Surface Of Amyloid Fibrils Research Articles

The Rigid Core and Flexible Surface of Amyloid Fibrils Probed by Magic-Angle-Spinning NMR Spectroscopy of Aromatic Residues.

Aromatic side chains are important reporters of the plasticity of proteins, and often form important contacts in protein-protein interactions. We studied aromatic residues in the two structurally homologous cross-β amyloid fibrils HET-s, and HELLF by employing a specific isotope-labeling approach and magic-angle-spinning NMR. The dynamic behavior of the aromatic residues Phe and Tyr indicates that the hydrophobic amyloid core is rigid, without any sign of "breathing motions" over hundreds of milliseconds at least. Aromatic residues exposed at the fibril surface have a rigid ring axis but undergo ring flips on a variety of time scales from nanoseconds to microseconds. Our approach provides direct insight into hydrophobic-core motions, enabling a better evaluation of the conformational heterogeneity generated from an NMR structural ensemble of such amyloid cross-β architecture.

Der starre Kern und die flexible Oberfläche von Amyloidfibrillen – Magic‐Angle‐Spinning NMR Spektroskopie von aromatischen Resten

AbstractAromatische Seitenketten sind wichtige Indikatoren für die Plastizität von Proteinen und bilden oft entscheidende Kontakte bei Protein‐Protein‐Wechselwirkungen. Wir untersuchten aromatische Reste in den beiden strukturell homologen cross‐β Amyloidfibrillen HET‐s und HELLF mit Hilfe eines spezifischen Ansatzes zur Isotopenmarkierung und Festkörper NMR mit Drehung am magischen Winkel. Das dynamische Verhalten der aromatischen Reste Phe und Tyr deutet darauf hin, dass der hydrophobe Amyloidkern starr ist und keine Anzeichen von “atmenden Bewegungen” auf einer Zeitskala von Hunderten von Millisekunden zeigt. Aromatische Reste, die exponiert an der Fibrillenoberfläche sitzen, haben zwar eine starre Ringachse, weisen aber Ringflips auf verschiedenen Zeitskalen von Nanosekunden bis Mikrosekunden auf. Unser Ansatz bietet einen direkten Einblick in die Bewegungen des hydrophoben Kerns und ermöglicht eine bessere Bewertung der Konformationsheterogenität, die aus einem NMR‐Strukturensemble einer solchen Cross‐β‐Amyloidstruktur hervorgeht.

Electroabsorption and Stark Fluorescence Spectroscopies of Thioflavin T.

Thioflavin T (ThT) is a typical fluorescent marker for detecting the formation of amyloid fibrils, because its fluorescence intensity increases by more than 2 orders of magnitude upon complexation with the fibrils. Strong electrostatic fields on protein surfaces are known to be a significant factor in chemical reactions and biological functions. Therefore, ThT bound to amyloid fibrils must experience strong electric fields. This study employed electroabsorption and Stark fluorescence spectroscopies to clarify the effects of external electric fields on the photophysics of ThT. The absorption spectrum shows two bands ascribed to locally excited (LE) and charge transfer (CT) states. Coupling between the LE and CT states is enhanced in the presence of an external electric field, resulting in fluorescence quenching. The electric field strength of the amyloid fibril surface was inferred from the fluorescence quenching efficiency of ThT.

Disulfide-Mediated Elongation of Amyloid Fibrils of α-Synuclein For Use in Producing Self-Healing Hydrogel and Dye-Absorbing Aerogel

