Aggregation Behavior of Bovine κ- and β-Casein Studied with Small Angle Neutron Scattering, Light...

Aggregation Behavior of Bovine κ- and β‑Casein Studied with SmallAngle Neutron Scattering, Light Scattering, and CryogenicTransmission Electron MicroscopySofie Ossowski,*,†,‡ Andrew Jackson,‡ Marc Obiols-Rabasa,† Carl Holt,# Samuel Lenton,§,⊥

Lionel Porcar,§ Marie Paulsson,∥ and Tommy Nylander*,†

†Division of Physical Chemistry, and ∥Department of Food Technology, Engineering and Nutrition, Lund University, P.O. Box 124,SE-221 00 Lund, Sweden‡European Spallation Source ESS AB, Box 176, 221 00 Lund, Sweden#School of Life Sciences, University of Glasgow, Glasgow, G12 8QQ, United Kingdom§Institute Laue-Langevin, BP 1566 rue Jules Horowitz, 380 42 Cedex 9 Grenoble, France⊥EPSAM, Keele University, Keele, Staffordshire ST5 5BG, England

ABSTRACT: In the native bovine casein micelle the calciumsensitive caseins (αS1-, αS2- and β-casein) sequester amorphouscalcium phosphate in nanometer-sized clusters, whereas thecalcium-insensitive κ-casein limits the growth of the micelle. Inthis paper, we further investigate the self-association of κ- andβ-casein, which are two of the key proteins that control thesubstructure of the milk casein micelle, using neutron and lightscattering techniques and cryogenic transmission electronmicroscopy. Results demonstrate that κ-casein can, apart fromthe known self-assembly, form amyloid-like fibrils already at temperatures of 25 °C when subject to agitation. This extendedaggregation behavior of κ-casein is inhibited by β-casein, as reported by others. These findings have implications for the structureand stability of casein micelles. The neutron scattering data was used to gain information on the self-assembly structure of κ-casein. β-Casein shows similar self-association behavior as κ-casein, but unlike κ-casein, the self-association exhibits temperaturedependence within the studied temperatures (6 and 25 °C). Here, we will discuss our extended study of the known self-assemblyof casein in the context of the fibrillation of κ-casein.

1. INTRODUCTIONThe biological function of the casein micelles in milk is toprovide nutritious milk containing high concentrations ofcasein, calcium, and phosphate for the neonate without causingpathological calcification or amyloidosis of the mother'smammary gland.1−3 Casein micelles and their substructurehave been studied for decades and a number of different modelshave been proposed including the Submicelle model4,5 and theCalcium Phosphate Nanocluster model,6−8 recently reviewedby Dalgleish.9 But as yet, the details of the substructure are notfully resolved.Casein micelles form through self-association of caseins and

calcium phosphate, and can be regarded as lyophilic colloidsthat undergo physical changes such as aggregation if conditionsare changed.10,11 The main proteins constituting the caseinmicelle are αS1-, αS2-, β-, and κ-casein, together with calciumphosphate in the form of nanoclusters.1,6,7,11 αS1-, αS2, and β-Caseins are calcium binding proteins, whereas κ-casein is lesscalcium sensitive and is believed to be responsible for colloidalstability of the casein micelle system.9,11 The calcium-sensitivecaseins contain sequences with a high degree of phosphorylatedresidues, known as phosphate centers (PC). It is through thesePCs that the caseins can sequester amorphous calcium

phosphate stabilizing the calcium phosphate nanoclusters(CPN).1,6,7,11−15 The casein micelle has an open hydratedstructure with a radius of 30−300 nm.16,17 In this paper, wehave focused on some new observations regarding thefibrillation of κ-casein in relation to the self-association behaviorof the calcium-binding β-casein and the calcium-insensitive κ-casein, with the aim to include the calcium-sensitive αS1- andαS2-casein in future studies. The reason for focusing on the β-casein is that this is the most studied of the calcium sensitivecaseins with respect to self-assembly, temperature, and calciumbinding.1,6,11,18−21 Furthermore, it was previously reported thatβ-casein could inhibit κ-casein fibrillation,22,23 and it wastherefore of prime interest to us.κ-Casein self-assembles into nearly spherical particles

consisting of a hydrophilic core and more flexible hydrophilicouter layer, with an average molecular weight (MW) of 630kDa and average diameter of 13−18 nm at 25 °C.24,25 The κ-casein monomer has a MW of 19 kDa and a radius of 3−4nm.26 κ-Casein can be divided into two fragments, para κ-casein

Received: June 14, 2012Revised: August 26, 2012Published: August 27, 2012

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© 2012 American Chemical Society 13577 dx.doi.org/10.1021/la302416p | Langmuir 2012, 28, 13577−13589

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and the caseinomacropeptide (CMP), since addition of theenzyme chymosin cleaves at the Phe(106)-Met(107) bond andcuts off the more hydrophilic CMP part.11 The para κ-caseinpart consists of the first 105 amino acid residues including thetwo cysteine residues,27,28 through which intramoleculardisulfide bonding can lead to oligomerization.11,25 Thecaseinomacropeptide (CMP) part consists of the 64 C-terminalamino acids, which contains the carbohydrate moiety andamino acids subject to genetic substitutions.9,27,28 The CMP,being the most hydrophilic part of κ-casein, is believed to belocated on the surface of micellar κ-casein aggregates as well ason the surface of the casein micelles,29,30 extending some 5−10nm from the surface and thus providing steric stabilization ofthe micelles.9,30 At concentrations above the critical micelleconcentration (CMC), reported to be about 0.5 mg/mL, κ-casein self-associates.28

Native κ-caseins has been reported to polymerize extensivelyat 37 °C.22,23,31,32 Thorn et al.23 reported that κ-casein formedamyloid fibrils at 37 °C, observed with transmission electronmicroscopy (TEM) and X-ray fiber diffraction. Fibril formation,rather than association into spherical aggregates, seems to bepredominant for reduced κ-casein at elevated temper-atures,22,23,33,34 where the size of the fibrils ranges from 10 to16 nm in diameter and 50−300 nm in length.22,23 The ratiobetween fibrils and spherical aggregates can be regulated byadding reducing agents that prevent disulfide bonding andenhance the formation of fibrillar aggregates.23,31,33 Thorn andcolleagues23 reported a cross-β structure for these fibrillaraggregates, characteristic of amyloid fibrils. Amyloid fibrilsconsist of β-sheets in a highly structured arrangement furtherorganized into rope-like assemblies.34−36 In addition, fibrillationof reduced κ-casein was inhibited by the presence of αS- and β-casein, acting as molecular chaperons.22,23,37

