Bridging the Gap between the Nanostructural Organization and

Article pubs.acs.org/Langmuir

Bridging the Gap between the Nanostructural Organization and Macroscopic Interfacial Rheology of Amyloid Fibrils at Liquid Interfaces Sophia Jordens,† Patrick A. Rühs,‡ Christine Sieber,‡ Lucio Isa,§ Peter Fischer,*,‡ and Raffaele Mezzenga*,† †

Laboratory of Food and Soft Materials, Department of Health Sciences and Technology and ‡Laboratory of Food Process Engineering, Department of Health Sciences and Technology, ETH Zurich, 8092 Zurich, Switzerland § Laboratory for Interfaces, Soft matter and Assembly, Department of Materials, ETH Zurich, 8093 Zurich, Switzerland

ABSTRACT: The interfacial behavior of proteins and protein aggregates such as fibrils influences the bulk behavior of multiphase systems in foods, pharmaceuticals, and other technological applications. Additionally, it is an important factor in some biological processes such as the accumulation of amyloid fibrils at biological membranes in neurodegenerative diseases. Here, using β-lactoglobulin fibrils as a model system, we cover a large range of characteristic measuring length scales by combining atomic force microscopy, passive probe particle tracking, tensiometry, interfacial shear, and dilatational rheology in order to correlate the intricate structure of fibril-laden interfaces with their macroscopic adsorption kinetics and viscoelasticity. A subtle change in solution pH provokes pronounced changes in interfacial properties such as alignment, entanglement, multilayer formation, and fibril fracture, which can be resolved and linked across the various length scales involved.

1. INTRODUCTION The experimental characterization of complex liquid interfaces has advanced by great strides over the past few decades with the emergence of techniques specifically geared toward interfacial processes such as sum-frequency generation vibrational spectroscopy,1−3 ellipsometry,4−6 neutron reflectivity,7−10 X-ray reflectivity,11,12 and increasingly sophisticated imaging and rheology approaches.13−22 Taking each method individually, however, the accessible range of length scales is often rather limited. Because the overall characteristics of a material are often governed by effects on a length scale comparable to that of its components, it is crucial to choose an appropriate and complementary set of experimental techniques for the material at hand in order to understand which microscopic structures give rise to its macroscopic properties. In the food, pharmaceutical, and cosmetics industries, in particular, interfacial effects play a central role because many products are present in the form of emulsions or foams, where their overall properties are determined by two-dimensional (2D) phenomena on the microscopic level taking place at the interface.13,23,24 For such interface-driven systems, one there© 2014 American Chemical Society

fore has to know how the properties of surface-active components are altered by subtle changes in physicochemical conditions arising during production, processing, or use. With their amphiphilic character as a prerequisite,25 all proteins and aggregates thereof can be expected to adsorb to air−water or oil−water interfaces, highlighting the importance of understanding the interfacial properties of common food proteins. One of the most thoroughly studied globular food proteins is βlactoglobulin, the major whey protein in bovine milk26 and capable of forming amyloid fibrils in vitro.27 These fibrils are unbranched, multistranded, micrometer-long, semiflexible aggregates of peptides derived from the original protein monomer, with the characteristic arrangement of β strands running perpendicularly to the main fibril axis.28−32 Apart from their possible applications as structuring agents in foods,33,34 βlactoglobulin fibrils can be used as a model system to study amyloid fibrils formed by the deleterious misfolding of proteins Received: May 28, 2014 Revised: August 5, 2014 Published: August 6, 2014 10090

