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A survey, primarily based on work in the authors’ laboratory during the last 10 years, is provided of recent developments in NMR studies of exchange processes involving protein–ligand and protein–protein interactions. We start with a brief overview of the theoretical background of Dark state Exchange Saturation Transfer (DEST) and lifetime line-broadening (ΔR2) NMR methodology. Some limitations of the DEST/ΔR2 methodology in applications to molecular systems with intermediate molecular weights are discussed, along with the means of overcoming these limitations with the help of closely related exchange NMR techniques, such as the measurements of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion, exchange-induced chemical shifts or rapidly-relaxing components of relaxation decays. Some theoretical underpinnings of the quantitative description of global dynamics of proteins on the surface of very high molecular weight particles (nanoparticles) are discussed. Subsequently, several applications of DEST/ΔR2 methodology are described from a methodological perspective with an emphasis on providing examples of how kinetic and relaxation parameters for exchanging systems can be reliably extracted from NMR data for each particular model of exchange. Among exchanging systems that are not associated with high molecular weight species, we describe several exchange NMR-based studies that focus on kinetic modelling of transient pre-nucleation oligomerization of huntingtin peptides that precedes aggregation and fibril formation.

Amyloid fibrils are found in systemic amyloidosis diseases such as Alzheimer’s disease, Parkinson’s disease, and type II diabetes. Currently, these diseases are diagnosed by observation of fibrils or plaques, which is an ineffective method for early diagnosis and treatment of disease. The goal of this study was to develop a simple and quick method to predict the possibility and speed of fibril formation before its occurrence. Oligomers generated from seven representative peptide segments were first isolated and detected by ion-mobility mass spectrometry (IM-MS). Then, their assemblies were disrupted using formic acid (FA). Interestingly, oligomers that showed small ion intensity changes upon FA addition had rapid fibril formation. By contrast, oligomers that had large ion intensity changes generated fibrils slowly. Two control peptides (aggregation/no fibrils and no aggregation/no fibrils) did not show changes in their ion intensities, which confirmed the ability of this method to predict amyloid formation. In summary, the developed method correlated MS intensity ratio changes of peptide oligomers on FA addition with their amyloid propensities. This method will be useful for monitoring peptide/protein aggregation behavior and essential for their mechanism studies., Graphical abstract Image 1, Highlights • Co-populated oligomers are differentiated by IM-MS. • Oligomers that showed small ranges of fold change had rapid fibril formation. • Oligomers that had large ranges of fold change generated fibrils slowly. • Fold changes of oligomers are representative to their aggregation propensities.

A number of algorithms have been developed to predict the aggregation propensity of peptides and proteins, but virtually none have the ability to provide sequence-specific information on what physicochemical properties are most important in altering aggregation propensity. In this study, a chemometric approach using reduced amino acid properties is used to examine the aggregation behavior of a highly amyloidogenic peptide, Aβ(1-42). Specific residues are identified as being critical to the aggregation process. At each of these positions, the important physicochemical properties are identified that would either accelerate or inhibit fibril formation.

α-Synuclein (αSyn) is an intrinsically disordered protein that can form amyloid fibrils. Fibrils of αSyn are implicated with the pathogenesis of Parkinson's disease and other synucleinopathies. Elucidating the mechanism of fibril formation of αSyn is therefore important for understanding the mechanism of the pathogenesis of these diseases. Fibril formation of αSyn is sensitive to solution conditions, suggesting that fibril formation of αSyn arises from the changes in its inherent physico-chemical properties, particularly its dynamic properties because intrinsically disordered proteins such as αSyn utilize their inherent flexibility to function. Characterizing these properties under various conditions should provide insights into the mechanism of fibril formation. Here, using the quasielastic neutron scattering and small-angle x-ray scattering techniques, we investigated the dynamic and structural properties of αSyn under the conditions, where mature fibrils are formed (pH 7.4 with a high salt concentration), where clumping of short fibrils occurs (pH 4.0), and where fibril formation is not completed (pH 7.4). The small-angle x-ray scattering measurements showed that the extended structures at pH 7.4 with a high salt concentration become compact at pH 4.0 and 7.4. The quasielastic neutron scattering measurements showed that both intra-molecular segmental motions and local motions such as side-chain motions are enhanced at pH 7.4 with a high salt concentration, compared to those at pH 7.4 without salt, whereas only the local motions are enhanced at pH 4.0. These results imply that fibril formation of αSyn requires not only the enhanced local motions but also the segmental motions such that proper inter-molecular interactions are possible.