Due to their mechanical robustness, biocompatibility, and nanoscale size, amyloid fibrils (AFs) have been considered as a potential nanomaterial for biological applications. Unfortunately, however, AFs are usually not fully extended because of their pre-mature breakage, which hampers their use to generate biocompatible suprastructures, although the amounts of AFs could be amplified via their self-propagation property. Here, we have demonstrated the full extension of AFs of α-synuclein (αS) by introducing a cysteine residue to its C-terminus which prevents the shear-induced fragmentation of AFs via site-directed disulfide bond formation on the exposed surface of AFs. These heat- and cold-resistant elongated AFs were entangled into self-healable hydrogels through a mild disulfide-exchange process in the presence of tris(2-carboxyethyl) phosphine, which subsequently developed into dye-absorbing aerogels upon freeze-drying without collapsing the three-dimensional internal fibrillar network. The resulting αS aerogel with high porosity and increased surface area was shown to be capable of absorbing both hydrophilic and hydrophobic substances. In addition, the aerogel was further engineered with 8-arm polyethylene glycol containing a sulfhydryl group to increase its drug loading capacity and protease susceptibility for drug unloading. The elongated AFs, therefore, have been suggested to play a pivotal component for the development of bio-nano-matrix for diverse biological applications including drug delivery, tissue engineering, and environmental remediation. Statement of significanceDue to accurate protein self-assembly process, α-synuclein forms an amyloid fibril which are the major component of Lewy bodies. In general, α-synuclein amyloid fibrils break under thermal fluctuations as these nanofibrils elongate to reach certain length. In this study, we have demonstrated the full extension of α-synuclein amyloid fibrils by introducing a cysteine residue to its C-terminus by forming site-directed disulfide bonds on the exposed surface of amyloid fibrils for the first time. The resulting elongated amyloid fibrils were mechanically robust and stable. By using elongated amyloid fibrils, we have made self-healable amyloid fibril hydrogel and dye-absorbing aerogel.

Higher affinity of polyphenol to zein than to amyloid fibrils leading to nanoparticle-embed network wall scaffold to construct amyloid fibril-zein-EGCG hydrogels for coating of beef

Globally, 800 million are undernourished and 2 billion are deficient in micronutrients. A regular dietary intake of meat is one of effective strategies to fight against undernourishment and deficiency of micronutrients. However, meat is a typical perishable food; proper handling to extend the shelf life of meat is required. In the present study, homogenous hydrogels composed with high contents of lysozyme amyloid fibrils, zein and green tea polyphenol epigallocatechin gallate (EGCG) were developed via the orchestration of protein–protein interaction and polyphenol-protein interaction for coating of beef. The protein–protein interaction between amyloid fibrils and zein showed higher affinity than that of the polyphenol-protein interactions. In addition, polyphenol EGCG was found to be more inclined to bind with zein than to the amyloid fibrils. Thus, the amyloid fibrils performed as the scaffold, in which EGCG on one hand induced the zein aggregate nanoparticles and on the other hand deposited on the surface of amyloid fibrils, leading to the dense multi-pore network with the nanoparticle-embed wall. It served as the microstructure mechanism for the enhanced gel strength. Coating of fresh chilling beef with the amyloid fibril-zein-EGCG hybrid hydrogels effectively protected the freshness and tenderness through inhibiting the over-growth of microorganisms and oxidation of lipid. This study paves the way to develop functional edible biomaterials via polyphenol induced coordination of the protein–protein interaction and polyphenol-protein interaction, where polyphenol acts as the molecular glue. This strategy shows high application potentials in health promotion related fields, including edible coating to extend the shelf-life of fresh meat.

High Internal Phase Emulsions Stabilized with Polyphenol-Amyloid Fibril Supramolecules for Encapsulation and Protection of Lutein.

High internal phase emulsions (HIPEs), also called highly concentrated emulsions with a minimal internal phase volume fraction of 74%, have been paid increasing attention in the development of functional foods due to their high potential in loading with large amounts of hydrophobic nutriceuticals. In the present study, HIPEs stabilized by polyphenol-amyloid supramolecular filaments were prepared for encapsulation of olive oil and loading with lutein. Binding and stacking of the green tea polyphenol epigallocatechin gallate (EGCG) on the surface of amyloid fibrils fabricated from hen egg lysozyme resulted in the hybrid supramolecules, which assembled to form hydrogels. The amyloid fibril clusters shrouded by EGCG were observed in the microstructure of the hydrogels characterized by atomic force microscopy (AFM). HIPEs stabilized by the EGCG-amyloid fibril supramolecules showed the typical microstructure of highly packed polyhedral geometric oil droplets. The gel strength of the HIPEs stabilized by the hybrid supramolecules was greater than that of HIPEs stabilized by pure amyloid fibrils. The droplet size of the HIPEs first decreased and then increased with the increase of EGCG contents in the hybrid supramolecules, which was consistent with the corresponding emulsion morphologies obtained from the images of confocal laser scanning microscopy (CLSM). Aggregation of the protein-based nanofibrils appeared in the continuous phase at higher EGCG contents. The droplet size of the HIPEs decreased with the increase of the amyloid fibril concentration, accompanied by more packed and homogenously dispersed lipid droplets, as shown in the CLSM images. A high loading content of lutein of up to 10 mg/mL in the prepared HIPEs was realized, and the stability of lutein against ultraviolet irradiation, heat, iron, and hydrogen peroxide was promoted significantly. In addition, encapsulation with the HIPEs prevented the oxidization of olive oil, and this effect was enhanced with the increase of the EGCG content in the hybrid supramolecules ranging from 0 to 0.25 wt %. The protection function of the HIPEs might be ascribed to the membrane of interfacial amyloid fibrils and the crowded oil droplet environment, both of which could shield the pro-oxidation factors.

