Β-lactoglobulin Amyloid Fibrils Research Articles

Food amyloid fibrils are safe nutrition ingredients based on in-vitro and in-vivo assessment

Food protein amyloid fibrils have superior technological, nutritional, sensorial, and physical properties compared to native monomers, but there is as yet insufficient understanding of their digestive fate and safety for wide consumption. By combining SDS-PAGE, ELISA, fluorescence, AFM, MALDI-MS, CD, microfluidics, and SAXS techniques for the characterization of β-lactoglobulin and lysozyme amyloid fibrils subjected to in-vitro gastrointestinal digestion, here we show that either no noticeable conformational differences exist between amyloid aggregates and their monomer counterparts after the gastrointestinal digestion process (as in β-lactoglobulin), or that amyloid fibrils are digested significantly better than monomers (as in lysozyme). Moreover, in-vitro exposure of human cell lines and in-vivo studies with C. elegans and mouse models, indicate that the digested fibrils present no observable cytotoxicity, physiological abnormalities in health-span, nor accumulation of fibril-induced plaques in brain nor other organs. These extensive in-vitro and in-vivo studies together suggest that the digested food amyloids are at least equally as safe as those obtained from the digestion of corresponding native monomers, pointing to food amyloid fibrils as potential ingredients for human nutrition.

β-Lactoglobulin amyloid fibrils supported Fe(III) to activate peroxydisulfate for organic pollutants elimination

In this study, Fe(III) was successfully loaded on the β-Lactoglobulin amyloid fibrils (Fe(III)@β-LGAF) by the in-situ method for efficiently activating peroxydisulfate (PDS) to remove organics in water. Methyl orange (MO) was used as the model contaminant to explore the influences of key parameters in the Fe(III)@β-LGAF/PDS system, including the Fe(III)@β-LGAF dose, PDS dosage, initial MO concentration, and beginning pH value. The results indicated that 94.2% MO was eliminated within 20 min under the optimized conditions of 1 g/L Fe(III)@β-LGAF, 1 mmol/L PDS, 30 mg/L MO, and pH 5. The synthesized Fe(III)@β-LGAF manifested a certain excellent stability and applicability for the PDS activation and other typical organic pollutants degradation. Various characterization methods were applied to characterize the properties of Fe(III)@β-LGAF before and after the oxidation reaction. The radical scavenging test and electron paramagnetic resonance analyses testified that singlet oxygen was the dominant active species in the synergistic process, which exhibited a stronger selective oxidation capacity than the hydroxyl and sulfate radicals. Hence, the synthetic material Fe(III)@β-LGAF could provide a new reference for the heterogeneous Fenton-like reaction.

Covalent β-lactoglobulin-maltodextrin amyloid fibril conjugate prepared by the Maillard reaction

The surface modification of β-lactoglobulin amyloid fibrils (AFs) was investigated by performing the Maillard reaction with the free anomeric carbon of the maltodextrin in water at pH 9.0 and 90 °C. The bonding of maltodextrin to fibrils was confirmed by determining the free amino group content and the presence of final products from the Maillard reaction. The secondary structure of AFs was preserved as observed by circular dichroism analysis. Atomic force microscopy evidenced that prolonged heat treatment caused hydrolysis of the attached polysaccharide and consequently lowered the height of the fibrils from 8.0 nm (after 1 h) to 6.0 nm (after 24 h), which led to the reduction of hydrophilicity of resulting conjugate. Increasing the reaction time, however, resulted in the improvement of colloidal stability and decrease in turbidity ascribed to the increment of glycation degree, as well as, a decrease in the isoelectric point of the protein-based supramolecular object.

Assessing the Binding Performance of Amyloid-Carbon Membranes toward Heavy Metal Ions.