Amyloid fibril formation has long been studied because of the variety of proteins that are capable of adopting this structure despite sharing little sequence homology. This makes amyloid fibrils a challenging focus for inhibition studies because the peptides and proteins that form amyloid fibrils cannot be targeted based on a sequence motif. Most peptide inhibitors that target specific amyloidogenic proteins rely heavily on sequence recognition to ensure that the inhibitory peptide is able to bind its target. This approach is limited to targeting one amyloidogenic protein at a time. However, there is increasing evidence of cross-reactivity between amyloid-forming polypeptides. It has therefore become more useful to study the similarities between these proteins that goes beyond their sequence homology. Indeed, the observation that amyloidogenic proteins adopt similar secondary structures along the pathway to fibril formation opens the way to an interesting investigation: the development of inhibitors that could be universal amyloid traps. The review below will analyze two specific amyloidogenic proteins, α-synuclein and human amylin, and introduce a small number of peptides that have been shown to be capable of inhibiting the amyloidogenesis of both of these very dissimilar polypeptides. Some of the inhibitory peptide motifs may indeed, be applicable to Aβ and other amyloidogenic systems., Graphical abstract Image 1, Highlights • hAM and α-syn share similarities during amyloidogenesis but no sequence homology. • Small molecules such as resveratrol are capable of inhibiting both hAM and α-syn. • Cyclo-WW2 can inhibit both peptides despite no sequence homology to either.

d-amino acid-containing proteins have been found in several human tissues, and the spontaneous accumulation of d-amino acids in proteins is thought to be involved in age-dependent diseases including dementia. Tau, a microtubule-associated protein, is a major component of neurofibrillary tangles in Alzheimer's disease. Site-specific amino acid D-isomerization in Tau has been observed in the brains of patients with Alzheimer's disease. Here, we conducted amino acid D-isomerization at specific sites in microtubule-binding repeat peptides of Tau (Tau R2 and R3) and examined the effects on Tau structure and fibril formation. Our results demonstrate that amino acid D-isomerization in Tau R2 peptides decreased the rates of β-sheet transition and fibril formation compared with those of the wild-type peptide composed of all l-amino acids. In contrast, Tau R3 peptides that had undergone amino acid D-isomerization at either Asp314, Ser316, or Ser324 showed increased rates of β-sheet transition and fibril formation compared with those of the wild-type Tau R3 peptide.

The physical and microstructural properties of four straight run asphalt binders were examined and compared in combination with short term aging (RTFOT) and long-term (PAV) laboratory aging. RTFOT aging was conducted at temperatures of 123, 143 and 163 °C. The physical testing parameters included penetration, softening point and Dynamic Shear Rheology (DSR) complex shear modulus and phase angle at 10 °C. The binders selected came from four different sources and had the same penetration grading (70/100). They all showed an increase in stiffness with aging, including with the increase in RTFOT temperatures and especially with PAV aging. The microstructural evolution of the binder was examined by Environmental Scanning Electron Microscopy (ESEM) on aged binders at 123 and 163 °C. The physical changes with aging corresponded to an evolution in the binders’ ‘fibril’ microstructure under ESEM, as a result of electron beam exposure, with the microstructure getting denser with PAV aging. This densification (fibril area) of the microstructure was quantified with image analysis for the virgin and RTFOT aged samples, and the fibril formation time was also measured. The asphalt binders showed varied ESEM ‘fingerprints’ and aged in different ways. The ESEM ‘formation time’ and ‘fibril area’ of the binders generally showed good correlation with the physical properties, although this was not the case for all of the binders due to their unique aging characteristics.