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Article align calculated on the real AFM image is fit as the sum Simage 2D (d) = aS2d (d) random random + (1 − a)S2D (d). Here, S2D (d) was calculated on an artificial image with fully randomly oriented chains, created using all relevant input parameters (interfacial fibril density, bending length, and contour length distribution) from the real image.22 2.3. Passive Probe Particle Tracking. The sample cell used for interfacial passive probe particle tracking at the medium chain triglycerides (MCT)−water interface consisted of an inner metal ring (2 mm inner diameter) placed within an outer plastic ring (6 mm inner diameter), both of which were glued onto a coverslip with UVcurable glue (NOA81, Norland Products, USA). A 4 μL aliquot of the aqueous phase containing 0.02 w/v% 1 μm FITC-labeled FluoSquere amine-modified tracer beads (Life Technology, Switzerland) was pipetted into the metal ring, and its surface was pinned to the upper edge of the ring before covering it with 200 μL MCT (Cognis, Germany). As the interfacial conformation of fibrils is expected to be qualitatively similar at air−water and MCT−water interfaces,22 passive probe particle tracking was performed at the oil−water interface to avoid evaporation and reduce drift. Time-lapse image sequences of tracers adsorbed at the interface were taken with a Zyla (Andor, U.K.) camera on an Axio Observer D1 microscope (Zeiss, Germany) equipped with an LD Plan-NeoFluar 63×/0.75 objective (Zeiss, Germany) at 500 ms intervals for a maximum of 1000 images. Tracers at the interface stay trapped indefinitely due to adsorption energies greatly exceeding thermal energy and never move away from the focal plane of the microscope, as opposed to tracers in the bulk that undergo 3D diffusion. By focusing the objective at the interface, only tracers at the interface were followed, as already detailed in the literature.39 To reduce vibrational noise, the microscope was placed onto a Halcyonics i4 isolation system (Accurion, Germany). Image analysis was done with standard as well as custom-written software in IDL (ITT Visual Information Solutions).46 2.4. Interfacial Shear and Dilatational Rheology. Interfacial shear rheology was performed against air on a rheometer (Physica MCR501, Anton Paar, Germany) equipped with biconical disk geometry.21,47 To investigate the development of the interfacial layer, a time sweep was performed using a 0.01 w/w% protein solution at constant strain γ = 0.1% and frequency ω = 1 Hz. To determine the network properties upon increasing strain, an amplitude sweep was performed after 15 h with increasing strain from 0.01 to 30% at a constant frequency of ω = 1 Hz. The determination of the interfacial tension and interfacial dilatational viscoelasticity was performed against air on a profile analysis tensiometer (PAT-1, Sinterface Technologies, Germany). With the setup, a drop is formed at the end of a capillary and the resulting drop shape is fitted by the Young−Laplace equation.48 To observe protein adsorption kinetics and interfacial layer buildup, a 12 h time sweep was performed by forming a drop with a constant area. After 12 h, the drop area A was oscillated at a constant frequency of 0.01 Hz with increasing amplitudes of surface area deformations ΔA of 1, 2, 4.5, 6, 8, 10, and 15%.

in humans and animals, such as those accumulating in Alzheimer’s disease or type II diabetes.35−38 Recently, βlactoglobulin fibrils have been studied in our group not only in the bulk but also at liquid interfaces,21,22,39−41 where they were found to adsorb and align into 2D liquid-crystalline domains at pH 2 (the pH at which the well-characterized fibrils are formed) and to exhibit rich linear and nonlinear interfacial rheological behavior at varying ionic strengths and pH. Fibrils formed from β-lactoglobulin have been shown to possess net charges similar to those of the native protein, with a maximum linear charge density of +20e per monomer at pH 2.42 An increase in pH results in a decrease from this average surface charge to +10e at pH 3.8 and to neutrality at the isoelectric point of around pH 5.2.42 Due to this lower repulsion among fibrils at pH values greater than 2, it is expected that fibrils can approach each other more closely, leading to a different liquid-crystalline behavior when confined to 2D. By employing in parallel three direct and independent experimental techniques covering length scales from the nanometer to millimeter range, we are able to correlate the microscopic structure of fibril-laden interfaces visualized by atomic force microscopy (AFM) and passive probe particle tracking on the mesoscale with their macroscopic properties analyzed by drop tensiometry and interfacial shear rheology. As shown previously, the presence of peptides in fibril solutions, which exist in equilibrium with the fully formed fibrils,22,43,44 can interfere with interfacial tensiometry.41 To account for this, we compare the macroscopic adsorption and rheological behavior of fibrils and β-lactoglobulin monomers. We show that a small change in the solution’s pH has dramatic consequences for the adsorption and interfacial behavior of βlactoglobulin fibrils: a slight increase in pH prevents the formation of 2D nematic phases known to form at pH 222,39 which give rise to a highly elastic rheological response of the interface. A higher solution pH of 3 promotes the development of inhomogeneous, disordered, and weaker layers. By extending the study to long-time measurements, age-induced changes in the interfacial moduli of the adsorbed material are correlated to multilayer formation and fibril breakage as elucidated by AFM.

