideal ‘building block’ for studying the generalmolecular mechanisms underlying amyloid aggregation. bLGconsists of 162 amino acid residues, with a molecular weight ofabout 18 kDa and a globular shape with a radius of about1.5 nm.19 Under physiological conditions (pH z 6.5, salinity z10 mM, temperature z 38 �C), native bLG is mainly found indimers and binds Cu2+ ions.20 At low salinity and pH < 3, bLGbecomes predominantly monomeric while keeping its native

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Fig. 1 Model of periodic multi-strand fibril produced by the aggregation ofaperiodic protofilaments: fibrils of the nth order are twisted bundles containing nprotofilaments.18 h and pn are respectively the thickness of a protofilament (1st

order) and the period of nth order fibrils.

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folding.21 Under such conditions, denaturation by heating in ametal-free solution leads to the formation of protolamentswith nm-sized diameter which can further aggregate to formtwisted ‘ribbon-like’ brils of higher order (Fig. 1) depending onthe monomer concentration, denaturation temperature andincubation time.22–24 Fibrillation proceeds from bLG moleculesand small hydrolyzed fragments of various lengths25,26 throughthree different processes: nucleation of seeds, followed by brilelongation and 'thickening' into higher order brils.22

Using Differential Scanning Calorimetry (DSC), opticalspectrophotometry, Electronic Paramagnetic Resonance (EPR)and Atomic Force Microscopy (AFM) we have found clearevidence that the presence of Cu2+ ions in solution at pH 2favors the denaturation of the protein upon heating, shortensthe “lag time” associated with seed nucleation, and acceleratesthe elongation and thickening of brils without producingbranching. Interestingly, EPR showed that copper, despitedeeply affecting bLG denaturation and aggregation, does notbind permanently to protein monomers or fragments, seeds ormature brils, and does not signicantly alter the brilmorphology at pH 2. Therefore, copper acts as a catalytic agentduring the process going from protein denaturation to brilnucleation and elongation. Copper ions and, possibly, othertransition metals may provide efficient control of the aggrega-tion of brils and other proteic brillar nano-objects producedin vitro.

Materials and methodsProtein solutions

Bovine bLG (variant A, from Sigma-Aldrich) with a molecularweight of 18.3 kDa was dissolved in puried water (Millipore Q,

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resistivity 18.2 MU$cm at 25 �C), adjusted to pH 2 by addingHCl. The protein was solubilised by stirring for 1.5 h at 4 �C witha Teon-coated magnetic stir-bar on a stir-plate set to 200 rpm.The solution was then dialyzed for 3 days against the samesolvent using a Spectra-Por membrane with a molecular weightcut-off of 3.5 kDa. The dialysis membranes were prepared asin ref. 26. The solvent volume outside the membrane was 100times the volume inside the membrane. Aer dialysis, theprotein concentration was in the mM range, as measured byUV-Vis spectrophotometry using an extinction coefficient 3278 ¼17600 M�1 cm�1. When present in solution, CuCl2 was addedwith the same molar concentration as the bLG protein(bLG : Cu2+ ¼ 1 : 1, equimolar solution), or ten times thisconcentration (bLG : Cu2+ ¼ 1 : 10).

Differential scanning calorimetry

Differential scanning calorimetry (DSC) measurements wereperformed on a VP-DSC MicroCalorimeter (MicroCal, Inc.) witha cell volume of 0.52 mL and a temperature resolution of 0.1 �C.To determine the baseline, we carried out at least four scanswith pure solvent at pH 2 in both the sample and reference cells.Protein samples at a concentration of 0.05 mM were extensivelydegassed before measurements and le equilibrating in thecalorimeter cell for 30 min at 20 �C before starting a measure-ment. DSC scans were obtained by heating the samples from20 to 100 �C at a rate of 90 �C h�1. The reversibility of thethermal transitions was checked by decreasing the temperatureto 20 �C before rescanning the sample. The data were analysedusing the Origin soware package (MicroCal). The solventbaseline was subtracted from the protein/solvent scan and thedata were normalized by the protein concentration. Finally, thepre- and post-transition baselines were tted by a cubic func-tion to obtain the excess heat capacity, Cpex.