β-casein exists as monomers in solution at low temperatures(4−10 °C), with elevated temperatures leading to association ofthe protein into micellar-like aggregates having a flexible,hydrated conformation with a radius of 10−12 nm.18,19 de Kruifet al.20 found the radius of gyration for β- and κ-casein micellesto be 8 nm, with β-casein forming more dynamic and lesscompact micelles compared to κ-casein. Concentrations abovethe CMC (about 0.5 mg/mL) would give rise to self-association into close to spherical aggregates.11,19 This processhas been thought to be analogous to surfactant micelles in thatit can be considered to be an equilibrium reversible process.21

Neutron and magnetic resonance spectroscopy measurementspropose that the associated state of β-casein, as well as κ-casein,has high specific volume, i.e., highly hydrated assembly incontrast to the interior of surfactant micelles, with open, flexiblepolypeptide chains and little or no interparticle cross-linking ofthe micelles.20 The hydrophobic C-terminal seems to beoriented toward the interior of the micellar aggregates, creatinga less hydrated core,18,19 with a softer exterior composed of thehydrophilic N-terminal.19 In the monomeric form at 4 °C, theradius of gyration (Rg) for the flexible chain is 4.6 nm.18

Phosphorylated β-casein possesses the ability to bind calciumions and calcium phosphate to its phosphate centers, which ishighly important for the association of the casein micelle.9

Our strategy to resolve the casein micellar substructure is toconstruct assemblies of key components of the casein micellesuch as κ-casein, the calcium binding β-casein and the calciumphosphate nanocluster (CPN) and to study the assembledstructure by a combination of scattering techniques. Weperformed preliminary investigations of this model system of

casein micelle (consisting of β-casein, κ-casein, and CPN) usingsmall angle neutron scattering and found evidence of extensiveaggregation. These results raised questions on the influence andpossible aggregation behavior of the individual components β-casein and κ-casein, which are known to individually self-assemble forming micellar-like structures. In addition, temper-ature was believed to exert a strong influence on the self-association and the stability of the formed aggregates as well asκ-caseins tendency to form fibrillar-type structures.In the present investigation, we have focused on the self-

association behavior of β- and κ-casein and the formation oflarger aggregates of caseins, e.g., κ-casein fibrils. The importantinfluence of temperature, concentration, and agitation on theself-assembly and fibrillation processes are the key parametersthat have been considered. We will also discuss the relationbetween the known self-assembly of casein and the fibrillationof κ-casein. For this purpose, we have used a combination ofreciprocal and direct space techniques such as 3D multianglecross-correlation light scattering, small-angle neutron scattering(SANS), and cryogenic transmission electron microscopy(cryo-TEM).

2. EXPERIMENTAL SECTION2.1. Sample Preparation. Pure κ-casein (MW = 19 kDa) and β-

casein (MW = 24 kDa) was obtained by Dr. Carl Holt (former atHannah Research Institute, Ayr, Scotland). Native κ-casein wasprepared from 4 L of bulk skim milk; precipitation at 30 °C by 8 mMCaCl2 in the presence of 3.5 M urea and in a final volume of 5 Lyielded a crude κ-casein fraction. This was recovered by pelleting,dissolved at pH 7.0 in deionized water by titration with NaOH andfreeze-dried, prior to chromatographic fractionation following themethod by Davies and Law.38 The peak corresponding to thenonglycosylated, monophosphorylated form39 was collected for use inexperiments. The freeze−dried proteins were carefully dissolved inbuffer to a final concentration of 5 mg/mL and 10 mg/mL and the pHwas adjusted to 6.9, in order to emulate the concentration andconditions found in milk. The buffer used was according to Vreemanet al.27,28 (0.01 M EDTA, 0.001 M DTT, and 0.1 M NaCl) dispersedin H2O (Milli-Q) or in D2O (>99%, Sigma-Aldrich Sweden AB,Stockholm) at room temperature. The reason for the isotopicreplacement of water will be discussed in section 2.2. When measuringthe pH in the D2O-based samples, referred to hence as pD, the well-known relationship pD = pH + 0.4 was used.40 In order to alkylate thetwo cysteine residues and to break any preformed hydrophobicinteractions, κ-casein was treated with a molar excess of DTT(dithiothreitol) and 8 M urea (Sigma-Aldrich Sweden AB, Stockholm)overnight, followed by exhaustive dialysis (against a weak buffercontaining 0.5 mM Na2HPO4, 0.5 mM NaH2PO4, pH 7) to removethe urea. A final dialysis step was done against ultrapure water beforefreeze−drying the protein.

Samples were left to incubate under agitation at 6 °C for 16−20 h,and then filtered with sterile 0.2 μm Anotop 10 filters (Whatman Ltd.,Sweden) prior any measurements, followed by 16−20 h ofequilibration in the sample cells used for the small angle neutronscattering (SANS) and light scattering (LS) experiments at 25 °C.Selected samples for the LS measurements were also incubated underagitation at 25 °C from the start, and the results were compared withthe 6 °C samples, in order to study the important effects of agitation aswell as temperature during incubation.

Cryogenic transmission electron microscopy (Cryo-TEM) sampleswere equilibrated at 6, 25, and 37 °C simultaneously for 16−20 hunder agitation (unless stated otherwise). The cryo-TEM sampleswere not filtered prior to the measurements, in order to ensure anundisturbed aggregation process and sufficient quantity of protein inthe samples.

2.2. Small Angle Neutron Scattering (SANS). The SANSmeasurements were performed at the D22 instrument at the Institute

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Laue Langevin ILL, Grenoble, France. Measurements were made usingan incident neutron wavelength, λ, of 6 Å and detector-sampledistances of 1.4 m, 5.6 m, and 17.0 m, covering the q-range 0.003−0.4Å−1. H2O samples were loaded in a rectangular or banjo Hellma QSquartz cells with a 1 mm path-length; D2O samples were placed in asimilar cell with a 2 mm path-length. Measurements were performed at6 and 25 °C, using a water bath to control the temperature. Data wascollected within the q-range 0.003−0.4 Å−1, where the scatteringvector q is defined by eq 141

πλ

θ= · ⎜ ⎟⎛⎝

⎞⎠q

4sin

2 (1)

with θ being the scattering angle and λ the neutron wavelength.Since neutrons interact mainly with the nuclei of each element in

the sample, the scattering amplitude depends on the nucleus and istherefore different for isotopes of the same element. The scatteringlength density (SLD) is the sum of coherent scattering lengths over allnuclei in a given volume.42 Hydrogen and its isotope deuterium havevery different neutron scattering lengths, and therefore throughchemical or biological synthesis, the neutron contrast of materials canbe changed. Furthermore, the isotopic contrast of the aqueous solventcan be changed by using H2O or D2O or mixtures thereof, whichmeans that components with different SLD can be made more or lessvisible for neutrons depending on the solvent contrast. Table 1 statesthe different SLD for the components used in this work; thecalculation of the SLD for the proteins/protein segments are based onthe approach given by Jacrot.43

