Flow-assisted assembly of nanostructured protein microfibers.

Some of the most remarkable materials in nature are made from proteins. The properties of these materials are closely connected to the hierarchical assembly of the protein building blocks. In this perspective, amyloid-like protein nanofibrils (PNFs) have emerged as a promising foundation for the synthesis of novel bio-based materials for a variety of applications. Whereas recent advances have revealed the molecular structure of PNFs, the mechanisms associated with fibril-fibril interactions and their assembly into macroscale structures remain largely unexplored. Here, we show that whey PNFs can be assembled into microfibers using a flow-focusing approach and without the addition of plasticizers or cross-linkers. Microfocus small-angle X-ray scattering allows us to monitor the fibril orientation in the microchannel and compare the assembly processes of PNFs of distinct morphologies. We find that the strongest fiber is obtained with a sufficient balance between ordered nanostructure and fibril entanglement. The results provide insights in the behavior of protein nanostructures under laminar flow conditions and their assembly mechanism into hierarchical macroscopic structures.

Changes in secondary structure of α-synuclein during oligomerization induced by reactive aldehydes.

The oxidative stress-related reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) have been shown to promote formation of α-synuclein oligomers in vitro. However, the changes in secondary structure of α-synuclein and the kinetics of the oligomerization process are not known and were the focus of this study. Size exclusion chromatography showed that after 1 h of incubation, HNE induced the formation of an oligomeric α-synuclein peak with a molecular weight of about ∼2000 kDa, which coincided with a decreasing ∼50 kDa monomeric peak. With prolonged incubation (up to 24 h) the oligomeric peak became the dominating molecular species. In contrast, in the presence of ONE, a ∼2000 oligomeric peak was exclusively observed after 15 min of incubation and this peak remained constant with prolonged incubation. Western blot analysis of HNE-induced α-synuclein oligomers showed the presence of monomers (15 kDa), SDS-resistant low molecular (30-160 kDa) and high molecular weight oligomers (≥260 kDa), indicating that the oligomers consisted of both covalent and non-covalent protein. In contrast, ONE-induced α-synuclein oligomers only migrated as covalent cross-linked high molecular-weight material (≥300 kDa). Both circular dichroism (CD) and Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy showed that the formation of HNE- and ONE-induced oligomers coincided with a spectral change from random coil to ß-sheet. However, ONE-induced α-synuclein oligomers exhibited a slightly higher degree of ß-sheet. Taken together, our results indicate that both HNE and ONE induce a change from random coil to ß-sheet structure that coincides with the formation of α-synuclein oligomers; albeit through different kinetic pathways depending on the degree of cross-linking.

A hexameric peptide barrel as building block of amyloid-ß protofibrils.

Oligomeric and protofibrillar aggregates formed by the amyloid-ß peptide (Aß) are believed to be involved in the pathology of Alzheimer's disease. Central to Alzheimer pathology is also the fact that the longer Aß42 peptide is more prone to aggregation than the more prevalent Aß40 . Detailed structural studies of Aß oligomers and protofibrils have been impeded by aggregate heterogeneity and instability. We previously engineered a variant of Aß that forms stable protofibrils and here we use solid-state NMR spectroscopy and molecular modeling to derive a structural model of these. NMR data are consistent with packing of residues 16 to 42 of Aß protomers into hexameric barrel-like oligomers within the protofibril. The core of the oligomers consists of all residues of the central and C-terminal hydrophobic regions of Aß, and hairpin loops extend from the core. The model accounts for why Aß42 forms oligomers and protofibrils more easily than Aß40 .

Structural conversion of the spidroin C-terminal domain during assembly of spider silk fibers.

The major ampullate Spidroin 1 (MaSp1) is the main protein of the dragline spider silk. The C-terminal (CT) domain of MaSp1 is crucial for the self-assembly into fibers but the details of how it contributes to the fiber formation remain unsolved. Here we exploit the fact that the CT domain can form silk-like fibers by itself to gain knowledge about this transition. Structural investigations of fibers from recombinantly produced CT domain from E. australis MaSp1 reveal an α-helix to ß-sheet transition upon fiber formation and highlight the helix No4 segment as most likely to initiate the structural conversion. This prediction is corroborated by the finding that a peptide corresponding to helix No4 has the ability of pH-induced conversion into ß-sheets and self-assembly into nanofibrils. Our results provide structural information about the CT domain in fiber form and clues about its role in triggering the structural conversion of spidroins during fiber assembly.

Ultralight aerogels via supramolecular polymerization of a new chiral perfluoropyridine-based sulfonimidamide organogelator.