2. EXPERIMENTAL SECTION 2.1. Fibril Formation. Fibrils were formed from native βlactoglobulin according to a previously published protocol.28 Briefly, a 10 w/w% solution of β-lactoglobulin (TU Munich, Germany)45 was dialyzed at 4 °C against pH 2 Milli-Q water in tubes with a molecular weight cutoff (MWCO) of 6−8 kDa (Spectrum Laboratories, USA) to remove ions. A 2 w/w% solution of this dialyzed protein was heated under constant agitation at 90 °C for 5 h to form fibrils. To remove unconverted peptides, the solution was dialyzed at 4 °C in membrane tubes with a MWCO of 100 kDa (Spectrum Laboratories, USA) against pH 3 Milli-Q water for 5 days. For experiments conducted at pH 2, the pH was lowered by the addition of 1 M HCl. 2.2. Atomic Force Microscopy and Image Analysis. AFM samples of the β-lactoglobulin fibril layer adsorbed at the air−solution interface were prepared by horizontal transfer using a modified Langmuir−Schaefer protocol onto a freshly cleaved piece of mica, solvent exchange with ethanol, and drying with pressurized air.22 Imaging was performed on a Nanoscope VIII multimode scanning probe microscope (Veeco Instruments) in tapping mode in air. After the fibrils’ xy coordinates were extracted from AFM images with a custom-written tracking routine, the length-scale-dependent 2D order parameter S2D(d), where d is the box size, can be calculated to obtain the amount of alignment a on the image of interest.22 To account for the presence of both aligned and randomly oriented fibrils, Simage 2D (d)

3. RESULTS AND DISCUSSION As shown in previous work, β-lactoglobulin fibrils adsorb and accumulate over time at air−water and MCT−water interfaces.21,22,39,49 Here, they align into distinct 2D nematic domains at bulk concentrations that are up to 400 times lower than reported for the 3D analogue at pH 2.50,51 While atomic force microscopy imaging and passive probe particle tracking have so far been conducted only at pH 2,22,39 interfacial rheology measurements with changing solution physicochemical conditions after fibril adsorption to the interface show that this is a highly pH-responsive system.21 By combining the above-mentioned techniques, we are now able to investigate the structure−property relationship of β-lactoglobulin fibrils at liquid interfaces. 3.1. Interfacial Structure on the Single-Molecule Length Scale. Starting from a bulk concentration of 0.01 w/ 10091

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Figure 1. AFM images of the evolution of the air−water interfacial layer of β-lactoglobulin fibrils adsorbed from a 0.01 w/w% fibril solution at pH 2 after (A) 1, (B) 2, and 5 h (C). (D) Higher-magnification image after 5 h showing multilayer formation. (E) Higher-magnification 3D height projection showing the aligned fibril layer that was directly at the liquid interface (darker fibrils; direction indicated by orange arrow) running horizontally and another aligned layer that adsorbed underneath and orthogonally to the first layer (lighter fibrils; direction indicated by red arrow). (F) Higher-magnification image after 5 h showing fibril breakage. All scale bars correspond to 1 μm.

Figure 2. AFM images of the evolution of the air−water interfacial layer of β-lactoglobulin fibrils adsorbed from a 0.01 w/w% fibril solution at pH 3 after (A) 1, (B) 3, and (C) 22 h, showing large entanglements and inhomogeneous coverage at long adsorption times. All scale bars correspond to 1 μm.

w%, AFM imaging of fibrils adsorbed to the air−water interface for 1 and 2 h shows a very dense layer of highly aligned fibrils bending collectively into 2D nematic domains (Figure 1A,B). Calculating the length-scale-dependent 2D order parameter S2D(d) on these images allows an estimation of the amount of alignment a on the interface.22 We find a ≈ 0.7, indicating that 70% of the interface is covered in aligned fibrils while 30% is due to disorder, i.e., isotropically arranged filaments. Fibrils lying on top (as seen in the AFM images) of the nematically aligned fibrils can already be seen after short adsorption times (Figure 1A and ref 22.). These fibrils, which are actually adsorbed below the topmost interfacial fibril layer in the original sample, grow in number over time and finally also align into a second, less-dense layer of nematic fibril domains (Figure 1D,E). Because of this, the fibrils that had adsorbed to the air− water interface first might be constrained in their in-plane