Prior to the incubation at 6 °C while mixing (as described above),SANS samples were dialyzed against the dilution buffer for 12 h at 6°C. The buffer dialysate was measured using the same method as forthe sample and subtracted from the protein scattering data.Transmission measurements were made on all samples and buffers,as well as empty cell, and used for data reduction. Direct beammeasurements were used in the reduction to put the data on absolutescale.2.3. SANS Data Analysis. Data reduction from the scattering

pattern was performed using the Matlab script application GRASPdesigned for reduction of SANS data from ILL.44 Buffer subtractionand normalization with concentration was done in IGOR Pro using theNCNR analysis package provided by NIST (National Institute ofStandards and Technology), United States.45 A first modelindependent analysis was done using Kratky plots, where Q2*I(Q)was plotted against Q, in order to reveal to what extent the caseinswere aggregated or monomeric unfolded state.46 Fitting of thenormalized data was performed using SANSview by combining twomodels: the Ellipsoid model with polydispersity on the ellipse radii andthe Debye model. Applying the Ellipsoid model with polydispersity onits own did not produce any satisfactory fits to the data, nor did onlythe core shell Ellipsoid model with polydispersity. The reason for thisis likely to be the presence of monomeric casein, as expected above theCMC, and a diffuse, very hydrated outer layer. Therefore, a well-

defined ellipsoid model alone cannot accurately describe the caseinassemblies, and for this reason, the Debye model was used togetherwith the Ellipsoid model. The Debye model expresses the form factorfor a polymer chain (Gaussian chain), which can either be in solutionor attached to a surface, and gives the radius of gyration (Rg) of thepolymer chain.47 The Ellipsoid model provides the form factor,normalized by the particle volume, for an ellipsoid with uniformscattering length density. The model yields the radius along therotation axis of the ellipsoid Ra, and the radius perpendicular to therotation axis Rb.

48 The Rg of the ellipsoid is then calculated with eq 2.

=+

RR R

4ga

2b

2

(2)

The total intensity (I) measured by SANS can be expressed as theproduct of the form factor, P(q), and the structure factor, S(q), as in eq3.49

ρ= · Δ · · +I q NV P q S q B( ) ( ) ( ) ( )2 2 (3)

B is the incoherent background, N is the number particleconcentration, V is their volume, and Δρ is the difference in scatteringlength density between the solvent and the scattering particles. Theform factor is related to the shape of the scattering particles, whereasthe structure factor is an effect of particle ordering and revealsinformation about interparticle interferences. For very dilute samples,concentration <1%, the structure factor is negligible and eq 3 can bewritten as eq 449

ρ= · Δ · +I q NV P q B( ) ( ) ( )2 2 (4)

The concentrations used in our experiments can be considered tobe very dilute, and hence the structure factor is insignificant and eq 4applies. Since two independent models are used for the fits, the totalintensity is given by eq 5

ρ ρ= · Δ · + · Δ · +⎡⎣ ⎤⎦ ⎡⎣ ⎤⎦I q NV P q NV P q B( ) ( ) ( ) ( ) ( )2 2ellipsoid

2 2debye

(5)

2.4. Dynamic and Static Light Scattering. Measurements wereperformed using a 3D multiangle cross-correlation light scatteringinstrument,50 which extends the 3D cross-correlation schemedescribed before,51−54 and allows measurement at four anglessimultaneously. The angular range used for the measurements was20−140°, which corresponds to a q-range of 0.0055−0.030 nm−1 (532nm of laser wavelength). This instrument was recently used andproven to be suitable to study casein micelle systems, where size,structure, and stability upon acidification were investigated.10

From dynamic measurements, the relaxation rate Γ was obtainedand used to determine the diffusion coefficient Dc of the aggregates(eq 6)55

= ΓD

qc 2(6)

The hydrodynamic radius, Rh, was obtained from the Stokes−Einstein equation, assuming dilute suspensions of monodispersespheres (eq 7)

πη=R

k TD6h

b

0 (7)

where kb is the Boltzmann constant, T is the temperature, and η is thedynamic viscosity of the solvent.55

Static light scattering (SLS) measurements were performed toinvestigate the presence of aggregates. Normalized intensities wereobtained as described in eq 856

= −I qI q

I

I q

I( )

( ) ( )norm

sample

0,sample

background

0,background (8)

where Inorm(q) is the normalized intensity, Isample(q) is the averagedscattered intensity of the sample, Ibackground(q) is the averaged scattered

Table 1. Scattering Length Density (SLD) for the DifferentComponentsa

SLD (10−6 Å−2)

dispersed in H2O dispersed in D2O

β-casein, tot 1.74 2.82κ-casein, tot 1.79 3.01κ-casein, parab 1.84 3.13κ-casein, CMPc 1.70 2.82κβ-casein mixd 1.76 2.88H2O −0.56D2O 6.35

aCalculations based on Jacrot.43 bAmino acid residues 1−105. cAminoaid residues 106−169. dAssuming β-casein/κ-casein molar ratio of 2.2.

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intensity of the sample cell and the solvent, and Io,sample and Io,backgroundare the incoming intensities for sample and background, respectively.The normalized intensities were brought to absolute scale (excessRayleigh ratio) by eq 956,57

θθ

θθΔ =

Δ· ·

⎛⎝⎜

⎞⎠⎟R

I

IR

nn

( )( ( ))

( ( ))( )sample

tolueneref

solvent

toluene

2

(9)

where Rref(θ) is a constant (the Rayleigh ratio of toluene, 2.79 × 10−5

cm−1 at 20 °C),58 ΔIsample(θ) the averaged scattered intensity from thesample after background subtraction, Itoluene(θ) the scattered intensityfrom toluene, and nsolvent and ntoluene are the refractive index of thesolvent and the toluene, respectively. No transmission correction wasmade, since the transmission of the sample was very close to that ofthe solvent.2.5. Cryogenic Transmission Electron Microscopy (Cryo-

TEM). Samples for cryo-TEM were prepared by using a controlledenvironment vitrification system (CEVS), described by Bellare et al.59

The sample (5 μL of 5 mg protein/mL) was added to a lacey carbonfilm, supported by a copper grid, followed by gentle blotting with filterpaper. This resulted in a thin liquid film (20−200 nm) on the grid,which was plunge-frozen in liquid ethane (at −180 °C) and stored inliquid nitrogen until transferring into the cryo-TEM. Plunge-freezingensures rapid vitrification of the sample while minimizing theformation of ice crystals. Imaging of the sample was undertaken in aPhilips CM 120 BioTWIN Cryo microscope, with a cryo holder of thetype Oxford CT3500 and a postcolumn energy filter (Gatan GIF 100).Images were recorded digitally with a CCD camera under low doseconditions.