As the most abundant protein in the extracellular matrix, collagen has become widely studied in the fields of tissue engineering and regenerative medicine. Of the various collagen types, collagen type I remains the most commonly utilised in laboratory studies. In tissues, collagen type I forms into fibrils that provide an extended fibrillar network. In tissue engineering and regenerative medicine, little emphasis has been placed on the nature of the network that is being formed. Various factors could affect the network structure, including the method used to extract collagen form the native tissue, since this may remove the telopeptides, and the nature and extent of any chemical modifications and crosslinking moieties. The structure of any fibril network impacts cellular proliferation and differentiation, as well as the overall modulus of hydrogels. In this study, the network forming properties of two distinct forms of collagen (telo- and atelo- collagen), and their methacrylated derivatives were compared. The presence of the telopeptides facilitated fibril formation for unmodified samples, but this benefit was substantially reduced by subsequent methacrylation, leading to a loss in the native self-assembly potential. Furthermore, the impact of methacrylation of the collagen which enables rapid crosslinking and makes it suitable for use in 3D printing was investigated. The crosslinking of the methacrylated samples (both telo- and atelo-) was seen to improve the fibril-like network compared to non-crosslinked samples. This contrasted with samples of methacrylated gelatin which showed little, if any, fibrillar or ordered network structure whether or not they were crosslinked.

Two types of special structures, homogeneous and secondary nuclei, form during fibril formation. The structural and functional properties of amyloid fibrils in whey protein concentrate (WPC) with different ratios of added homogeneous nuclei to secondary nuclei were investigated. Thioflavin T fluorescence analysis and kinetic equations indicated that two types of nuclei could accelerate WPC fibrillation compared with WPC self-assembling into amyloid fibrils, thereby reducing the lag time and increasing the number of fibrils. However, there were considerable differences in the nucleation-inducing capability of WPC fibrillation between homogeneous and secondary nuclei. The number of fibrils formed by adding homogeneous nuclei was higher than that obtained with secondary nuclei, the increase in the Th T fluorescence intensity induced by homogeneous nuclei was 1.83-fold much than secondary nuclei. Meanwhile, secondary nuclei yielded a 2.71-fold faster aggregation rate of WPC than homogeneous nuclei, particularly during the first hour of thermal treatment (protein mass ratio of nuclei to WPC 1:1). The gelation time of WPC after secondary nuclei addition was shorter, from 10 h (WPC (2.0/6.5)) to 4 h (WPC + HN) to 2 h (WPC + SN); however, the gel microstructure of WPC after the addition of homogeneous nuclei was denser, yielding a preferred water holding capacity.

The AXH domain of protein Ataxin 1 is thought to play a key role in the misfolding and aggregation pathway responsible for Spinocerebellar ataxia 1. For this reason, a molecular level understanding of AXH oligomerization pathway is crucial to elucidate the aggregation mechanism, which is thought to trigger the disease. This study employs classical and enhanced molecular dynamics to identify the structural and energetic basis of AXH tetramer stability. Results of this work elucidate molecular mechanisms behind the destabilizing effect of protein mutations, which consequently affect the AXH tetramer assembly. Moreover, results of the study draw attention for the first time, to our knowledge, to the R638 protein residue, which is shown to play a key role in AXH tetramer stability. Therefore, R638 might be also implicated in the AXH oligomerization pathway and stands out as a target for future experimental studies focused on self-association mechanisms and fibril formation of full-length ATX1.

Serum amyloid A (SAA) is actively involved in such pathological processes as atherosclerosis, rheumatoid arthritis, cancer and Alzheimer's disease by its aggregation. One of the factors that can attenuate its aggregation and so affects its physiological role is its interactions with glycosminoglycans (GAGs), linear anionic periodic polysaccharides. These molecules located in the extracellular matrix of the cell are highly variable in their chemical composition and sulfation patterns. Despite the available experimental evidence of SAA-GAG interactions, no mechanistic details at atomic level have been reported for these systems so far. In our work we aimed to apply diverse computational tools to characterize SAA-GAG complexes formation and to answer questions about their potential specificity, energetic patterns, particular SAA residues involved in these interactions, favourable oligomeric state of the protein and the potential influence of GAGs on SAA aggregation. Molecular docking, conventional and replica exchange molecular dynamics approaches were applied to corroborate the experimental knowledge and to propose the corresponding molecular models. SAA-GAG complex formation was found to be electrostatics-driven and rather unspecific of a GAG sulfation pattern, more favorable for the dimer than for the monomer when binding to a short GAG oligosaccharide through its N-terminal helix, potentially contributing to the unfolding of this helix, which could lead to the promotion of the protein aggregation. The data obtained add to the specific knowledge on SAA-GAG systems and deepen the general understanding of protein-GAG interactions that is of a considerable value for the development of GAG-based approaches in a broad theurapeutic context.