Chiral Selectivity of Secondary Nucleation in Amyloid Fibril Propagation

AbstractChirality is a fundamental feature of asymmetric molecules and of critical importance for intermolecular interactions. The growth of amyloid fibrils displays a strong enantioselectivity, which is manifested as elongation through the addition of monomers of the same, but not opposite, chirality as the parent aggregate. Here we ask whether also secondary nucleation on the surface of amyloid fibrils, of relevance for toxicity, is governed by the chirality of the nucleating monomers. We use short amyloid peptides (Aβ20‐34 and IAPP20‐29) with all residues as L‐ or all D‐enantiomer in self and cross‐seeding experiments with low enough seed concentration that any acceleration of fibril formation is dominated by secondary nucleation. We find a strong enantio‐specificity of this auto‐catalytic process with secondary nucleation being observed in the self‐seeding experiments only. The results highlight a role of secondary nucleation in strain propagation.

Chiral Selectivity of Secondary Nucleation in Amyloid Fibril Propagation.

Chirality is a fundamental feature of asymmetric molecules and of critical importance for intermolecular interactions. The growth of amyloid fibrils displays a strong enantioselectivity, which is manifested as elongation through the addition of monomers of the same, but not opposite, chirality as the parent aggregate. Here we ask whether also secondary nucleation on the surface of amyloid fibrils, of relevance for toxicity, is governed by the chirality of the nucleating monomers. We use short amyloid peptides (Aβ20‐34 and IAPP20‐29) with all residues as L‐ or all D‐enantiomer in self and cross‐seeding experiments with low enough seed concentration that any acceleration of fibril formation is dominated by secondary nucleation. We find a strong enantio‐specificity of this auto‐catalytic process with secondary nucleation being observed in the self‐seeding experiments only. The results highlight a role of secondary nucleation in strain propagation.

Structural insights into α-synuclein monomer–fibril interactions

Protein aggregation into amyloid fibrils is associated with multiple neurodegenerative diseases, including Parkinson's disease. Kinetic data and biophysical characterization have shown that the secondary nucleation pathway highly accelerates aggregation via the absorption of monomeric protein on the surface of amyloid fibrils. Here, we used NMR and electron paramagnetic resonance spectroscopy to investigate the interaction of monomeric α-synuclein (α-Syn) with its fibrillar form. We demonstrate that α-Syn monomers interact transiently via their positively charged N terminus with the negatively charged flexible C-terminal ends of the fibrils. These intermolecular interactions reduce intramolecular contacts in monomeric α-Syn, yielding further unfolding of the partially collapsed intrinsically disordered states of α-Syn along with a possible increase in the local concentration of soluble α-Syn and alignment of individual monomers on the fibril surface. Our data indicate that intramolecular unfolding critically contributes to the aggregation kinetics of α-Syn during secondary nucleation.

Targeting the Amyloid-β Fibril Surface with a Constrained Helical Peptide Inhibitor.