3. RESULTS AND DISCUSSIONThe self-association behavior of κ-casein and β-casein wasstudied by SANS, cryo-TEM, and light scattering. In particular,we aimed to understand under which conditions largeraggregates and fibrils could form, and how this fibrillationcould be retarded. Preliminary studies of the mixed κ-casein andβ-casein system aimed to contribute to the understanding ofthe casein micelle substructure.We note that the scattering curves, especially for κ-casein,

feature an upturn in the scattering at low q with inconsistentslope and shape for the different data, and hence believed to becaused by larger aggregates of various size and shape.Therefore, the lowest q-range was not included in the fit tomodels. For the reason of getting consistency in the fits used tomodel the experimental data the same q-range was applied forall samples investigated in this study, independent of whetherwe observe an upturn or not.3.1. κ-Casein self-Associates to Ellipsoidal Micellar

Aggregates. κ-Casein scattering curves were analyzed byplotting Q2*I(Q) vs Q, known as Kratky plots, in order toemphasize structures at low intensity.46 Figure 1 illustratesKratky plots for κ-casein 5 mg/mL and 10 mg/mL, 6 and 25 °Cin D2O buffer. The scattering curves propose rather well-defined protein assemblies,46 as expected since we are wellabove the CMC reported for κ-casein (0.5 mg/mL).28 Samplesin H2O buffer show similar results but has been omitted for thesake of brevity.Scattering length densities (SLD) and volume fractions for

the κ-casein were deduced on the basis of calculations byJacrot.43 For SLD values, see Table 1. Assumptions made are inagreement with the general accepted features of κ-casein, suchas the hydrophilic part of κ-casein (referred to as the CMP)being located on the surface where it extends some 5−10 nminto its surroundings, with the rest of the κ-casein located in thecore.9,28 With this, SANS data were fitted to the best modelsuited.

The best and most consistent fits for the differentconcentrations, temperatures, and buffers used to disperse κ-casein were obtained when applying a polydisperse Ellipsoidmodel in combination with a Debye model, as shown in Figure2 and Figure 3 with the fitting parameters summarized in Table2. Figure 2c illustrates the fit results when only using thepolydisperse Ellipsoid model, without combination with theDebye model, clearly providing a poor fit in the higher q region.The fitting parameters yield a radius of gyration of the ellipsoid,Rg, of approximately 5.7 nm, and solvent content about 63%. Itshould be noted that the polydispersity in radii (Ra and Rb) ofthe ellipsoid is 10−20%, which can be interpreted as a dilutesurface layer extending a few nanometers from the ellipsoid.The values are not significantly affected by the exchange ofD2O/H2O buffer; Rg(mean D2O) = 5.8 nm and Rg(mean H2O) = 5.6nm, together with 62% solvent for D2O compared to 64% forH2O. The radii, composed of Ra and Rb according to eq 2, showminor size dependence on temperature. For instance when thetemperature is increased from 6 to 25 °C, Ra (mean 6 °C) increasesfrom 4.6 nm to 4.8 nm for Ra (mean 25 °C), while Rb decreasesslightly from 10.4 nm for Rb (mean 6 °C) to 10.2 nm forRb (mean 25 °C). Hence, the aggregates tend to become morespherical with increased temperature, but the changes arealmost within the error in the size estimate. Polymerization ofκ-casein into micellar aggregates is insensitive to temperaturesbetween 1 and 32 °C, implying that hydrophobic interactionsare not the primary driving force for aggregation.27

Furthermore, altering the concentration of κ-casein (5 mg/mL and 10 mg/mL, respectively) does not significantly affectthe size of the ellipsoid, as shown in Table 2. A study by deKruif et al.20 reported a Rg = 8 nm for the proposed quasispherical κ-casein micelles seen with SANS, while in anotherstudy by de Kruif and May,60 SANS results fitted with a hardsphere model indicated a dense core of 6−7 nm in Rg with afluffy outer layer about 8 nm thick containing very littlescattering material. On the basis of the dimension of theellipsoid, we can calculate the volume of the κ-casein aggregates(Vellipsoid = 4π/3*Ra*Rb

2), and based on the degree of hydrationand the molecular volume 24 nm3 of the κ-casein monomer,43

we arrive at aggregation number of 30−35 (see Table 2). Thisis consistent with literature data.The Debye model fitting parameters give a mean radius of

gyration for a polymer chain (Gaussian chain) of about 5.1 nm.Values for κ-casein dispersed in D2O based buffers are, just as

Figure 1. Kratky plot for κ-casein dispersed in D2O buffer, Q2*I(Q)against Q (Å−1). 5 mg/mL, 6 °C (▲), 5 mg/mL, 25 °C (■), 10 mg/mL, 6 °C (⧫), and 10 mg/mL, 25 °C (●).

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for the ellipsoid, not significantly different compared to thevalues recorded in the H2O based buffers; Rg Debye (mean D2O) =5.2 nm and Rg Debye (mean H2O) = 4.9 nm (Table 2). Here, wenote that the Debye model does not reveal whether thepolymer chain is linked to the assembly or if it accounts for thefree monomers in solution. The Rg Debye = 5.1 nm valuecorresponds well to the CMP length of 5−10 nm,9 described asa hydrophilic polyelectrolyte brush protruding from the surfaceof the casein micelle by de Kruif.61 As discussed before, theDebye model does not differentiate between bound polymerchains and those that are free in solution. The data can beinterpreted as a result of the monomers being in solution withan average Rg value of 5.1 nm. Here, it should be noted thatabove the critical micelle concentration (CMC), reported to be0.5 mg/mL for κ-casein,11,19,28 the monomer concentrationshould be constant and equal to 0.5 mg/mL (corresponding toa volume fraction of 0.4 × 10−3, calculations based on Jacrot43

and Table 1). As reported in Table 2, the volume fractioncalculated from the Debye function scales with the proteinconcentration, and hence, we expect that the scattering mainlyarise from the presence of a dilute hairy layer, protruding some5 nm from the ellipsoidal core.Dynamic light scattering on the samples gives a hydro-

dynamic radius Rh of 11−16 nm, dispersed in H2O as well asD2O (Table 3). If a 5−10-nm-thick shell, as reported here andfound in the literature,9,29,30,60,61 were to be added to the κ-casein micelle from the fitted SANS data (Rg = 6 nm), thiswould give an apparent total radii of 11−16 nm correspondingwell to the measured Rh. de Kruif and May60 reported on ahydrodynamic radius determined by DLS of 11.5 nm, and aproposed core shell model comprising a dense core of 6−7 nmwith a diffuse outer layer, probably consisting of the CMP partof κ-casein, protruding 14.7 nm from the center. A schematicpicture of the κ-casein micelle based on our SANS and DLSdata is shown in Figure 4.The scattering curves for κ-casein show a clear upturn in the

lowest q-region (Figure 2 and Figure 3), especially noticeablefor the D2O based buffers mainly due to the higher contrastprovided by D2O. The observed upturn is believed to be due tothe presence of larger aggregates. It is difficult to say anythingabout the aggregates, which could be due to an isotope effect,and since they could not be fully understood based purely onthe SANS data, they were further studied using othertechniques such as cross-correlation light scattering and cryo-TEM.