Monomers, homogeneous nuclei and protofibrils are intermediate products during β-lactoglobulin (β-lg) fibril formation, they have different abilities to resist disturbance by other protein agents. The results showed that when β-lg monomer coassembly with α-Lactalbumin (α-la)/к-Casein (к-CN), it would affect β-lg self-assembly to fibril. While homogeneous nuclei and protofibrils coassembly with α-la/к-CN had little effect on fibril formation. Moreover, the result of forming fibril was quite different when β-lg monomers coassembled with α-la and к-CN. Notably, α-la increased the fibril diameter, and к-CN clearly inhibited fibril formation. The reason for the differences was the different binding sites. α-la and к-CN had different effects on β-lg's α-helical structure: α-la did not destroy the β-lg's α-helical structure thus had little effect on fibril formation. While к-CN especially its hydrophobic area, interacted with β-lg via –S-S- during the aggregation, it destroyed β-lg's α-helical structure thus resulted in an inability to form fibrils. This study would expand the application value of dairy protein-based products aggregated at low pH.

Bacteria within Curli biofilms are protected from environmental pressures (e.g., disinfectants, antibiotics), and this is responsible for intractable infections. Understanding aggregation of the major protein component of Curli, CsgA, may uncover disease-associated amyloidogenesis mechanisms. Here, we report the application of pulsed hydrogen–deuterium exchange and mass spectrometry (HDX-MS) to study CsgA aggregation, thereby obtaining region-specific information. By following time-dependent peptide signal depletion, presumably a result of insoluble fibril formation, we acquired sigmoidal profiles that are specific for regions (region-specific) of the protein. These signal-depletion profiles not only provide an alternative aggregation measurement, but also give insight on soluble species in the aggregation. The HDX data present as bimodal isotopic distributions, one representing a highly disordered species whereas the other a well-structured one. Although the extents of deuterium uptake of the two species remain the same with time, the relative abundance of the lower mass, less-exchanged species increases in a region-specific manner. The same region-specific aggregation properties also pertain to different aggregation conditions. Although CsgA is an intrinsically disordered protein, within the fibril it is thought to consist of five imperfect β-strand repeating units (labeled R1–R5). We found that the exterior repeating units R1 and R5 have higher aggregation propensities than do the interior units R2, R3, and R4. We also employed TEM to obtain complementary information of the well-structured species. The results provide insight on aggregation and a new approach for further application of HDX-MS to unravel aggregation mechanisms of amyloid proteins.

The bovine whey protein β-lactoglobulin will self-assemble into amyloid-like nanofibrils when heated at pH ≤ 3 and temperatures ≥75 °C. These fibrils have diameters

The conformational ensemble of intrinsically disordered proteins, such as α-synuclein, are responsible for their function and malfunction. Misfolding of α-synuclein can lead to neurodegenerative diseases, and the ability to study their conformations and those of other intrinsically disordered proteins under varying physiological conditions can be crucial to understanding and preventing pathologies. In contrast to well-folded peptides, a consensus feature of IDPs is their low hydropathy and high charge, which makes their conformations sensitive to pH perturbation. We examine a prominent member of this subset of IDPs, α-synuclein, using a divide-and-conquer scheme that provides enhanced sampling of IDP structural ensembles. We constructed conformational ensembles of α-synuclein under neutral (pH ~ 7) and low (pH ~ 3) pH conditions and compared our results with available information obtained from experimental smFRET, SAXS (small-angle X-ray scattering), and NMR studies. Specifically, α-synuclein has been found to in a more compact state at low pH conditions and the structural changes observed are consistent with those from experiments. We also characterize the conformational and dynamic differences between these ensembles and discussed the implication on promoting pathogenic fibril formation. We find that under low pH conditions, neutralization of negatively charged residues leads to compaction of the C-terminal portion of α-synuclein while internal reorganization allows α-synuclein to maintain its overall end-to-end distance under both pH conditions. We also observe different levels of intra-protein interaction between three domains of α-synuclein at varying pH and a shift towards more hydrophilic interactions with decreasing pH.