Chiral and enantiopure perfluorinated sulfonimidamides act as low-molecular weight gelators at low critical gelation concentration (<1 mg mL-1) via supramolecular polymerization in nonpolar organic solvents and more heterogenic mixtures, such as biodiesel and oil. Freeze-drying of the organogel leads to ultralight aerogel with extremely low density (1 mg mL-1). The gelation is driven by hydrogen bonding resulting in a helical molecular ordering and unique fibre assemblies as confirmed by scanning electron microscopy, CD spectroscopy, and computational modeling of the supramolecular structure.

Stabilization of neurotoxic Alzheimer amyloid-beta oligomers by protein engineering.

Soluble oligomeric aggregates of the amyloid-beta peptide (Abeta) have been implicated in the pathogenesis of Alzheimer's disease (AD). Although the conformation adopted by Abeta within these aggregates is not known, a beta-hairpin conformation is known to be accessible to monomeric Abeta. Here we show that this beta-hairpin is a building block of toxic Abeta oligomers by engineering a double-cysteine mutant (called Abetacc) in which the beta-hairpin is stabilized by an intramolecular disulfide bond. Abeta(40)cc and Abeta(42)cc both spontaneously form stable oligomeric species with distinct molecular weights and secondary-structure content, but both are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Abeta aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in Abetacc oligomers. Stable oligomers are expected to become highly toxic and, accordingly, we find that beta-sheet-containing Abeta(42)cc oligomers or protofibrillar species formed by these oligomers are 50 times more potent inducers of neuronal apoptosis than amyloid fibrils or samples of monomeric wild-type Abeta(42), in which toxic aggregates are only transiently formed. The possibility of obtaining completely stable and physiologically relevant neurotoxic Abeta oligomer preparations will facilitate studies of their structure and role in the pathogenesis of AD. For example, here we show how kinetic partitioning into different aggregation pathways can explain why Abeta(42) is more toxic than the shorter Abeta(40), and why certain inherited mutations are linked to protofibril formation and early-onset AD.

Protein Nanofibrils from Fava Bean and Its Major Storage Proteins: Formation and Ability to Generate and Stabilise Foams.

Protein nanofibrils (PNFs) have potential for use in food applications as texture inducers. This study investigated the formation of PNFs from protein extracted from whole fava bean and from its two major storage proteins, globulin fractions 11S and 7S. PNFs were formed by heating (85 °C) the proteins under acidic conditions (pH 2) for 24 h. Thioflavin T fluorescence and atomic force microscopy techniques were used to investigate PNF formation. The foaming properties (capacity, stability, and half-life) were explored for non-fibrillated and fibrillated protein from fava bean, 11S, and 7S to investigate the texturing ability of PNFs at concentrations of 1 and 10 mg/mL and pH 7. The results showed that all three heat-incubated proteins (fava bean, 11S, and 7S) formed straight semi-flexible PNFs. Some differences in the capacity to form PNFs were observed between the two globulin fractions, with the smaller 7S protein being superior to 11S. The fibrillated protein from fava bean, 11S, and 7S generated more voluminous and more stable foams at 10 mg/mL than the corresponding non-fibrillated protein. However, this ability for fibrillated proteins to improve the foam properties seemed to be concentration-dependent, as at 1 mg/mL, the foams were less stable than those made from the non-fibrillated protein.

Food protein-derived amyloids do not accelerate amyloid ß aggregation.

The deposition of proteins in the form of amyloid fibrils is closely associated with several serious diseases. The events that trigger the conversion from soluble functional proteins into insoluble amyloid are not fully understood. Many proteins that are not associated with disease can form amyloid with similar structural characteristics as the disease-associated fibrils, which highlights the potential risk of cross-seeding of disease amyloid by amyloid-like structures encountered in our surrounding. Of particular interest are common food proteins that can be transformed into amyloid under conditions similar to cooking. We here investigate cross-seeding of amyloid-ß (Aß), a peptide known to form amyloid during the development of Alzheimer's disease, by 16 types of amyloid fibrils derived from food proteins or peptides. Kinetic studies using thioflavin T fluorescence as output show that none of the investigated protein fibrils accelerates the aggregation of Aß. In at least two cases (hen egg lysozyme and oat protein isolate) we observe retardation of the aggregation, which appears to originate from interactions between the food protein seeds and Aß in aggregated form. The results support the view that food-derived amyloid is not a risk factor for development of Aß pathology and Alzheimer's disease.

Hydrophobicity and conformational change as mechanistic determinants for nonspecific modulators of amyloid ß self-assembly.