Amyloid-carbon hybrid membranes have exceptional performance in removing heavy metal ions from water because of the presence of multiple binding sites on the amyloid fibrils, but the binding process is still not fully understood. To understand the mechanisms of amyloid-metal ion binding, we perform adsorption isotherms on a model system given by β-lactoglobulin amyloid fibrils and four representative heavy metal ions: chromium (Cr), nickel (Ni), silver (Ag), and platinum (Pt). Furthermore, to get a comprehensive thermodynamic picture of the binding process between amino acid residues and heavy metals, we here use isothermal titration calorimetry on native β-lactoglobulin monomers and amyloid fibrils exposed to the two model metal ions, that is, silver and chromium. A conclusive thermodynamic insight on the binding process emerges by direct measurements of enthalpy and entropy changes, association binding constant, and average number of binding sites of the protein monomer and amyloid fibril. As a result of the strong amyloid binding affinity between amino acids and metal ions, when the protein is converted into amyloid fibrils and assembled into membranes, the resulting amyloid-activated carbon hybrids remove all the tested heavy metals with efficiencies beyond 99%. Importantly, the efficiency remains stable during several consecutive cycles, demonstrating a high adsorption capacity and a long lifetime and reusability of the membranes. The recovery of adsorbed precious metal ions converted into elemental metals is shown to be a general feature of these membranes, with platinum and silver successfully recovered from saturated hybrid membranes by a simple thermal reduction. The separation performance, evaluated on real electroplating industrial wastewater containing chromium and nickel, is found to exceed 99% at a permeability as high as 2.92 × 10-16 m2, that is, at least 4 orders of magnitude higher than typical nanofiltration membranes, conclusively validating the technology under stringent real conditions.

Amyloid Fibrils Length Controls Shape and Structure of Nematic and Cholesteric Tactoids.

Amyloid fibrils offer the possibility of controlling their contour length, aspect ratio, and length distribution, without affecting other structural parameters. Here we show that a fine control in the contour length distribution of β-lactoglobulin amyloid fibrils, achieved by mechanical shear stresses of different levels, translates into the organization of tactoids of different shapes and morphologies. While longer fibrils lead to highly elongated nematic tactoids in an isotropic continuous matrix, only sufficiently shortened amyloid fibrils lead to cholesteric droplets. The progressive decrease in amyloid fibrils length leads to a linear decrease of the anchoring strength and homogeneous tactoid → bipolar tactoid → cholesteric droplet transitions. Upon fibrils length increase, we first find experimentally and predict theoretically a decrease of the cholesteric pitch, before full disappearance of the cholesteric phase. The latter is understood to arise from the decrease of the energy barrier separating cholesteric and nematic phases over thermal energy for progressively longer, semiflexible fibrils.

Continuous Isotropic-Nematic Transition in Amyloid Fibril Suspensions Driven by Thermophoresis

The isotropic and nematic (I + N) coexistence for rod-like colloids is a signature of the first-order thermodynamics nature of this phase transition. However, in the case of amyloid fibrils, the biphasic region is too small to be experimentally detected, due to their extremely high aspect ratio. Herein, we study the thermophoretic behaviour of fluorescently labelled β-lactoglobulin amyloid fibrils by inducing a temperature gradient across a microfluidic channel. We discover that fibrils accumulate towards the hot side of the channel at the temperature range studied, thus presenting a negative Soret coefficient. By exploiting this thermophoretic behaviour, we show that it becomes possible to induce a continuous I-N transition with the I and N phases at the extremities of the channel, starting from an initially single N phase, by generating an appropriate concentration gradient along the width of the microchannel. Accordingly, we introduce a new methodology to control liquid crystal phase transitions in anisotropic colloidal suspensions. Because the induced order-order transitions are achieved under stationary conditions, this may have important implications in both applied colloidal science, such as in separation and fractionation of colloids, as well as in fundamental soft condensed matter, by widening the accessibility of target regions in the phase diagrams.