3.2. Agitation Induces the Formation of Larger κ-Casein Aggregates. The SANS data of the κ-casein samplesin D2O show clear upturns at low q (Figure 2) indicating thepresence of larger aggregates in solution. The presence of thelarger aggregates are not apparent in the light scattering dataand the upturn in scattering intensity at low q is also much lesspronounced in H2O (Figure 3). However, when κ-casein isincubated for longer than 20 h (at 25 °C), larger aggregates areformed, as seen in Table 3. Figure 5 illustrates the increase inscattering intensity for κ-casein measured with light scatteringafter 4 days at 25 °C, compared to the reference incubation of20 h at 25 °C. The formation of larger aggregates (observed byan increase in forward scattering) is strongly enhanced whenthe sample is stirred during incubation. Recent studies of theeffect of shear on protein aggregation suggest that proteins withslightly perturbed structures act as seeds for aggregation,62

followed by the linear association of these aggregatesaccelerated by shear.62,63

Figure 2. Intensity (I) vs scattering vector (q) as used in the SANSexperiments for κ-casein dissolved in D2O buffer. (a) 5 mg/mL at 6 °C(Δ) with corresponding fit (−) and 25 °C (○) with corresponding fit(--); (b) 10 mg/mL at 6 °C (Δ) with corresponding fit (−) and 25 °C(○) with corresponding fit (--); and (c) higher q-region for sample 10mg/mL, 25 °C (○) with corresponding fit (--) and illustration of thefit results when only using the ellipsoidal model (●). The q range usedfor the fits in (a) and (b) is 0.01−0.4 Å−1 (black lines).

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3.3. Fibrillation of κ-Casein and the Morphology ofthe Formed Aggregates. The morphology of the formedaggregates was additionally examined using cryo-TEM. In thisstudy, we used a κ-casein concentration of 5 mg/mL, where κ-casein is expected to be in the micellar state. κ-Casein reducedby DTT has previously been reported to form fibrils uponincubation at 37 °C31,23 for 12 h, with a diameter of 12−16 nmand a length of 50−300 nm.23 These results were confirmed by

our study with the observation of threadlike structures in thecryo-TEM after incubation of the sample at 37 °C for 20 h(Figure 6). The fibrils have a diameter of approximately 10−20nm and are 50−200 nm long. Fibrils of similar thickness andlength have also been observed when incubating reduced κ-casein at 25 °C for 20 h (while stirring), Figure 6. This confirmsthe tendency of κ-caseins to form larger aggregates apart fromthe self-association of κ-casein into micelles as also observed

Figure 3. Intensity (I) vs scattering vector (q) as used in the SANS experiments for κ-casein dissolved in H2O buffer. (a) 5 mg/mL at 6 °C (Δ) withcorresponding fit (−) and 25 °C (○) with corresponding fit (--); and (b) 10 mg/L at 6 °C (Δ) with corresponding fit (−) and 25 °C (○) withcorresponding fit (--). The q range used for the fits is 0.01−0.4 Å−1 (black lines).

Table 2. Values Obtained from SANS Data Fitted to the Polydisperse Ellipsoid Model in Combination with the Debye Model

Ellipsoid Debye

Raa (nm) Rb

b (nm) Rgc (nm) solvent in core (%) aggreg. no.d Rg Debye (nm) vol. fraction ×10−3e

κ-cn, D2O buffer5 mg/mL, 6 °C 4.7 10.5 5.8 ± 0.2 61.6 34 5.1 1.5 ± 0.35 mg/mL, 25 °C 5.0 10.2 5.7 ± 0.1 61.5 35 5.2 1.5 ± 0.110 mg/mL, 6 °C 4.8 10.7 5.8 ± 0.1 62.4 36 5.2 3.0 ± 0.210 mg/mL, 25 °C 5.1 10.4 5.8 ± 0.1 60.9 37 5.5 3.0 ± 0.2

κ-cn, H2O buffer5 mg/mL, 6 °C 4.5 10.2 5.6 ± 0.9 64.1 29 4.9 0.5 ± 0.25 mg/mL, 25 °C 4.5 10.1 5.5 ± 0.8 64.3 28 4.9 0.5 ± 0.210 mg/mL, 6 °C 4.5 10.1 5.5 ± 0.4 63.7 29 4.9 1.0 ± 0.510 mg/mL, 25 °C 4.5 10.3 5.6 ± 0.4 62.9 31 4.9 1.0 ± 0.5

β-cn, D2O buffer5 mg/mL, 6 °C 3.8 10.3 5.5 ± 0.1 63.6 20 1.8 0.5 ± 0.55 mg/mL, 25 °C 5.2 10.5 5.9 ± 0.1 65.0 28 1.4 0.2 ± 0.510 mg/mL, 6 °C 4.0 10.0 5.4 ± 0.1 62.1 21 1.9 1.0 ± 0.110 mg/mL, 25 °C 5.3 10.4 5.8 ± 0.1 62.5 30 1.3 0.3 ± 0.1

κβ-cn, D2O buffer10 mg/mL, 25 °C 5.3 10.6 5.9 ± 0.1 64.0 1.1 0.3 ± 0.1

κβ-cn, H2O buffer10 mg/mL, 25 °C 4.3 11.1 5.9 ± 0.1 63.5 1.3 1.0 ± 0.8

aPolydispersity for Ra is in the range 10−15% for all samples. bPolydispersity for Rb is in the range 15−20% for all samples. cResults with ± standarddeviation as propagated from the fit derived errors in Ra and Rb

dAggregation number calculations based on the volume of the ellipsoidal κ- and β-casein aggregates (Vellipsoid = 4π/3*Ra*Rb

2), the degree of hydration and the molecular volume for the κ-casein (24.1 nm3) and β-casein monomers(30.5 nm3), respectively.43 eFor reference, calculated volume fractions of monomers based on the CMC 0.5 mg/mL11,19,28 corresponds to 0.4 × 10−3

for κ- as well as β-casein (calculations based on Jacrot43 and Table 1). It should be noted that CMC values are reported to change with temperature,pH, and buffer.