Amyloid-β (Aβ) oligomers are well-known toxic molecular species associated with Alzheimer's disease. Recent discoveries of the ability of amyloid fibril surfaces to convert soluble proteins into toxic oligomers suggested that these surfaces could serve as therapeutic targets for intervention. We have shown previously that a short helical peptide could be a key structural motif that can specifically recognize the K16-E22 region of the Aβ40 fibril surface with an affinity at the level of several micromolar. Here, we demonstrate that in-tether chiral center-induced helical stabilized peptides could also recognize the fibril surfaces, effectively inhibiting the surface-mediated oligomerization of Aβ40. Moreover, through extensive computational sampling, we observed two distinct ways in which the peptide inhibitors recognize the fibril surface. Apart from a binding mode that, in accord with the original design, involves hydrophobic side chains at the binding interface, we observed much more frequently another binding mode in which the hydrophobic staple interacts directly with the fibril surface. The affinity of the peptides for the fibril surface could be adjusted by tuning the hydrophobicity of the staple. The best candidate investigated here exhibits a submicromolar affinity (∼0.75 μM). Collectively, this work opens an avenue for the rational design of candidate drugs with stapled peptides for amyloid-related disease.

α-Helical Motif as Inhibitors of Toxic Amyloid-β Oligomer Generation via Highly Specific Recognition of Amyloid Surface.

SummaryAmyloid fibril surfaces can convert soluble proteins into toxic oligomers and are attractive targets for intervention of protein aggregation diseases. Thus far, molecules identified with inhibitory activity are either large proteins or flat cyclic compounds lacking in specificity. The main design difficulty is flatness of amyloid surfaces and the lack of knowledge on binding interfaces. Here, we demonstrate, for the first time, a rational design of alpha-helical peptide inhibitors targeting the amyloid-beta 40 (Aβ40) fibril surfaces, based on our in silico finding that a helical fragment of Aβ40 interacts in a unique way with side-chain arrays on the fibril surface. We strengthen the fragment's binding capability through mutations and helicity enhancement with our Terminal Aspartic acid strategy. The resulting inhibitor shows micromolar affinity for the fibril surface, effectively impedes the surface-mediated oligomerization of Aβ40, and mitigates its cytotoxicity. This work opens up an avenue to designing aggregation modulators for amyloid diseases.

Exploring the binding sites and proton diffusion on insulin amyloid fibril surfaces by naphthol-based photoacid fluorescence and molecular simulations

The diffusion of protons along biological surfaces and the interaction of biological structures with water are fundamental areas of interest in biology and chemistry. Here, we examine the surface of insulin amyloid fibrils and follow the binding of small molecules (photoacids) that differ according to the number and location of their sulfonic groups. We use transient fluorescence combined with a spherically-symmetric diffusion theory to show that the binding mode of different photoacids determines the efficiency of proton dissociation from the photoacid and the dimensionality of the proton’s diffusion. We use molecular dynamics simulations to examine the binding mode and mechanism of the photoacids and its influence on the unique kinetic rates and diffusion properties of the photoacid’s dissociated proton, where we also suggest a proton transfer process between one of the photoacids to proximal histidine residues. We show that the photoacids can be used as fluorescent markers for following the progression of amyloidogenic processes. The detailed characterisation of different binding modes to the surface of amyloid fibrils paves the way for better understanding of the binding mechanism of small molecules to amyloid fibrils.

Binding Modes of Thioflavin T on the Surface of Amyloid Fibrils Studied by NMR.

The mechanism for the interaction of thioflavin T (ThT) with amyloid fibrils at the molecular level is not known. Here, we used (1) H NMR spectroscopy to determine the binding mode of ThT on the surface of fibrils from lysozyme and insulin. Relayed rotating-frame Overhauser enhancements in ThT were observed, indicating that the orientation of ThT is orthogonal to the fibril surface. Importantly, the assembly state of ThT on both surfaces is different. On the surface of insulin fibrils, ThT is oligomeric, as indicated by rapid (1) H spin-lattice relaxation rate in the rotating frame (R1ρ ), presumably due to intermolecular dipole-dipole interactions between ThT molecules. In contrast, ThT on the surface of lysozyme fibrils is a monomer, as indicated by slower (1) H R1ρ . These results shed new light into the mechanism for the enhancement of ThT fluorescence and may lead to more efficient detectors of amyloid assemblies, which have escaped detection by ThT in monomer form.

Oligo(p-phenylene ethynylene) Electrolytes: A Novel Molecular Scaffold for Optical Tracking of Amyloids.