Determination of the elastic modulus of β-lactoglobulin amyloid fibrils by measuring the Debye-Waller factor

Although amyloid fibrils are associated with amyloidoses, they are now being considered as novel biomaterials for industrial use due to their structural stability in the matured state. Therefore, the physical characteristics of these materials need to be clarified prior to their industrial application. In the present study, the mechanical properties of amyloid fibrils precursored by β-lactoglobulin were investigated. Previous studies have examined the stiffness or modulus values of these fibrils using atomic force microscopy. However, the modulus values reported, even for amyloid fibrils from the same precursor proteins, range over three orders of magnitude, from a few MPa to GPa, depending on the experimental methods employed under specific loading conditions. We determined the elastic modulus of amyloid fibrils by measuring spontaneous thermal fluctuations in the material, the Debye-Waller factor. This method does not require any contact between the probe and material or any loading. The vibrational modes of a fibril were considered in order to estimate mechanical parameters. The modulus value determined along the fibril axis for single amyloid fibrils was slightly smaller than those reported in the literature. The smaller modulus value suggests the existence of less ordered proto-fibrils in our specimen, which was confirmed by the AFM images.

Self-assembled amyloid fibrils with controllable conformational heterogeneity.

Amyloid fibrils are a hallmark of neurodegenerative diseases and exhibit a conformational diversity that governs their pathological functions. Despite recent findings concerning the pathological role of their conformational diversity, the way in which the heterogeneous conformations of amyloid fibrils can be formed has remained elusive. Here, we show that microwave-assisted chemistry affects the self-assembly process of amyloid fibril formation, which results in their conformational heterogeneity. In particular, microwave-assisted chemistry allows for delicate control of the thermodynamics of the self-assembly process, which enabled us to tune the molecular structure of β-lactoglobulin amyloid fibrils. The heterogeneous conformations of amyloid fibrils, which can be tuned with microwave-assisted chemistry, are attributed to the microwave-driven thermal energy affecting the electrostatic interaction during the self-assembly process. Our study demonstrates how microwave-assisted chemistry can be used to gain insight into the origin of conformational heterogeneity of amyloid fibrils as well as the design principles showing how the molecular structures of amyloid fibrils can be controlled.

Universal behavior in the mesoscale properties of amyloid fibrils.

Amyloid fibrils are ubiquitous proteinaceous aggregates occurring invivo and invitro, with an invariant structural fingerprint at the molecular length scale. However, interpretation of their mesoscopic architectures is complicated by diverse observable polymorphic states. We here present a constitutive model for amyloid fibrils based on the minimization of the total energy per fibril. The model is benchmarked on real amyloid fibrils studied by atomic force microscopy. We use multistranded β-lactoglobulin amyloid fibrils as a model system exhibiting a rich polymorphism. The constitutive model quantitatively recapitulates the main mesoscopic topological features of amyloid fibrils, that is, the evolution of fibril periodicity as a function of the ionic strength of the solution and of the fibril width. A universal mesoscopic structural signature of the fibrils emerges from this picture, predicting a general, parameter-free law for the periodicity of the fibrils, that depends solely on the number of protofilaments per fibril. These predictions are validated experimentally and conclusively highlight the role of competing electrostatic and elastic contributions as the main players in the establishment of amyloid fibrils structure.

Magnetic-Responsive Hybrids of Fe3O4 Nanoparticles with β-Lactoglobulin Amyloid Fibrils and Nanoclusters

We report on the synthesis and magnetic-responsive behavior of hybrids formed by dispersing negatively charged iron oxide (Fe3O4) magnetic nanoparticles in positively charged β-lactoglobulin protein solutions at acidic pH, followed by heating at high temperatures. Depending on the pH used, different hybrid aggregates can be obtained, such as nanoparticle-modified amyloid fibrils (pH 3) and spherical nanoclusters (pH 4.5). We investigate the effect of magnetic fields of varying strengths (0-5 T) on the alignment of these Fe3O4-modified amyloid fibrils and spherical nanoclusters using a combination of scattering, birefringence and microscopic techniques and we find a strong alignment of the hybrids upon increasing the intensity of the magnetic field, which we quantify via 2D and 3D order parameters. We also demonstrate the possibility of controlling magnetically the sol-gel behavior of these hybrids: addition of salt (NaCl, 150 mM) to a solution containing nanoparticles modified with β-lactoglobulin amyloid fibrils (2 wt % fibrils modified with 0.6 wt % Fe3O4 nanoparticles) induces first the formation of a reversible gel, which can then be converted back to solution upon application of a moderate magnetic field of 1.1 T. These hybrids offer a new appealing functional colloidal system in which the aggregation, orientational order and rheological behavior can be efficiently controlled in a purely noninvasive way by external magnetic fields of weak intensity.