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with SANS and light scattering. Here, we stress again that thesamples studied with the scattering techniques were, unless

stated otherwise, incubated in the measuring cells and thereforenot stirred. Hill et al.62 reported on the enhanced fibrilformation of whey protein β-lactoglobulin when samples wereexposed to shear, with amyloid-like fibrils observed after 18 h.With this in mind, it should be noted that κ-casein incubated at25 °C without stirring might not form mature fibrils after only20 h. However, samples of κ-casein incubated without stirringat 25 °C did show fibrils of similar or longer sizes as reportedabove after 1 month. In addition, no fibrils could be observedupon incubation of κ-casein at 20 °C for 20 h without stirring(Figure 6). A control sample, which was passed through a 0.2μm filter performed prior incubation (25 °C, 20 h, stirring),showed similar, or even more structured, fibrils compared tothe nonfiltered samples. Hence, the fibrillation does not seemto be induced by any large protein aggregates already present inthe sample (Figure 6).κ-Casein dispersed in D2O buffer was also examined with

cryo-TEM, undergoing the same incubation procedures as forH2O dispersion, and showed similar or even somewhat moreextended fibrils as for κ-casein dispersed in H2O buffer (datanot shown).

3.4. β-casein self-associates to ellipsoidal micellaraggregates. Figure 7a shows Kratky plots for β-casein in D2Oat 25 °C. Concentrations ranging from 1 mg/mL to 10 mg/mLshow protein association behavior, while the 0.1 mg/mL sampleshows protein monomeric behavior.46 This result is consistentwith those reported elsewhere,19,64 reporting CMC values ofabout 0.5 mg/mL for β-casein.11,19 Figure 7b shows 5 mg/mLand 10 mg/mL β-casein at different temperatures (6 and 25°C), and when comparing to κ-casein (Figure 1) one can seethat the temperature effect is greater for β-casein with asignificant difference in Q2*I(Q) for 6 °C compared to 25 °C.Samples in H2O buffer (5 mg/mL and 10 mg/mL) showssimilar results, and has been omitted, whereas coherentscattering in H2O is too weak to record the scattering curvesfor such low concentrations as 0.1 mg/mL.As shown above for κ-casein, the most consistent fits for the

different concentrations of β-casein and temperatures wereobtained when applying a polydisperse ellipsoid core model incombination with a Debye model, see Figure 8. Table 2summarizes the values obtained from the fits for the varioussamples. Samples are fitted within the same q-range as κ-casein

Table 3. Hydrodynamic Radius (Rh) of κ-Casein and β-Casein, Measured by DLS Using a Multiangle Cross-Correlation Instrument

incubation parameters

temperature time agitation Rh (nm)

κ-cn, 1 mg/mL H2O buffer6 °C 20 h y 18.1a

6 °C 6 days yb 18.2a

25 °C 20 h y 28.6a

25 °C 6 days yc 24.3a

κ-cn, 5 mg/mL H2O buffer6 °C 1 h - 10.725 °C 1 h - 16.125 °C 20 h - 13.625 °C 20 h y 30.6a

25 °C 4 days - 22.625 °C 20 h y 29.6

κ-cn, 1 mg/mL D2O buffer25 °C 20 h y 36.1a

κ-cn, 5 mg/mL D2O buffer6 °C 1 h - 15.425 °C 1 h - 14.825 °C 20 h - 12.225 °C 20 h y 57.7a

β-cn, 5 mg/mL H2O buffer25 °C 1 h - 13.8

β-cn, 5 mg/mL D2O buffer25 °C 1 h - 13.5

aRh values are for 90° only, and not for the full range (20−140°).bSamples were agitated for the first 20 h at 6 °C, followed bynonagitated incubation at 6 °C. cSamples were agitated for the first 20h at 25 °C, followed by nonagitated incubation at 25 °C.

Figure 4. Schematic illustration of an ellipsoidal micellar κ-caseinaggregate: (a) 2D model with mean values for Ra, and Rb; and (b) 3Dmodel.

Figure 5. Absolute light scattering intensity vs scattering vector (q) ofκ-casein, 5 mg/mL incubation at 25 °C. Unstirred sample, incubationfor 20 h (⧫); unstirred sample, incubation for 3 days (▲); and stirredsample, incubation for 20 h (●).

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for consistency. Due to the beamtime restraints, we could notrecord the scattering patterns for β-casein samples dispersed inH2O buffers. In contrast to the results for κ-casein theellipsoidal radius as well as the Rg values for the polymer chainin the Debye model show a clear dependence upon increasingthe temperature from 6 to 25 °C. This is in accordance withtemperature dependent self-assembly of β-casein reportedpreviously.18,19,64 The Rg of the ellipsoid increases from 5.5nm at 6 °C to 5.8 nm at 25 °C (mean values from Table 2 for 6and 25 °C respectively). However, the increase in size of theellipsoid is not symmetric since Ra increases from 3.9 to 5.3 nmwhen increasing the temperature from 6 to 25 °C, while Rbonly increases slightly from 10.2 to 10.5 nm. This implies thatthe aggregates tend to become slightly more spherical whenincreasing the temperature. No significant effect on the size ofthe micellar β-casein aggregates is observed when increasing theconcentration from 5 mg/mL to 10 mg/mL. In addition thefitting gave a constant solvent content in the micellar core ofabout 63% in this concentration range. Calculations based onthe dimension of the ellipsoid gives the volume of the β-caseinaggregates (Vellipsoid = 4π/3*Ra*Rb

2), and based on the degreeof hydration and the molecular volume 30.5 nm3 of the β-caseinmonomer43 we arrive at aggregation number of 20 for 6 °C and30 for 25 °C (see Table 2).As observed for κ-casein, no distinct core−shell model could

provide a realistic and satisfactory fit, and hence the combinedDebye-Ellipsoid model was used. The parameters of the fit ofthe Debye model for β-casein suggest a slight temperaturedependence of Rg Debye in contrast to what has been observedfor κ-casein. In fact Rg Debye values increased with temperaturefrom 1.4 nm at 6 °C to 1.9 nm at 25 °C (Table 2). In addition,

the volume fraction, calculated according to the Debyemodel,47 decreased significantly when raising the temperaturefor β-casein (Table 2). This is consistent with the observationthat the CMC is shifted to higher concentrations whendecreasing the temperature and in fact some reports suggeststhat no β-casein micelles are formed at 4 °C.19,67,68 It isnoteworthy that the corresponding volume fraction values forκ-casein does not seem to be affected by these temperatures,consistent with previous findings which reported that self-association of κ-casein is not temperature dependent as it is forβ-casein.18,19 Volume fraction values are significantly higher forκ-casein (dispersed in D2O) and the Debye model predictsRg Debye = 5.1 nm for κ-casein compared to β-caseins Rg Debye =1.4 nm (6 °C) and 1.9 nm (25 °C).Dynamic light scattering measurements of 5 mg/mL β-casein