Finding new optical probes to detect and track amyloid protein aggregates is key to understanding and defeating the myriad of neurodegenerative and other diseases associated with these misfolded proteins. Herein we report that a series of fluorescent, soluble oligo(p-phenylene ethynylene)s (OPEs) are able to detect amyloids in vitro by massive binding-activated superluminescence, with low micromolar affinity and high selectivity for the amyloid conformer. The OPEs track the kinetics of amyloid fibril formation from native hen egg white lysozyme (HEWL) similarly to thioflavin T (ThT), and the dependence of binding affinity on OPE length supports the theory of a linear binding groove. We hypothesize, based on spectral properties, induced circular dichroism, and previous work in analogous systems, that the fluorescence turn-on mechanism is a combination of the reduction of static solvent-mediated quenching at the ethyl ester end groups of the phenylene ethynylene fluorophore and the formation of chiral J-type aggregates templated on the amyloid fibril surface.

Structure and Assembly of Functional Amyloids

The ability of proteins to self‐assemble into beta‐sheet‐rich aggregates called amyloid fibrils is considered to be universal, although certain polypeptide sequences have a particularly high propensity to adopt these conformations. In many cases the formation of amyloid fibrils is deleterious and associated with the progression of disease, but there are also examples of proteins for which the cross‐beta structure represents the functional conformation. Ure2 is the protein determinant of the yeast prion [URE3]. Ure2 consists of an N‐terminal prion‐inducing domain that is disordered in the native state, whereas the C‐terminal functional domain has a globular fold with structural similarity to glutathione transferase enzymes. The C‐terminal domain shows enzymatic activity in both the soluble and fibrillar forms of Ure2. We have used a variety of biophysical approaches to investigate the structure of Ure2 fibrils and their mechanism of assembly. We have also created chimeric constructs where the prion domain is genetically fused to other enzymes of different sizes and architectures. These chimeric polypeptide constructs spontaneously self‐assemble into nanofibrils with fused active enzyme subunits displayed on the amyloid fibril surface. We can measure steady‐state kinetic parameters for the appended enzymes in situ within fibrils, and compare these for the identical protein constructs in solution. We are currently investigating the use of microfluidic techniques to form enzymatically‐active microgel particles from the chimeric self‐assembling protein nanofibrils. These approaches allow amyloid fibrils to be used as a scaffold for enzyme immobilization, facilitating recycling and reuse of the appended enzymes in functional assays, both in batch processes and for microflow chemistry.

Self healing hydrogels composed of amyloid nano fibrils for cell culture and stem cell differentiation

Amyloids are highly ordered protein/peptide aggregates associated with human diseases as well as various native biological functions. Given the diverse range of physiochemical properties of amyloids, we hypothesized that higher order amyloid self-assembly could be used for fabricating novel hydrogels for biomaterial applications. For proof of concept, we designed a series of peptides based on the high aggregation prone C-terminus of Aβ42, which is associated with Alzheimer's disease. These Fmoc protected peptides self assemble to β sheet rich nanofibrils, forming hydrogels that are thermoreversible, non-toxic and thixotropic. Mechanistic studies indicate that while hydrophobic, π–π interactions and hydrogen bonding drive amyloid network formation to form supramolecular gel structure, the exposed hydrophobic surface of amyloid fibrils may render thixotropicity to these gels. We have demonstrated the utility of these hydrogels in supporting cell attachment and spreading across a diverse range of cell types. Finally, by tuning the stiffness of these gels through modulation of peptide concentration and salt concentration these hydrogels could be used as scaffolds that can drive differentiation of mesenchymal stem cells. Taken together, our results indicate that small size, ease of custom synthesis, thixotropic nature makes these amyloid-based hydrogels ideally suited for biomaterial/nanotechnology applications.

Self-Assembly of Amyloid Fibrils That Display Active Enzymes.