movement and pushed more toward the hydrophobic phase, causing them to fracture into shorter but still aligned fibrils, as shown in Figure 1F. Starting from a solution with the same initial fibril concentration but at a slightly higher pH of 3, we find a very different behavior: The fibrils still adsorb to the air−water interface, but they no longer align into well-defined nematic domains (Figure 2A). Instead, they can be seen to entangle and overlap on most of the interface, while only very few fibrils align parallel to each other locally at longer times (Figure 2B,C). Because the adsorption layer is no longer 2D and the interface is highly heterogeneously covered, S2D(d) cannot be used for a meaningful evaluation of the order in these images. 3.2. Mesoscale Structure. Since a similar microstructure is expected at air−water and oil−water interfaces,22 a direct comparison of AFM, rheological, and tensiometric measure10092

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Figure 3. Passive probe particle tracking spatial maps of the MCT−0.01 w/w% β-lactoglobulin fibril solution interface (A) at pH 2 after 54 min and (B) at pH 3 after 64 min. (C) Mean square displacements ⟨x2 + y2⟩ versus time for each tracer particle in the image sequence used in A. The solid black reference line in the upper left corner of panel C has a slope of 1, indicating purely diffusive behavior. (D) Decomposition of ⟨x2 + y2⟩ into ⟨x2⟩ (main direction of motion, dashed line) and ⟨y2⟩ (transverse direction of motion, dotted line) versus time for the anisotropically moving tracer highlighted in A. (E) Mean square displacements ⟨x2 + y2⟩ versus time for each tracer particle in the image sequence used in B.

Figure 4. Adsorption kinetics at the air−water interface obtained from tensiometry (A) and interfacial elastic moduli G′i from time sweep interfacial shear rheology (ω = 1 Hz, γ = 0.1%) (B) of β-lactoglobulin monomers at pH 2 (blue), fibrils at pH 2 (red), and fibrils at pH 3 (orange) at a bulk concentration of 0.01 w/w%.

confirmed by the individual tracer particle’s mean square displacements (Figure 3C,E). All tracers at the fibril-covered interface at pH 3 exhibit subdiffusive motion because only small cages in the entangled fibril layer are probed, and the mean square displacements remain very limited compared to the pH 2 case (Figure 3E). In contrast, at pH 2, even though the interfacial fibril layer is dense, the tracers still have the freedom to move along the director of the domains. This is reflected in their short-time diffusive behavior in 1D and subdiffusive motion at longer times (Figure 3C). Those tracers which are able to follow the local director of the nematic domain in its immediate vicinity for longer distances show an overall higher mean square

ments performed at the air−water interface with passive probe particle tracking at the MCT−water interface is feasible. The latter technique can help to reveal the structure of the fibrilcovered interface over areas larger than possible with AFM, and we find that the patterns seen in AFM are also discernible on this larger scale (Figure 3). At pH 2, tracer particles apparently trapped and immobile among the fibrils adsorbed at the interface (for example, in between nematic domains with different directors) coexist with some forced to move anisotropically along the local director of a nematic fibril domain (Figure 3A). At the disordered interface between MCT and the pH 3 fibril solution (Figure 3B), the tracers are caged within the disordered fibril network. These observations are 10093

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Figure 5. (A) Dilatational storage and loss moduli Ei′ and Ei″ obtained from dilatational rheology and (B) interfacial elastic and viscous moduli Gi′ and Gi″ from strain sweep interfacial shear rheology (ω = 1 Hz and γ = 0.01−30%) of the air−water interfacial layer of β-lactoglobulin monomers at pH 2 (blue), fibrils at pH 2 (red), and fibrils at pH 3 (orange) at an initial bulk concentration of 0.01 w/w%.