Gelation, Phase Behavior, and Dynamics of β-Lactoglobulin Amyloid Fibrils at Varying Concentrations and Ionic Strengths

We have investigated the thermodynamic and dynamic behavior of multistranded β-lactoglobulin protein fibrils in water, by combining static, dynamic, and depolarized dynamic light scattering (SLS, DLS, DDLS), small angle neutron scattering (SANS), rheology, and cryogenic transmission electron microscopy (cryo-TEM). We focus on the region of the phase diagram at which ionic strength and concentration changes induce transitions in gelation and lyotropic liquid crystalline behavior. An increase in ionic strength, induced by NaCl salt, progressively causes the phase transitions from nematic (N) to gel (G) phases; a further increase causes the transition to a translucent phase and to a macroscopic phase separation, respectively. An increase in fibril concentration induces first a phase transition from an isotropic (I) to a nematic phase (N); a further increase induces the formation of a gel phase. The protein gel strength is investigated by rheology measurements. SANS and osmotic compressibility calculated by SLS measurements clearly capture the main features of the IN transition of β-lactoglobulin protein fibrils. The form and structure factors measured by scattering experiments are analyzed by the polymer reference interaction site model (PRISM). Dynamics of the protein fibrils at different concentrations, measured by polarized and depolarized dynamic light scattering, show both individual and collective diffusion after the isotropic-nematic transition. Above this transition, cryo-TEM images further demonstrate the alignment of the protein fibrils, which is quantified by a 2D order parameter. This work discusses comprehensively, both experimentally and theoretically, the thermodynamics and dynamic features of β-lactoglobulin amyloid fibrils in a vast region of the concentration-ionic strength phase diagram.

Morphology and Persistence Length of Amyloid Fibrils Are Correlated to Peptide Molecular Structure

The formation of amyloid fibrils is a self-assembly process of peptides or proteins. The superior mechanical properties of these fibrils make them interesting for materials science but constitute a problem in amyloid-related diseases. Amyloid structures tend to be polymorphic, and their structure depends on growth conditions. To understand and control the assembly process, insights into the relation between the mechanical properties and molecular structure are essential. We prepared long, straight as well as short, worm-like β-lactoglobulin amyloid fibrils and determined their morphology and persistence length by atomic force microscopy (AFM) and the molecular conformation using vibrational sum-frequency generation (VSFG) spectroscopy. We show that long fibrils with near-100% β-sheet content have a 40-times higher persistence length than short, worm-like fibrils with β-sheet contents below 80%.

Amyloid-mediated synthesis of giant, fluorescent, gold single crystals and their hybrid sandwiched composites driven by liquid crystalline interactions

We report for the first time on the templating effect of β-lactoglobulin amyloid-like fibrils to synthesize gold single crystals of several decades of μm in dimensions. The gold single crystals were produced by reducing an aqueous solution of chloroauric acid by β-lactoglobulin amyloid protein fibrils. Atomic force microscopy, conventional and scanning transmission electron microscopy, electron diffraction and optical microscopy techniques were combined to characterize the structure of the gold crystals. The single-crystalline features of these macroscopic gold crystals are witnessed by their distinctive hexagonal and triangular shape and are confirmed by selected area electron diffraction (SAED). UV–vis absorption spectrum, recorded after a reaction time of 6h at the heating temperature of 55°C showed a surface plasmon resonance peak at 540nm. With the increase of reaction time to 24h, the absorption spectrum peaks shift to a very broad and higher wavelength region extending up to near infrared region. Remarkably, these single crystalline gold crystals show auto fluorescence when illuminated to UV lamp. Further increase in β-lactoglobulin amyloid fibrils concentration above the isotropic-nematic transition, drives the formation of gold single crystals microplates stacking together and self-assembling into new hierarchical, layered protein-gold hybrid composites.