Electron paramagnetic resonance

Conventional EPR spectra were recorded for 0.1 mM solutionsof bLG in puried water at pH 2 containing CuCl2 at 1 : 1 or1 : 10 molar ratios, and for protein-free CuCl2 reference solu-tions at the same concentrations and pH. The samples wereplaced in EPR-grade quartz tubes (inner diameter 4 mm),heated for 4 h at 80 �C to induce protein denaturation andbrillation24 (for protein solutions), then rapidly frozen byplunging the tube into a nger Dewar containing liquidnitrogen. Finally, the samples were inserted into a standardrectangular cavity (Bruker ER4201 TE102) for EPR measure-ments at a temperature of �196 �C. The measurements wereperformed on an ESP 300 X-band spectrometer (Bruker, Karls-ruhe, Germany) equipped with an ESP 1600 data acquisitionsystem.

Absorption spectroscopy and turbidimetry

Protein concentration was measured by UV-Vis absorptionspectroscopy at room temperature in 1 cm path length quartzcuvettes using a JASCO 7850 spectrophotometer. The turbidityof 0.05 mM bLG solutions with or without Cu2+ was determinedbymeasuring light attenuation due to scattering at a wavelength

Soft Matter, 2013, 9, 2412–2419 | 2413

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of 400 nm that is not absorbed by bLG. The turbidity wasmeasured as a function of time while heating the proteinsolution at a constant temperature of 80 �C (accuracy � 0.5 �C)in a TPU-436 Peltier thermostated cell holder. The samples wereinserted in the pre-heated cell holder at time zero and reachedthermal stability in about 1 minute.

Fig. 2 DSC scans of bLG in purified water at pH 2 in the absence of metal ions(black line) and in the presence of Cu2+ ions at equimolar concentration (blue line)or ten times the equimolar concentration (red line). Cpex is the excess heatcapacity. The scan rate was 90 �C h�1.

Atomic force microscopy

Diluted bLG solutions (1 mM), free of metals or containing Cu2+

at 1 : 1: and 1 : 10 molar ratios, were heated in glass vials for 4hours at 80 �C at pH 2. Aer incubation, the protein solutionwas further diluted to a nal concentration of 0.05 mM usingpuried water at pH 2. Droplets of such a solution weredeposited on a freshly cleaved mica surface to completely coverthe surface so as to avoid the formation of a liquid–mica–aircontact line where the protein solute may accumulate due tothe “coffee stain” effect.27 We let the solutes adsorb for 7–10minutes before abundantly rinsing with puried water. Thesample was then le drying in air overnight before starting theAFM imaging of the deposit le on the surface. To avoidcontamination by ambient particles and molecules, sampleswere prepared in a clean laminar ow hood. We obtainedtopographic images of the sample surface in air using a Multi-mode AFM (Veeco-Bruker) operating in tapping mode. We usedAFM silicon cantilevers (RTESPA by Veeco, resonance frequencyaround 300 kHz) bearing a conical tip with a nal radius ofcurvature of about 10 nm.

To determine the bril height and periodicity, the AFMimages were analyzed by using the freely distributed analysissoware AfmFibrils (by Bart Vermolen, University of Twente),running under MATLAB. We selected isolated brils orcontinuous bril portions that did not overlap (cross) or runclosely side by side with other brils, and did not split. Theprogram automatically interpolated a continuous line along thelongitudinal axis of the bril (portion) to determine the heightvariation z(s) as a function of the distance s along the line. Thebaseline, z¼ 0, was chosen to be the average height measured inthe randomly rough empty regions between brils. For eachbril we determined its length l ¼ Ð

sds and thickness (averagedheight) h ¼ (1/l)

Ðz(s)ds. When the bril (portion) showed a

periodic modulation of height, we calculated the fast Fouriertransform (FFT) of z(s) and identied the peak whose spatialfrequency corresponded to the period p. From AFM images wecould also measure the lateral width w of a bril, e.g., as thecross-sectional width of a bril at height h/2. However, suchmeasurement is known to overestimate the real value of w dueto the effect of shape convolution between the apex of the AFMconical tip and the bril,28 and it was not pursued further.