samples at 25 °C gave Rh = 14 nm for both buffers (Table 3),consistent with the findings of OConnell et al.19 and de Kruifand Grinberg64 with Rh = 12 nm for β-casein micelles. Thelatter study suggests that β-casein self-association can bedescribed as a temperature dependent sequential association,where the aggregation number increases with temperature untilit levels off at about 40 °C. These observations are confirmed inthe study by Mikheeva et al.,21 where β-casein self-association isproposed to follow Kegeles65 shell model. This model considersthe micellization process to occur as a series of intermediates ofgrowing size, where the addition of monomers is a cooperativeprocess. In our experiments the SANS data gave Rg = 5.8 nmfor the ellipsoid aggregates at 25 °C, comparable with the Rg =8 nm for the quasi spherical micelles reported by de Kruif etal.20 If we assume that a diffuse and dilute 4.6 nm layer of β-casein, a value corresponding to Rg for the monomeric β-

Figure 6. Cryo-TEM images for κ-casein, 5 mg/mL. The scale bar is 200 nm. (a) No fibrils after incubation at 6 °C, 20 h agitation; (b) fibrils afterincubation at 25 °C, 20 h agitation; (c) fibrils after incubation of filtered samples (0.2 μm) at 25 °C, 20 h agitation; (d) no fibrils after incubation at20 °C, 20 h; (e) fibrils after incubation at 25 °C, 1 month; and (f) fibrils after incubation at 37 °C, 20 h.

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casein,18 protrudes from the surface of the micelle, the totalradius of the micelle is about 10.4 nm. It should be noted thatthe hydrophilic N-terminal of β-casein adsorbed on a

hydrophobic surface forms a diffuse hairy layer, protrudingabout 5−10 nm from the surface.66

Finally it should be noted that the scattering curves for β-casein (Figure 8), fitted within the same q-range as κ-casein forconsistency, do not reveal an upturn in the lower q-region asobserved for κ-casein (Figure 2). This indicates that unlike κ-casein, β-casein does not form larger aggregates, which wasfurther confirmed by cryo-TEM where no fibrils are observedfor β-casein (Figure 10).

3.5. β-Caseins Inhibit Aggregation and Fibrillation ofκ-Casein. The interaction between β-casein and κ-casein at thesame ratio as in bovine milk, that is at a 2.2-fold molar excess ofβ-casein, was also studied. The SLD of the complex, assumingthat both proteins κ- and β-casein are involved in the complexat the ratio they are added to the solution, was calculated basedthe Jacrot approach43 and values are given in Table 1. It shouldbe noted that the SLDs of the two proteins are rather similar soan error in the estimate of the composition does notsignificantly affect the result. As shown before, SANS datagave the best fit when applying a polydisperse ellipsoid coremodel in combination with a Debye model. Figure 9 illustratesfits for a total casein concentration of 10 mg/mL, incubated at25 °C in D2O (H2O data not shown), and samples are fittedwithin the same q-range as κ-casein for consistency. Table 2summarizes the values obtained from the fits for the samples.The ellipsoid radius was similar to the values obtained for the

κ-casein and β-casein micelles values, with Rg (mean) = 5.9 nmand a micellar core solvent content of about 63.5% (Table 2).The ellipsoid radii are as stated earlier composed of Ra and Rb

according to eq 2, with Ra H2O = 4.3 nm compared to Ra D2O =5.3 nm, and Rb H2O (mean) = 11.1 nm compared to Rb D2O (mean) =10.6 nm. Andrews et al.18 and Evans at al.67 report on D2Ogiving rise to somewhat larger aggregates for β-casein, implyingthat this could be an effect upon β-casein behavior whendispersed in heavy water that might apply for this protein mix.The Rg Debye value = 1.2 nm and the corresponding volume

fractions, obtained form the Debye model, are similar to those

Figure 7. Kratky plot for β-casein dispersed in D2O buffer, Q2*I(Q)against Q (Å−1). a) 0.1 mg/mL, 25 °C (⧫), 1 mg/mL, 25 °C (▲), 5mg/mL, 25 °C (■) and 10 mg/mL, 25 °C (●); and b) 5 mg/mL, 6°C (⧫), 5 mg/mL, 25 °C (■), 10 mg/mL, 6 °C (▲) and 10 mg/mL,25 °C (●).

Figure 8. Intensity (I) vs scattering vector (q) as used in the SANS experiments for β-casein dissolved in D2O buffer. a) 5 mg/mL at 6 °C (Δ) withcorresponding fit (−) and 25 °C (○) with corresponding fit (--); and b) 10 mg/mL at 6 °C (Δ) with corresponding fit (−) and 25 °C (○) withcorresponding fit (--). The q range used for the fits are 0.01−0.4 Å−1 (black lines).

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reported for β-casein in D2O at 25 °C. This could be explainedwith the 2.2-fold molar excess of β-casein used, causing β-caseinto be the dominant protein in this assembly. It could also be thecase that the CMC is altered significantly when mixing thesetwo proteins. It should, however, be noted that the derivederrors in the fit are quite high for these samples. In addition,future studies will make use of a buffer containing calciumphosphate, since this would be more suitable for a properanalysis of the κβ-casein mix.The scattering curves for the κβ-casein mix (Figure 9) do not

show a clear upturn in the lowest q-region as for κ-casein(Figure 2) but rather resemble the scattering curves observedfor β-casein (Figure 8), indicating that β-casein inhibits anyextensive aggregation behavior of κ-casein. In order to confirmthese results, the κβ-casein mix was studied with cryo-TEM,after incubation at 37 °C for 20 h, and as reported by Thorn et

al.23 and Sood et al.,33 β-casein inhibits fibril formation of κ-casein (Figure 10). Pure β-casein samples were studied withcryo-TEM as well, after incubation at 37 °C for 20 h, and didnot show any fibrils or larger aggregates compared to κ-caseinthat forms fibrils under the same conditions (Figure 10).