Single-step direct measurement of amyloid fibrils stiffness by peak force quantitative nanomechanical atomic force microscopy

We present an original application of a new atomic force microscopy mode called peak force tapping for the investigation of the mechanical properties of β-lactoglobulin amyloid fibrils. The values of Young’s modulus obtained by this technique are in perfect agreement with the indirect evaluation of fibrils stiffness obtained by combining polymer physics and topological statistical analysis on fibrils’ structural conformations. This technique shows great promise in the estimation of the elastic properties of nanostructured objects relevant in biology, soft matter, and nanotechnology.

Adjustable twisting periodic pitch of amyloid fibrils

We present compelling experimental evidence supported by theoretical arguments demonstrating that the periodic twisting pitch in protein amyloid fibrils arises from the fine balance between competing electrostatic energy and torsional elastic energy stored along the fibrils contour length. To construct the present picture we have used increasing ionic strengths to progressively screen the electrostatic interactions and observed the corresponding pitch variations in mature β-lactoglobulin amyloid fibrils using single-molecule atomic force microscopy (AFM). Because the ionic strength is changed afterfibrils formation, this does not affect the mechanism by which fibrils grow up to their mature structure. For each individual population of the multi-stranded fibrils family, the pitch is found to increase systematically with the salt content, leading to the gradual untwisting of the fibrils and to the establishment of a controllable pitch up to virtually infinite values.

Unravelling adsorption and alignment of amyloid fibrils at interfaces by probe particle tracking

We report the first direct, non-invasive experimental evidence of a 2D isotropic–nematic transition for highly anisotropic nanoparticles at liquid–liquid interfaces by using passive fluorescent particle tracking. In order to illustrate the potential of this approach on systems of high real practical and biological relevance, we select as a model anisotropic nanoparticles β-lactoglobulin amyloid fibrils of varying aspect ratios. Upon nanoparticle adsorption at the interface, we follow, in real time and as a function of fibril length and bulk concentration, the development of a 2D nematic phase by studying the anisotropy in probe particle traces. We furthermore demonstrate the long-range nature of the nematic phase by calculating order parameters for the traces as high as 0.8 over 102 to 103μm2 areas. The presented route is independent of the system investigated, and thus these findings open a new, general strategy for the experimental assessment of 2D structural changes at anisotropic fluid–fluid interfaces.

Shear-induced structure and mechanics of β-lactoglobulin amyloid fibrils

Alzheimer first identified amyloid plaques during autopsies of the brains of dementia patients in 1905. Considerable effort has since been directed to understanding the composition, structure and formation mechanism of amyloid fibrils. A variety of proteins have now been shown to self-assemble into amyloid fibrils. Furthermore, the association between misfolded proteins and a wide range of diseases is now established. Here we examine the effect of shear on the formation, structure and mechanical properties of amyloid fibrils. Atomic force microscopy is used to both image and measure the nanomechanical properties of β-lactoglobulin (β-Lg) amyloid fibrils generated in controlled (Couette) and uncontrolled shear flows (stirring). Controlled shear-induced fibrils with ‘beaded’ morphologies have lower mechanical strengths than fibrils formed in uncontrolled shear which show twisted ribbon like morphologies. The mechanical strengths of amyloid fibrils are determined by fibril structure and vary depending on the type of shear exposure during in vitro formation. These findings may have implications regarding the different toxicities of amyloid fibrils and linear aggregates that form in vivo.