Fig. 3 EPR spectra, recorded at �196 �C, for a solution of bLG in purified water(pH 2) containing Cu2+ at equimolar concentration (line a) before and (line b)after incubation at 80 �C for 4 h. Line c is the spectrum obtained for a protein-freesolution of CuCl2 in purified water at the same pH. The spectra have been shiftedvertically for clarity.

ResultsbLG denaturation and brillation in solution with copperions

Calorimetry is a powerful technique to obtain informationabout the conformational stability of a protein in solution.Fig. 2 shows DSC scans obtained for metal-free bLG and

2414 | Soft Matter, 2013, 9, 2412–2419

bLG : Cu2+ solutions. The excess heat capacity, Cpex, showed anendothermic peak at a temperature Tm ¼ 76.6 �C, regardless ofthe presence of Cu2+ ions. Such peak can be described by a two-state reversible process of protein denaturation N4 U betweenthe native, N, and unfolded state of the protein, U, which iscommon to many single-domain proteins at low pH.29 Theenthalpy of the transition, corresponding to the area under theCpex curve in Fig. 2, was higher for pure bLG solution (DH ¼351� 1 kJ) than for bLG solutions containing Cu2+ (DH ¼ 339�1 kJ). The curve obtained for equimolar bLG : Cu2+ solutionoverlapped with the curve obtained for a solution at a 1 : 10molar ratio. These results indicate that the protein structure

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Fig. 4 Absorbance due to scattering as a function of the heating time for asolution of bLG in purified water (pH 2) heated at a constant temperature of80 �C. Black squares, blue dots and red triangles indicate respectively pure bLGsolution, bLG : Cu2+ solution at equimolar concentration and bLG : Cu2+ solutionwith a 1 : 10 molar ratio. Data dispersion was contained within the shaded area.

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was destabilized by the interaction with Cu2+ ions regardless ofthe concentration and are in line with the results obtained athigher pH.20 EPR spectroscopy (Fig. 3) provides further insightinto metal–protein interactions, as the paramagnetic resonanceof Cu2+ ions is very sensitive to the surrounding environment,particularly to protein complexation. EPR spectra of equimolarbLG : Cu2+ solutions recorded at �196 �C before (Fig. 3, line a)and aer (Fig. 3, line b) 4 h incubation at 80 �C were comparedto the spectrum obtained for (protein-free) CuCl2 aqueoussolutions.

EPR spectra allowed us to evaluate the characteristicmagnetic parameters of the spin Hamiltonian: the componentsof the g tensor for the coupling between electron spin andmagnetic eld, and the components of the A tensor for thehyperne coupling between electron spin and nuclear spin (I ¼3/2). All spectra in Fig. 3 are characteristic of a Cu2+–H2Ocomplex with an axial symmetry (gzz ¼ gt > gxx z gyy ¼ g||): fourhyperne resonance peaks in the parallel region (low magneticeld) centered at g|| ¼ 2.336 and separated by a hypernecoupling constant A|| ¼ 15 mT.30,31 The peak asymmetry in theparallel region is due to structural heterogeneity of the coppercomplexes induced during the freezing of the samples.30 Theperpendicular region around 316 mT contains four hypernelines that overlap due to the small values of the hypernecoupling constant, with gt ¼ 2.089. According to the A|| vs. g||correlation map for copper complexes, such values of themagnetic parameters are consistent with four oxygen atomsinside the metal coordination sphere.32 Heating of the equi-molar bLG : Cu2+ sample during incubation reduces the struc-tural heterogeneity of the copper complexes resulting in aslightly different overlap aer re-freezing, more evident in theperpendicular region (Fig. 3). The use of cryprotectant agentssuch as glucose or glycerol avoids freezing-induced inhomoge-neity,31 but it would also affect the aggregation process.33 Thesimilarity of the spectra indicates that Cu2+ ions do not bindpermanently to bLGmolecules, fragments or aggregates neitherbefore nor aer incubation at 80 �C.