pH 3 are depicted in Figure 4B. For β-lactoglobulin fibrils at pH 2, the interfacial elastic modulus Gi′ increases at the beginning, reaching a peak after 1-2 h and decreases afterward. This behavior can be explained by the structural evolution of the interfacial fibril film observed in the AFM images (Figure 1). With increasing adsorption time more and more fibrils adsorb at the interface, and for time scales over 2 h, multilayers with broken fibrils were observed, weakening the interfacial layer. This fracture of fibrils decreases the interfacial elasticity. When the interfacial viscoelasticities of the monomers and the fibrils are compared, a much stronger interfacial layer can be observed for the fibrils than for the monomers. Although both systems contain the same protein material, the fibrils are longer and therefore possess a better ability to form a network than the monomers. However, fibrils at pH 3 show a completely different adsorption layer buildup compared to those at pH 2. A three-fold-lower value of G′i is measured after a 15 h time sweep at pH 3. Even though more overlapping was observed in the AFM images of fibrils at pH 3 (Figure 2), at long times, the surface coverage is heterogeneous, with highly entangled fibrils coexisting with more sparsely covered areas (Figure 2C). Thus, the network is weaker than at pH 2, where multilayer formation and more uniform coverage were found. 3.4. Macroscopic Rheological Behavior. To further characterize the protein monomer and fibril adsorption layers, the interfacial tension response during dilatational strain sweeps was measured after full layer formation in order to obtain elastic and viscous moduli Ei′ and Ei″ (Figure 5A). At both pH 2 and 3, fibrils have similar viscous and elastic moduli but the monomers have a much smaller loss modulus and also the smallest elastic modulus. All three systems have similar final surface tensions. As observed in refs 22 and 41, a multilayer is formed with the peptides directly at the interface and the fibrils below or adsorbed at the interface together. Therefore, the peptides, which adsorb faster than the fibrils due to their smaller size, are the driving force for the interfacial tension reduction, but the fibrils determine the interfacial viscosity, which is higher for the fibril systems (independent of the presence of peptides) than for the monomers. Since the monomers are much smaller than the fibrils, they show fewer interactions and thus a smaller E″i . For all systems containing fibrils, the linear viscoelastic regime could be observed up to a strain of 4%, which is in agreement with the findings of Rühs et al.41 investigating β-lactoglobulin fibrils, peptides, and monomers in water against MCT. Above this strain, structural rearrangements seem to interfere, causing nonlinear behavior. The monomers exhibit linear behavior up

displacement. Decomposing the mean square displacement into the motion occurring in the principal direction ⟨x2⟩ and into the transverse direction of motion ⟨y2⟩ shows that this high degree of mobility stems almost exclusively from the motion along the local director (Figure 3D). Albeit anisotropic, the motion of the tracers at the interface remains purely diffusive along the principal direction of motion (before caging effects take place due to the surrounding elastic protein network),39 indicating that the anisotropy stems from the microstructure of the local environment and not from bulk convection. 3.3. Macroscopic Adsorption Behavior. Moving on to the next larger length scale, the effect of a change in solution pH on the interfacial behavior of fibrils is analyzed by pendant drop tensiometry. As mentioned above, it is worth comparing the adsorption behavior at the solution surface of the two fibril systems to that of β-lactoglobulin monomers at pH 2 to account for the presence of nonfibrillar protein material in the solutions (Figure 4A). The surface tension is reduced by all βlactoglobulin systems, reaching a constant value after 8 h. A pronounced difference can be observed when comparing the adsorption behavior of β-lactoglobulin fibrils and β-lactoglobulin monomers as shown in Figure 4A. This can be explained by the presence of peptides in the fibril solutions, which are much smaller than the protein monomer and therefore adsorb faster, decreasing the surface tension more rapidly and thus dominating the adsorption behavior. The monomers, on the other hand, present a slower decrease in surface tension, finally resulting in the lowest surface tension of all investigated systems. As the monomers adsorb at the interface in their native state, they slowly unfold at the interface. This process takes more time but finally leads to a lower surface tension.27 Compared to fibrils at pH 2, fibrils at pH 3 show a slower adsorption rate at the interface, indicating a lower free peptide content at this higher pH, where fibrils have a lower linear charge density,42 and inferring that the peptide−fibril equilibrium is shifted to the fibril side at pH 3. This is supported by the AFM images, where the fibrils were seen to be more resistant to bending and fracture at pH 3 than at pH 2 (Figures 1 and 2), as well as by supramolecular polyelectrolyte theories, for which a breaking of the fibrillar structure can be expected at very large linear charge densities. The accumulation of proteins or fibrils at the interface leads to the buildup of an adsorption layer which can be measured with interfacial shear rheology. The transient interfacial storage moduli during a 15 h time sweep (ω = 1 Hz, γ = 0.1%) for βlactoglobulin monomers and β-lactoglobulin fibrils at pH 2 and 10094