The link between many neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, and the aberrant folding and aggregation of proteins has prompted a comprehensive search for small organic molecules that have the potential to inhibit such processes. Although many compounds have been reported to affect the formation of amyloid fibrils and/or other types of protein aggregates, the mechanisms by which they act are not well understood. A large number of compounds appear to act in a nonspecific way affecting several different amyloidogenic proteins. We describe here a detailed study of the mechanism of action of one representative compound, lacmoid, in the context of the inhibition of the aggregation of the amyloid ß-peptide (Aß) associated with Alzheimer's disease. We show that lacmoid binds Aß(1-40) in a surfactant-like manner and counteracts the formation of all types of Aß(1-40) and Aß(1-42) aggregates. On the basis of these and previous findings, we are able to rationalize the molecular mechanisms of action of nonspecific modulators of protein self-assembly in terms of hydrophobic attraction and the conformational preferences of the polypeptide.

Hierarchical propagation of structural features in protein nanomaterials.

Natural high-performance materials have inspired the exploration of novel materials from protein building blocks. The ability of proteins to self-organize into amyloid-like nanofibrils has opened an avenue to new materials by hierarchical assembly processes. As the mechanisms by which proteins form nanofibrils are becoming clear, the challenge now is to understand how the nanofibrils can be designed to form larger structures with defined order. We here report the spontaneous and reproducible formation of ordered microstructure in solution cast films from whey protein nanofibrils. The structural features are directly connected to the nanostructure of the protein fibrils, which is itself determined by the molecular structure of the building blocks. Hence, a hierarchical assembly process ranging over more than six orders of magnitude in size is described. The fibril length distribution is found to be the main determinant of the microstructure and the assembly process originates in restricted capillary flow induced by the solvent evaporation. We demonstrate that the structural features can be switched on and off by controlling the length distribution or the evaporation rate without losing the functional properties of the protein nanofibrils.

Robust Assembly of Cross-Linked Protein Nanofibrils into Hierarchically Structured Microfibers.

Natural, high-performance fibers generally have hierarchically organized nanosized building blocks. Inspired by this, whey protein nanofibrils (PNFs) are assembled into microfibers, using flow-focusing. By adding genipin as a nontoxic cross-linker to the PNF suspension before spinning, significantly improved mechanical properties of the final fiber are obtained. For curved PNFs, with a low content of cross-linker (2%) the fiber is almost 3 times stronger and 4 times stiffer than the fiber without a cross-linker. At higher content of genipin (10%), the elongation at break increases by a factor of 2 and the energy at break increases by a factor of 5. The cross-linking also enables the spinning of microfibers from long straight PNFs, which has not been achieved before. These microfibers have higher stiffness and strength but lower ductility and toughness than those made from curved PNFs. The fibers spun from the two classes of nanofibrils show clear morphological differences. The study demonstrates the production of protein-based microfibers with mechanical properties similar to natural protein-based fibers and provides insights about the role of the nanostructure in the assembly process.

Extracellular protein components of amyloid plaques and their roles in Alzheimer's disease pathology.

Alzheimer's disease (AD) is pathologically defined by the presence of fibrillar amyloid ß (Aß) peptide in extracellular senile plaques and tau filaments in intracellular neurofibrillary tangles. Extensive research has focused on understanding the assembly mechanisms and neurotoxic effects of Aß during the last decades but still we only have a brief understanding of the disease associated biological processes. This review highlights the many other constituents that, beside Aß, are accumulated in the plaques, with the focus on extracellular proteins. All living organisms rely on a delicate network of protein functionality. Deposition of significant amounts of certain proteins in insoluble inclusions will unquestionably lead to disturbances in the network, which may contribute to AD and copathology. This paper provide a comprehensive overview of extracellular proteins that have been shown to interact with Aß and a discussion of their potential roles in AD pathology. Methods that can expand the knowledge about how the proteins are incorporated in plaques are described. Top-down methods to analyze post-mortem tissue and bottom-up approaches with the potential to provide molecular insights on the organization of plaque-like particles are compared. Finally, a network analysis of Aß-interacting partners with enriched functional and structural key words is presented.

Protein nanofibrils and their use as building blocks of sustainable materials.

The development towards a sustainable society requires a radical change of many of the materials we currently use. Besides the replacement of plastics, derived from petrochemical sources, with renewable alternatives, we will also need functional materials for applications in areas ranging from green energy and environmental remediation to smart foods. Proteins could, with their intriguing ability of self-assembly into various forms, play important roles in all these fields. To achieve that, the code for how to assemble hierarchically ordered structures similar to the protein materials found in nature must be cracked. During the last decade it has been demonstrated that amyloid-like protein nanofibrils (PNFs) could be a steppingstone for this task. PNFs are formed by self-assembly in water from a range of proteins, including plant resources and industrial side streams. The nanofibrils display distinct functional features and can be further assembled into larger structures. PNFs thus provide a framework for creating ordered, functional structures from the atomic level up to the macroscale. This review address how industrial scale protein resources could be transformed into PNFs and further assembled into materials with specific mechanical and functional properties. We describe what is required from a protein to form PNFs and how the structural properties at different length scales determine the material properties. We also discuss potential chemical routes to modify the properties of the fibrils and to assemble them into macroscopic structures.