The kinetics of aggregation was followed by measuring theturbidity of the protein solutions. Fig. 4 shows the time-dependent absorbance of copper-free bLG solutions andbLG : Cu2+ solutions heated to a constant temperature of 80 �C,higher than Tm. The trend is similar and non-monotonic, with aminimum depending on the Cu2+ concentration. In the absenceof copper, the absorbance decreased for the rst 35 min,reached a minimum and started increasing again aer about50 min, eventually reaching an equilibrium value aer about150 min. In equimolar bLG : Cu2+ solutions, the minimumwas reached aer just a few minutes and was much narrower(short-lived) and shallower than for copper-free solutions.Moreover, the equilibrium absorbance was signicantly higherthan for copper-free solutions. In bLG : Cu2+ solutions at a 1 : 10molar ratio, a deeper minimum was reached at about the sametime as for equimolar solutions, but the rate of recovery wasslightly faster than for equimolar solutions. Aer 4 h of incu-bation, the absorbance was higher than for a copper-free andequimolar solution, and did not appear to have reached equi-librium yet.

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Such non-monotonic variation of the absorbance was due tomultiple processes of protein denaturation and aggregation,occurring sequentially or simultaneously, all of which wereaffected by the presence and concentration of Cu2+ ions. Thedecrease of absorbance at short incubation times is most likelydue to protein hydrolysis caused by the combination of hightemperature and low pH.25,26,34–36 The scattering cross-section ofa molecule with size d, much smaller than the optical wave-length l, is proportional to d6/l4. Therefore, the hydrolyzedfragments of a molecule scatter much less than the wholemolecule, lowering the absorbance of the solution. On the otherhand, the increase of absorbance at longer incubation times(Fig. 4) was due to aggregation of bLGmolecules and fragments.Increasing size of the scattering objects increases the absor-bance. At the minimum of absorbance, the effect of proteinaggregation balances the effect of hydrolysis. In metal-freesolutions, the growth of bLG brils is known to be limited by therate of self-assembly of oligomeric nuclei (seeds), whichproduces a characteristic “lag phase” in the early steps of brilgrowth and is observed as a delay between the beginning ofdenaturation and the rise of optical absorbance (Fig. 4).37 In thepresence of copper, such a delay was much shorter than in ametal-free solution and too rapid to be correctly measured,indicating that the lag phase was signicantly reduced.

AFM images of the protein deposit le on a mica substrateshow that bLG aggregates formed in metal-free solutions(Fig. 5a), equimolar bLG : Cu2+ solutions (Fig. 5b) andbLG : Cu2+ solutions at a 1 : 10 molar ratio (Fig. 5c) werepredominantly brillar and unbranched, with length widelydispersed l and different maximum values lmax (Table 1).Moreover, increasing the concentration of Cu2+ ions evidentlystimulated the rate of nucleation, as the number N of brils ofany length per unit area Amonotonically increased as the molarratio of Cu2+ over bLG increased. This result agrees with theshorter delay time and the overall faster rate of aggregation

Soft Matter, 2013, 9, 2412–2419 | 2415

Fig. 5 AFM images of fibrils obtained after 4 h incubation at 80 �C of (a) pure bLG solution, (b) bLG : Cu2+ equimolar solution and (c) bLG : Cu2+ solution with a 1 : 10molar ratio. The height scale was 10 nm.

Table 1 Surface density andmorphology of fibrils for different copper concentrations.N is the number of fibrils, A is the image area, L is the sum of fibril lengths, lmax isthe maximum fibril length, and hn is the height of the nth order fibril. Statistics for metal-free, 1 : 1 and 1 : 10 bLG : Cu2+ solutions are based on 40, 57 and 41 fibrils,respectively.

Solution N/A (mm�2) L/A (mm�2) L/N (mm) lmax (mm) h1 (nm) h2 (nm)

Metal-free 0.60 � 0.05 0.5 � 0.1 0.8 � 0.1 2.30 � 0.01 2.0 � 0.4 4.0 � 0.5bLG : Cu2+ 1 : 1 5.4 � 0.1 8 � 1 1.5 � 0.5 6.80 � 0.01 2.0 � 0.3 4.0 � 0.5bLG : Cu2+ 1 : 10 11.4 � 0.1 16 � 1 1.4 � 0.5 4.90 � 0.01 3.1 � 0.1 5.0 � 0.5

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observed in the presence of copper ions (Fig. 4). The maximumlength lmax (end-to-end or partial length measured among alllaments captured in AFM images), the cumulative length perunit area L/A ¼ Sli/A and the average length L/N of the laments(including partial lengths) signicantly increased going frommetal-free to equimolar copper solutions (Table 1).