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Scheme 1. Schematic Summary of the Three Protein Systems Investigated and Their Interfacial Structure, Adsorption Behavior, and Viscoelastic Properties Observed at Short and Long Adsorption Times

to higher strains, even at a 15% area increase, which is due to their different morphology and smaller size. The higher Ei′ of fibrils at pH 3 than at pH 2 can again be explained by observations made using AFM and passive probe particle tracking: due to the high surface charge density at pH 2, fibrils have fewer direct contact points, whereas at pH 3 more entangling occurs. Together with the absence of fibril breaking due to multilayer formation, this leads to a higher elasticity at pH 3. After the 15 h time sweeps in interfacial rheology, strain amplitude sweeps (ω = 1 Hz and γ = 0.01−30%) were performed to gain further insight into the interfacial network properties (Figure 5B). In all β-lactoglobulin adsorption layers, Gi′ is higher than Gi″ up to strains of 7%. For the monomers, simple strain-thinning behavior can be observed, with a linear regime at strains of up to 2%, as the monomers form only a weak interfacial layer with few interaction points. Both βlactoglobulin fibril systems show strain independence at shear deformations of up to 2−6%, after which G′i decreases with increasing strain. Due to the increased size and aspect ratio, fibrils interact more with each other, leading to a weak strain overshoot characterized by the local maximum in G″i at higher deformations. β-lactoglobulin fibrils at pH 2 show a stronger network elasticity due to less surface entanglement and strong multilayer formation. In summary, the strongest network formation (highest elastic moduli) was measured by interfacial shear rheology at pH 2. In contrast to this, the highest dilatational elastic moduli were measured for fibrils at pH 3. This apparent mismatch in the results of shear and dilatational rheology is due to completely different stress profiles at the interface and demonstrates the complexity of the phenomena involved. Directly at the interface of the expanding and compressing drop, several timedependent surface phenomena such as reorientation, relaxation, and order−disorder phase transitions might occur. Driven by concentration gradients and the free surface area, protein

adsorption from the multilayers observed in AFM or the subphase might interfere in the dilatational rheology.41,52,53

4. CONCLUSIONS We have attempted to link the microstructure of a complex liquid interface to its macroscopic properties by combining AFM, passive probe particle tracking, interfacial rheology, and tensiometry. By doing so for β-lactoglobulin amyloid fibrils as well as β-lactoglobulin monomers (Scheme 1), the consequences of a small increase in pH are discussed across the various length scales involved. At pH 2, the highly charged fibrils adsorb at air−water and oil−water interfaces, resulting in their 2D alignment into nematic domains as their interfacial concentration increases and a very elastic interface is created. As more fibrils adsorb into multilayers, fibril fracture in the topmost layer is observed in AFM and results in a decrease in interfacial elasticity and finally strain thinning above 2% deformation at long times. At pH 3, where the fibrils’ surface charge density is reduced by almost half, fibrils are more inclined to overlap at the interface, leading to the absence of extensive nematic domains and the buildup of a weaker elastic interfacial layer with strain-thinning behavior above 2% deformation. Because of this aggregation at a higher pH, the dilatational viscoelasticity of the less acidic fibril-covered interface is higher than that at pH 2. By comparing these results to the rheological and tensiometric data of βlactoglobulin monomers, we draw further conclusions concerning the fibrils’ propensity to fracture, finally giving a comprehensive picture of fibrillar interfacial behavior at varying physicochemical conditions across the length scales in order to help elucidate the structural characteristics underlying the overall material properties.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: peter.fi[email protected]. *E-mail: raff[email protected]. 10095

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.J. and P.A.R. acknowledge financial support from ETH Zurich (ETHIIRA TH 32-1), and L.I., from Swiss National Science Foundation grants PP00P2_144646/1 and PZ00P2_142532/1. Prof. Erich Windhab is acknowledged for inspiring discussions.

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