Structural characterisation of amyloid-like fibrils formed by an amyloidogenic peptide segment of ß-lactoglobulin.

Protein nanofibrils (PNFs) represent a promising class of biobased nanomaterials for biomedical and materials science applications. In the design of such materials, a fundamental understanding of the structure-function relationship at both molecular and nanoscale levels is essential. Here we report investigations of the nanoscale morphology and molecular arrangement of amyloid-like PNFs of a synthetic peptide fragment consisting of residues 11-20 of the protein ß-lactoglobulin (ß-LG11-20), an important model system for PNF materials. Nanoscale fibril morphology was analysed by atomic force microscopy (AFM) that indicates the presence of polymorphic self-assembly of protofilaments. However, observation of a single set of 13C and 15N resonances in the solid-state NMR spectra for the ß-LG11-20 fibrils suggests that the observed polymorphism originates from the assembly of protofilaments at the nanoscale but not from the molecular structure. The secondary structure and inter-residue proximities in the ß-LG11-20 fibrils were probed using NMR experiments of the peptide with 13C- and 15N-labelled amino acid residues at selected positions. We can conclude that the peptides form parallel ß-sheets, but the NMR data was inconclusive regarding inter-sheet packing. Molecular dynamics simulations confirm the stability of parallel ß-sheets and suggest two preferred modes of packing. Comparison of molecular dynamics models with NMR data and calculated chemical shifts indicates that both packing models are possible.

Protein Nanofibrils and Their Hydrogel Formation with Metal Ions.

Protein nanofibrils (PNFs) have been prepared by whey protein fibrillation at low pH and in the presence of different metal ions. The effect of the metal ions was systematically studied both in terms of PNF suspension gelation behavior and fibrillation kinetics. A high valence state and a small ionic radius (e.g., Sn4+) of the metal ion resulted in the formation of hydrogels already at a metal ion concentration of 30 mM, whereas an intermediate valence state and larger ionic radius (Co2+, Ni2+, Al3+) resulted in the hydrogel formation occurring at 60 mM. A concentration of 120 mM of Na+ was needed to form a PNF hydrogel, while lower concentrations showed liquid behaviors similar to the reference PNF solution where no metal ions had been introduced. The hydrogel mechanics were investigated at steady-state conditions after 24 h of incubation/gelation, revealing that more acidic (smaller and more charged) metal ions induced ca. 2 orders of magnitude higher storage modulus as compared to the less acidic metal ions (with smaller charge and larger radius) for the same concentration of metal ions. The viscoelastic nature of the hydrogels was attributed to the ability of the metal ions to coordinate water molecules in the vicinity of the PNFs. The presence of metal ions in the solutions during the growth of the PNFs typically resulted in curved fibrils, whereas an upper limit of the concentration existed when oxides/hydroxides were formed, and the hydrogels lost their gel properties due to phase separation. Thioflavin T (ThT) fluorescence was used to determine the rate of the fibrillation to form 50% of the total PNFs (t1/2), which decreased from 2.3 to ca. 0.5 h depending on the specific metal ions added.

Systematic evaluation of SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay.

OBJECTIVE: The COVID-19 pandemic poses an immense need for accurate, sensitive and high-throughput clinical tests, and serological assays are needed for both overarching epidemiological studies and evaluating vaccines. Here, we present the development and validation of a high-throughput multiplex bead-based serological assay. METHODS: More than 100 representations of SARS-CoV-2 proteins were included for initial evaluation, including antigens produced in bacterial and mammalian hosts as well as synthetic peptides. The five best-performing antigens, three representing the spike glycoprotein and two representing the nucleocapsid protein, were further evaluated for detection of IgG antibodies in samples from 331 COVID-19 patients and convalescents, and in 2090 negative controls sampled before 2020. RESULTS: Three antigens were finally selected, represented by a soluble trimeric form and the S1-domain of the spike glycoprotein as well as by the C-terminal domain of the nucleocapsid. The sensitivity for these three antigens individually was found to be 99.7%, 99.1% and 99.7%, and the specificity was found to be 98.1%, 98.7% and 95.7%. The best assay performance was although achieved when utilising two antigens in combination, enabling a sensitivity of up to 99.7% combined with a specificity of 100%. Requiring any two of the three antigens resulted in a sensitivity of 99.7% and a specificity of 99.4%. CONCLUSION: These observations demonstrate that a serological test based on a combination of several SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay.