This may be due to a faster elongation rate, a shorter lagphase (allowing brils to elongate for a longer time) or acombination of both. A tenfold increase of Cu2+ concentrationfrom the equimolar value did not increase lmax or L/N, indi-cating that the elongation rate was less sensitive to copper levelthan the nucleation rate. However, shear forces generated uponpipetting the protein solution may also cause the breakage oflong brils, thereby limiting the values of lmax and L/N.

Nanoscale bril morphology

Fig. 6a–b show AFM images of brils adsorbed on mica from ametal-free bLG solution. We notice two distinct brilmorphologies with thickness h1 ¼ 2.0 � 0.4 nm for the 1st typeand double this value, h2 ¼ 4.0� 0.5 nm for the 2nd type (Fig. 6cand Table 1). Fibrils of the 1st type showed a random modula-tion of height z along the longitudinal axis, comparable to theroughness of the substrate. Fibrils of the 2nd type showed aperiodic height modulation with period p2¼ 64� 1 nm and hada characteristic le-handed helicoidal appearance. The lattertype of bril was less frequently observed than the 1st type(Fig. 6c). The thickness was about 4 nm at points where twobrils of the 1st type crossed each other and about 6 nm at the

2416 | Soft Matter, 2013, 9, 2412–2419

crossing between the 1st and 2nd type brils. These ndingsagree very well with similar AFM observations of bLG brilsproduced by heat-denaturation at 90 �C, pH 2 and low ionicstrength (Fig. 1).18,22,38,39 Following the model by Adamciket al.,22 we identify bril of the 1st type as protolaments ofthickness h ¼ 2 nm, and brils of the 2nd type as double-strandbrils formed by two protolaments paired side to side andtwisted to form a sort of helicoidal ‘nano-ribbon’ (Fig. 1). Animmediate consequence of such a model is that the thickness ofbrils of nth order is hn ¼ nh and the thickness of a crossingbetween brils of orders p and q is hp,q¼ (p + q)h, as we observedexperimentally.

Fig. 7a–c shows the brils obtained in the bLG solutioncontaining Cu2+ ions at equimolar concentration. We observedthree types of brils corresponding to the 1st (protolament),2nd and 3rd order with thicknesses h1 ¼ 2.0� 0.3 nm, h2 ¼ 4.0�0.5 nm and h3 ¼ 6.0� 0.1 nm, respectively (Fig. 7d and Table 1).The majority of brils were of the 1st order, but the relativeabundance of 2nd order brils increased compared to metal-freesolutions and we observed two 3rd order brils. The period of2nd order brils was p2 ¼ 66 � 1 nm, close to the value found inthe absence of salt.22 A period p3 z 70 nm could be measuredfor only one 3rd order bril and, interestingly, was much closerto p2 than the corresponding value reported in the absence ofsalt,22 i.e. p3 ¼ 2p2. Possibly, such bril was formed by curling aprotolament around a preformed 2nd order bril of period p2,rather than by twisting a 3rd order at nano-ribbon. We alsoobserved a new type of aperiodic laments with thicknessaround 1 nm (labelled a in Fig. 7c), which may be due to the

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Fig. 6 (a and b) AFM images of fibrils of 1st and 2nd order obtained by heat-denaturation of bLG without metal ions and deposited on a mica surface. Theheight scale was 6 nm in all figures. (c) Distribution of heights h.

Fig. 7 (a and b) AFM images of bLG fibrils of the 1st, 2nd and 3rd order obtainedby heat-denaturation in the presence of Cu2+ at equimolar concentration. (b) alsoshows a crossing between the filament of 2nd and 3rd order. (c) Thin fibril of the a

type. The height scale was 10 nm. (d) Distribution of heights h.