4. GENERAL DISCUSSION

SANS results for κ-casein suggest that the protein self-assembles into micelle-like structures of hydrated, polydisperseellipsoid with Rg = 5.7 nm, probably surrounded by a low-density 5-nm-thick shell. The surface location of the CMP partof κ-casein, reported here to be in the same size range for pureκ-casein systems as for casein micelle systems, is believed togive colloidal stability to the casein micelle by increasing thesteric repulsive forces, and is believed to be approximately 5−10 nm thick.9,29 It is also noteworthy that we do not observeany firm evidence of a solvent isotope effect for the κ-caseinassembly or aggregation.β-Casein forms a similar type of micellar structure as κ-casein

according to SANS data, giving polydisperse, hydratedellipsoidal aggregates upon self-association surrounded by avery dilute outer layer. In contrast to the κ-casein case, the self-assembly of β-casein is temperature dependent, where theprotein is significantly less associated at lower temperature (6°C), consequently giving somewhat larger ellipsoidal aggregateswhen increasing the temperature (to 25 °C). Previously,Kajiwara et al.68 have reported that β-casein forms nonsphericalaggregates upon self-association, but they found that an oblatespheroid model gave better fits to their X-ray data.Interestingly, they reported that the particles became nearlyspherical when increasing the temperature from 5 to 25 °C. Inaddition, their measured hydrodynamic radius was greater thanthe radii calculated for the X-ray measurements, leading themto suggest the presence of a low mass density surface layercomparable to the suggested hairy layer of κ-casein on thesurface of native casein micelles.68 This is in line with theobservations made in this study, where Rh values for β-casein of14 nm (25 °C) were compared to the Rg = 5.9 nm (25 °C),given by the SANS data.κ-Casein is known to form amyloid-like fibrils when

incubated at 37 °C for more than 12 h22,23 in the size rangeof 12−16 nm thickness and 50−300 nm length.23 This wasconfirmed in this study where κ-casein fibrillated into 10−20-nm-thick fibrils, 50−200 nm long. In addition, we found that κ-casein can form similar type of fibrils when incubated at lower

Figure 9. Intensity (I) vs scattering vector (q) as used in the SANSexperiments for samples at 25 °C dispersed in D2O buffer,concentration 10 mg/mL. (a) κβ-casein mix (□) with correspondingfit (−); κ-casein (Δ) with corresponding fit (- -), and β-casein (○)with corresponding fit (−‑‑). The q range used for the fits are 0.01−0.4Å−1 (black lines).

Figure 10. Cryo-TEM images for (a) κβ-casein mix (5 mg/mL)no fibrils after incubation at 37 °C, 20 h; (b) β-casein (5 mg/mL)no fibrils afterincubation at 37 °C, 20 h; and (c) κ-casein (5 mg/mL)fibrils after incubation at 37 °C, 20 h.

Langmuir Article


temperatures, 25 °C, and that the process is sped up withagitation. Observations that mechanical agitation or shakingaccelerates fibril formation have been reported,69−71 which isconsistent with the rapid formation of fibrils at 25 °C underagitation observed in this study. In a different study, samples ofβ-lactoglobulin were shown to be most affected by stirringduring the first 5 h of incubation, leading to the acceleratedfibrillation compared to a slow fibril formation in the unstirredcontrol sample.62 Thorn et al.23 speculate that κ-casein goesfrom its more natural multimeric state to a monomericintermediate state prior to rearrangement into amyloid-likefibrils, where the dissociated κ-casein monomers are believed tobe very receptive to temperature-dependent fibrillation. Ecroydet al.34 propose that the dissociation of the oligomeric κ-caseinto an amyloidogenic forerunner is the rate-limiting step for thefibril formation. This dissociation might be influenced byagitation, which could explain the fibrillation upon 25 °C asreported in this study. Hill et al.62 report on shear-inducedaggregation of spheroids of β-lactoglobulin, which in turn act asseeds upon addition to the sample and enhancing fibrilformation. Lencki36 observed ThT fluorescent peaks at 485 nmfor κ-casein at concentrations above CMC, similar to the resultsreported by Thorn et al.,23 supporting the theory of κ-caseinforming fibril-like structures. In addition, Lencki36 investigatedthe presence of fibrillar structures in casein micelles undernative conditions and found indications of fibril-like structures;hence, fluorescence peak heights were on the same order ofmagnitude as for pure κ-casein, leading to the hypothesis thatfibril structures could be present in native micelles. Thisinteresting aspect, as well as the question of the effect ofcalcium on fibril formation, should be investigated further.The ability of β-casein to form fibrils similar to κ-casein at

uniform conditions was studied, and just as reported byLencki36 and Thorn et al.,23 β-casein did not undergofibrillation. In addition, β-casein inhibited aggregation intolarger aggregates as well as fibril formation of κ-casein, bothimportant observations with respect to the stability of caseinmicelles during processing, using shear and heat. The ability ofβ-casein to inhibit the fibrillation of κ-casein has been reportedelsewhere,23,33,37 and they found that β-casein acts as amolecular chaperone preventing the fibril formation of κ-caseincompletely, as reconfirmed by this study. Chaperones havebeen proposed to make the otherwise fibril-forming proteinfavor the formation of amorphous aggregates,72 probablythrough the exposed hydrophobic surfaces of the chaper-ones.37,72,73 Hence β-casein would “chaperone” unstablemonomers of κ-casein by adhering nonspecifically to theirhydrophobic surfaces. This would prohibit interactions betweenκ-casein molecules and prevent them from forming shared β-sheet structures crucial for associating into fibrillar structures.23

This is a very important feature of β-casein, and the structuresof the casein micelles are highly dependent on this interactionbetween β- and κ-casein, which prevents fibrillation.

5. CONCLUSIONSWe have demonstrated the tendency of κ-casein to formamyloid-like fibrils already at room temperature (25 °C) uponagitation and that β-caseins have the ability to inhibit fibrilformation. In addition, by using SANS and light scattering wehave obtained structural information about the self-associationof κ-casein into micellar aggregates during various conditions.Our data support a polydisperse ellipsoid model for the κ-caseinand β-casein micellar assemblies.

As the biological function of the casein micelles in milk is toprovide proteins with high nutritional value and highconcentrations of calcium and phosphate for the neonate, itis important that no pathological calcification or amyloidosisoccur. The findings in this study could also provide new leadsfor the understanding of technological aspects, where caseinsare subject to elevated temperature and shear forces. Duringprocessing, formation of larger aggregates might have bothnegative and positive effects on the structure of dairy productslike yogurt and cheese.

■ AUTHOR INFORMATIONCorresponding Author*E-mail [email protected], telephone +46 46 888 30 95;E-mail [email protected], telephone +46 46 222 8158.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe Institute Laue Langevin (ILL) in Grenoble, France isgreatly acknowledged for the beamtime given under proposalNo 9-10-1041. The European Spallation Source (ESS),Sweden, is acknowledged for financial support of this project,and Hakan Hahn, ESS Sweden for preparing 3D renderings ofthe models. We thank Gunnel Karlsson at Biomicroscopy Unit,Polymer and Materials Chemistry, Chemical Centre, LundUniversity, Lund, Sweden, for the cryo-TEM work. This workbenefited from software developed by the DANSE projectunder NSF award DMR-0520547.

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mailto:[email protected]

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Aggregation Behavior of Bovine κ- and β-Casein Studied with Small Angle Neutron Scattering, Light...

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