Detergent-like interaction of Congo red with the amyloid beta peptide.

Accumulating evidence links prefibrillar oligomeric species of the amyloid beta peptide (Abeta) to cellular toxicity in Alzheimer's disease, potentially via disruption of biological membranes. Congo red (CR) affects protein aggregation. It is known to self-associate into micelle-like assemblies but still reduces the toxicity of Abeta aggregates in cell cultures and model organisms. We show here that CR interacts with Abeta(1-40) in a manner similar to that of anionic detergents. Although CR promotes beta sheet formation and peptide aggregation, it may also solubilize toxic protein species, making them less harmful to critical cellular components and thereby reducing amyloid toxicity.

Dissecting the Structural Organization of Multiprotein Amyloid Aggregates Using a Bottom-Up Approach.

Deposition of fibrillar amyloid ß (Aß) in senile plaques is a pathological signature of Alzheimer's disease. However, senile plaques also contain many other components, including a range of different proteins. Although the composition of the plaques can be analyzed in post-mortem tissue, knowledge of the molecular details of these multiprotein inclusions and their assembly processes is limited, which impedes the progress in deciphering the biochemical mechanisms associated with Aß pathology. We describe here a bottom-up approach to monitor how proteins from human cerebrospinal fluid associate with Aß amyloid fibrils to form plaque particles. The method combines flow cytometry and mass spectrometry proteomics and allowed us to identify and quantify 128 components of the captured multiprotein aggregates. The results provide insights into the functional characteristics of the sequestered proteins and reveal distinct interactome responses for the two investigated Aß variants, Aß(1-40) and Aß(1-42). Furthermore, the quantitative data is used to build models of the structural organization of the multiprotein aggregates, which suggests that Aß is not the primary binding target for all the proteins; secondary interactions account for the majority of the assembled components. The study elucidates how different proteins are recruited into senile plaques and establishes a new model system for exploring the pathological mechanisms of Alzheimer's disease from a molecular perspective.

On the mechanism of nonspecific inhibitors of protein aggregation: dissecting the interactions of alpha-synuclein with Congo red and lacmoid.

Increasing evidence links the misfolding and aberrant self-assembly of proteins with the molecular events that underlie a range of neurodegenerative diseases, yet the mechanistical details of these processes are still poorly understood. The fact that many of these proteins are intrinsically unstructured makes it particularly challenging to develop strategies for discovering small molecule inhibitors of their aggregation. We present here a broad biophysical approach that enables us to characterize the mechanisms of interaction between alpha-synuclein, a protein whose aggregation is closely connected with Parkinson's disease, and two small molecules, Congo red and Lacmoid, which inhibit its fibrillization. Both compounds are found to interact with the N-terminal and central regions of the monomeric protein although with different binding mechanisms and affinities. The differences can be attributed to the chemical nature of the compounds as well as their abilities to self-associate. We further show that alpha-synuclein binding and aggregation inhibition are mediated by small oligomeric species of the compounds that interact with distinct regions of the monomeric protein. These findings provide potential explanations of the nonspecific antiamyloid effect observed for these compounds as well as important mechanistical information for future drug discovery efforts targeting the misfolding and aggregation of intrinsically unstructured proteins.

3D J-resolved NMR spectroscopy for unstructured polypeptides: fast measurement of 3J HNH alpha coupling constants with outstanding spectral resolution.

A powerful experiment for the investigation of conformational properties of unstructured states of proteins is presented. The method combines a phase sensitive J-resolved experiment with a (1)H-(15)N SOFAST-HMQC to provide a 3D spectrum with an E.COSY pattern originating from splittings due to (3)J(HNH alpha) and (2)J(NH alpha) couplings. Thereby an effectively homodecoupled (1)H-(15)N correlation spectrum is obtained with significantly improved resolution and greatly reduced spectral overlap compared to standard HSQC and HMQC experiments. The (3)J(HNH alpha) is revealed in three independent ways directly from the peak positions, allowing for internal consistency testing. In addition, the natural H(N) linewidths can easily be extracted from the lineshapes. Thanks to the SOFAST principle, the limited sweep width needed in the J-dimension and the short phase cycle, data accumulation is rapid with excellent sensitivity per time unit. The experiment is demonstrated for the intrinsically unstructured 14 kDa protein alpha-synuclein.