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copper-promoted brillation from small seeds that would notallow elongation into 1st order brils in the absence of copper.

Fig. 8 and Table 1 show the morphology and height distri-bution of brils obtained in bLG : Cu2+ solutions at a 1 : 10molar ratio. Also in this case, we identied brils of the 1st and2nd order as well as laments of a type. However, the thicknessof all bril types, including a, was larger than that obtained atzero and equimolar concentration of Cu2+ : h1 ¼ 3.1 � 0.1 nm,h2¼ 5.0� 0.1 nm and ha¼ 1.4� 0.1 nm. The period of 2nd orderbrils was p2 ¼ 66 � 2 nm, equal to the value measured forequimolar solutions.

Fibrils appeared to be deposited on a randomly roughbackground with a RMS roughness of 0.35 nm (metal-freesolutions), 0.27 nm (bLG : Cu2+ ¼ 1 : 1) or 0.14 nm(bLG : Cu2+ ¼ 1 : 10), whereas the roughness of a freshly cleavedmica substrate is lower than 0.1 nm.40 Such a rough backgroundis due to the adsorption of highly mobile molecular species withlow molecular weight present in the solution, including bLGmonomers and hydrolyzed protein fragments produced duringheat denaturation.25,26,34,35 The penetration of brils in therough background was negligible, otherwise the thicknesses hnand hp,q would have been offset from a multiple of h by thedepth of penetration. Moreover, protolaments were notsignicantly attened or deformed by the interactions with thesurface or other laments in multi-strand brils, otherwise thethicknesses hn and hp,q would have been offset by a value equalto the deformation. The apparent lateral width w on the AFM

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images of the brils also monotonously increased with the brilorder, but we could not determine the real width due to tip–sample shape convolution effects.

Finally, we point out that the thicknesses ha and h1 (Table 1)were always smaller than the diameter of 3 nm of the native(monomeric) bLG molecule.19 Proteins were strongly deformed(unfolded) from their native shape aer aggregation, or brilswere made of sub-monomeric fragments produced by hydro-lysis during denaturation, or both. Notice that protein hydro-lysis into shorter peptides is not an essential requirement forthe formation of bLG brils, whereas protein denaturation is.7

Discussion

Copper is known to play a key role during heat denaturation ofbLG at neutral pH. Previous EPR measurements have shownthat the preferential binding of Cu2+ ions to histidine residuesleads to a decrease of the denaturation temperature by 10 �C,whereas Zn2+ has a much weaker inuence.20 However, thiseffect does not appear to be relevant at pH 2, since EPR spectrawere unable to reveal any signicant binding of Cu2+ ions(Fig. 3) because both nitrogen atoms of histidine are protonatedand unavailable for binding.

Under denaturing conditions (heating), bLG protein mono-mers can form intermolecular disulphide bonds (–SS–) due tothe presence of a free sulphydryl group (–SH) of the Cys121

Soft Matter, 2013, 9, 2412–2419 | 2417

Fig. 8 (a–c) AFM images of fibrils obtained by heat-denaturation from abLG : Cu2+ solution with a 1 : 10 molar ratio. The height scale was 10 nm. (d)Distribution of heights h.

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residue and two intramolecular disulde bonds. Disuldebonds can be created by oxidation of two free sulphydryl groupsor transferred to a neighbouring sulphydryl group by sul-phydryl–disulde exchange reaction.41,42 Upon heating atneutral pH, copper ions specically promote sulphydryl oxida-tion over the exchange,43 without binding to the protein (Fig. 3).Under our experimental conditions, copper has the effect ofdestabilizing the protein monomer leading to a smallerenthalpy of denaturation (Fig. 2). Although our results do notprovide a clear understanding of the specic mechanisms ofcopper–bLG interaction, they indicate that the catalytic effect ofcopper on sulphydryl oxidation and disulphide exchange reac-tions deserves particular attention. Copper may destabilize bLGat pH 2 by interacting with the Cys121 exposed to the proteinsurface during denaturation, initiating sulphydryl oxidationand/or a chain of sulphydryl–disulphide exchange reactions.