Enzyme immobilization is an important strategy to enhance the stability and recoverability of enzymes and to facilitate the separation of enzymes from reaction products. However, enzyme purification followed by separate chemical steps to allow immobilization on a solid support reduces the efficiency and yield of the active enzyme. Here we describe polypeptide constructs that self-assemble spontaneously into nanofibrils with fused active enzyme subunits displayed on the amyloid fibril surface. We measured the steady-state kinetic parameters for the appended enzymes in situ within fibrils and compare these with the identical protein constructs in solution. Finally, we demonstrated that the fibrils can be recycled and reused in functional assays both in conventional batch processes and in a continuous-flow microreactor.

Surface Characterization of Insulin Protofilaments and Fibril Polymorphs Using Tip-Enhanced Raman Spectroscopy (TERS)

Amyloid fibrils are β-sheet-rich protein aggregates that are strongly associated with a variety of neurodegenerative maladies, such as Alzheimer’s and Parkinson’s diseases. Even if the secondary structure of such fibrils is well characterized, a thorough understanding of their surface organization still remains elusive. Tip-enhanced Raman spectroscopy (TERS) is one of a few techniques that allow the direct characterization of the amino acid composition and the protein secondary structure of the amyloid fibril surface. Herein, we investigated the surfaces of two insulin fibril polymorphs with flat (flat) and left-twisted (twisted) morphology. It was found that the two differ substantially in both amino acid composition and protein secondary structure. For example, the amounts of Tyr, Pro, and His differ, as does the number of carboxyl groups on the respective surfaces, whereas the amounts of Phe and of positively charged amino and imino groups remain similar. In addition, the surface of protofilaments, the precursors of the mature flat and twisted fibrils, was investigated using TERS. The results show substantial differences with respect to the mature fibrils. A correlation of amino acid frequencies and protein secondary structures on the surface of protofilaments and on flat and twisted fibrils allowed us to propose a hypothetical mechanism for the propagation to specific fibril polymorphs. This knowledge can shed a light on the toxicity of amyloids and define the key factors responsible for fibril polymorphism. Finally, this work demonstrates the potential of TERS for the surface characterization of amyloid fibril polymorphs.

Synchrotron-Infrared Microscopy Analysis of Amyloid Fibrils Irradiated by Mid-Infrared Free-Electron Laser

Amyloid fibrils are widely recognized as a cause of serious amyloidosis such as Alzheimer’s disease. Although dissociation of amyloid fibril aggregates is expected to lead to a decrease in the toxicity of the fibrils in cells, the fibril structure is robust under physiological conditions. We have irradiated amyloid fibrils with a free-electron laser (FEL) tuned to mid-infrared frequencies to induce dissociation of the aggregates into monomer forms. We have previously succeeded in dissociating fibril structures of a short peptide of the thyroid hormone by tuning the oscillation frequency to the amide I band, but the detailed structural changes of the peptide have not yet been determined at a high spatial resolution. Synchrotron-radiation infrared microscopy (SR-IRM) is a powerful tool for in situ analysis of minute structural changes of various materials, and in this study, the feasibility of SR-IRM for analyzing the microscopic conformational changes of amyloid fibrils after FEL irradiation was investigated. Reflection spectra of the amyloid fibril surface showed that the amide I peaks shifted to higher wave numbers after the FEL irradiation, indicating that the initial β-sheet-rich structure transformed into a mixture of non-ordered and turn-like peptide conformations. This result demonstrates that conformational changes of the fibril structure after the FEL irradiation can be observed at a high spatial resolution using SR-IRM analysis and the FEL irradiation system can be useful for dissociation of amyloid aggregates.

Conformational Analysis of Thioflavin T Bound to the Surface of Amyloid Fibrils

The interaction of small molecules with the surface of amyloid assemblies is important for the detection and inhibition of amyloid formation. Thioflavin T (ThT), a small molecular rotor, has been used for the detection of amyloid fibrils for over half a century. The basis for detection is simple in that in the presence of fibrils the fluorescence of ThT is dramatically enhanced. The mechanism for this enhancement is not well understood but may depend on the determination of the conformation of ThT bound to the fibril surface. Here, we first use solution-state (1)H NMR to show that the on-off binding of ThT to the surface of insulin amyloid fibrils correlates with the enhancement of ThT fluorescence. We then show that the conformation of surface-bound ThT is twisted. The implications of this result in light of recent experimental and computational studies of the binding of ThT to amyloid or amyloid-like assemblies are discussed.