The effect of copper is not limited to the denaturation phase(unfolding and hydrolysis) but was observed throughout theentire process of bLG aggregation, increasing the nucleationrate and shortening the lag phase, possibly increasing also theelongation rate (Fig. 4 and Table 1). Through the formation ofintermolecular disulde bridges, copper produces bLG aggre-gates of different size and shape depending on the conditionsduring heating (pH and type/concentration of salt),44 whichleads to the conversion of proteins into nanobrils.36 The effectof Cu2+ on the thickening rate is subtler and more difficult tostudy due to the infrequent occurrence of higher order brils.Addition of Cu2+ at equimolar concentration increased the

2418 | Soft Matter, 2013, 9, 2412–2419

abundance of 2nd order brils relative to 1st order brils andallowed the formation of a few 3rd order brils (Fig. 7). On theother hand, a tenfold increase of Cu2+ decreased the relativeabundance of 2nd order brils and eliminated 3rd order brils(Fig. 8). Notice that ‘mixed’ brils, such as 2nd order brilsending with 1st order ‘tails’ or having a 1st order segment in themiddle, were rarely observed. This indicates that newly elon-gated portions of a 1st order bril were immediately thickenedby addition of elementary units,45 rather than by pairing of twoindependently formed 1st order protolaments. Indeed, in thelatter case the pairing of protolaments with widely dispersedlengths l would frequently create mixed laments with limitedportions of the 2nd type and many tails and holes of the 1st type.

A number of considerations hint at a distinctive, rather thanaspecic, role of copper ions in the brillation of bLG. An excesscharge of +20e is present on bLG molecules at pH 2,21 favoringelectrostatic repulsion for separations larger than one Debyelength (about 3 nm).46 Increasing the ionic strength of oursolutions by adding 1–10 mM CuCl2 weakened the electrostaticbarrier by decreasing the Debye length and the protein chargedue to binding of Cl� counterions. However, the effect of CuCl2on the aggregation of bLG at low pH, even at a concentrationbelow 1 mM, is known to be dominant over an increase of NaClconcentration orders of magnitude higher.43 Therefore, at theionic strength of our experiments, Cl� anions and “double-layer” (i.e. mean-eld) electrostatic interactions are not expec-ted to play a relevant role. Instead, the brillation process ismostly driven by attractive forces acting at a relatively shortscale among contacting protein residues, such as disulphideand hydrogen bonds, van der Waals interactions, local electro-static attractions between charged residues, and other specicinteractions.47

Most likely, the effect of copper is not merely due to thepresence of a generic divalent cation that favors the formationof ion bridges. Cations of the IIa group (Mg2+, Ca2+ and Ba2+) areknown to speed up the aggregation of bLG and other wheyproteins more effectively than cations of the Ia group (Na+, Li+,and K+), but they also favor the formation of shorter and moreexible nanobrils than the ones produced without salt.24,48

Previous observations also suggest that metal Zn2+ ions affectthe time evolution and size of heat-induced bLG aggregatesdifferently from Cu2+, although experiments were performed atdifferent temperature, concentration, pH and ionic strength.20,49

Such difference may be related to the ability of Cu2+ to act as acatalyst in the sulphydryl oxidation, whereas Zn2+ does not.42

So far, the consequences of the presence of metallic cationson the aggregate morphology have been less investigated thanthe behavior in solution and further studies will be needed toaddress this issue. Our preliminary AFM observations (notshown here) indicate that brils created in equimolarbLG : Zn2+ and bLG : Fe2+ solutions are shorter and less abun-dant than for bLG : Cu2+ solutions, in line with previous reportson disease-related proteins showing a strong dependence ofbrillation rates on the specic type of metal ion.8–10,12–17 Futurework will contribute to clarify these aspects and may provideadditional ways to control the size and shape of the brillaraggregates produced.

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Acknowledgements

We thank the group of Prof. Vinod Subramaniam at theUniversity of Twente in Netherlands for helping us with thesoware analysis of the bril properties. C. Labate has beensupported by the European Commission and the CalabriaRegion through the European Social Fund. M. P. De Santowishes to acknowledge the nancial support from the EuropeanUnion programme FP7-PEOPLE-2009-IEF (project # 253087).

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