Our understanding of amyloid structures and the mechanisms by which disease-associated peptides and proteins self-assemble into these fibrillar aggregates, has advanced considerably in recent years. It is also established that amyloid fibrils are generally polymorphic. The molecular structures of the aggregation intermediates and the causes of molecular and structural polymorphism are less understood, however. Such information is mandatory to explain the pathological diversity of amyloid diseases. What is also clear is that not only protein mutations, but also the physiological milieu, i.e. pH, cosolutes, crowding and surface interactions, have an impact on fibril formation. In this minireview, we focus on the effect of the less explored physical parameters temperature and pressure on the fibrillization propensity of proteins and how these variables can be used to reveal additional mechanistic information about intermediate states of fibril formation and molecular and structural polymorphism. Generally, amyloids are very stable and can resist harsh environmental conditions, such as extreme pH, high temperature and high pressure, and can hence serve as valuable functional amyloid. As an example, we discuss the effect of temperature and pressure on the catalytic activity of peptide amyloid fibrils that exhibit enzymatic activity.

Methyl cellulose (MC) is a semiflexible cellulose ether derivative with a wide range of industrial applications, owing to its water solubility at low temperatures and thermoreversible gelation upon heating. The gelation mechanism of aqueous MC solutions has been debated for many years. However, in 2010, gelation was discovered to be concurrent with fibril formation upon heating, whereby the MC polymer chains self-assemble into fibrils with a remarkably consistent mean diameter, largely independent of polymer concentration, molecular weight, and temperature of gelation. This discovery has shed important light on the gelation mechanism, and initiated studies that lead to more intriguing questions about the fibrils themselves. This review emphasizes various developments since the discovery of fibril formation, while highlighting unanswered questions that require further investigation.

To increase the value of bester sturgeon (a hybrid of Huso huso × Acipenser ruthenus) and promote development of the sturgeon aquaculture industry, this study assessed the feasibility of using collagens from bester sturgeon by-products for industrial applications. We first found that individual stable characteristics in terms of yield, amino acid composition, thermal stability, and fibril-forming ability of bester skin collagen (SC), swim bladder collagen (SBC), and notochord collagen (NC) were comparable with those of the beluga and sterlet, the purebred parental species of bester sturgeon. In addition, seasonal variations in these factors were not evident, except for the fibril-forming ability of SC, which showed significant seasonal differences. In the summer, slower fibril formation with smaller individual variations was detected compared with winter for SC. Overall, the quality of products produced from bester sturgeon collagens was stable for industrial use, but batchwise certification of the fibril-forming ability of SC is essential if the products are to be used in fibril form, as in the case of collagen for tissue engineering scaffolds. Second, we determined that the optimal temperature for extracting SC, SBC, and NC was 4 °C, 4 °C, and 20 °C, respectively. We also found that the high endotoxin content of NC could be reduced by pretreatment with 80% ETOH-0.1 M NaOH and 0.1 M NaOH. This study suggests that industrial use of collagens from bester sturgeon is feasible.

Controlling the fibril-formation process of collagen in vitro to fabricate novel biomaterials is a new area in the field of collagen research. This study aimed to determine the effect of ultrasonication on collagen fibril formation and the properties of the resulting collagen gels. Native collagen, extracted from the skin of grass carp, self-assembled under ultrasonic conditions (at different ultrasonic power and duration). The self-assembly kinetics, fibrillar morphology, and physical and cell growth-promoting properties of the collagen gels were analyzed and compared. The results showed that the self-assembly rate of collagen was increased by ultrasonication at the nucleation stage. The resulting fibrils exhibited smaller diameters and D-periodicity lengths than that of the untreated collagen samples (p

Several lines of evidence from neuropathological studies, human genetics, in vitro aggregation studies and cellular and animal models support the hypothesis that aSyn plays a central role in the formation of Lewy pathologies. These are cytoplasmic proteinaceous and lipid-rich inclusions that represent key pathological hallmarks of Parkinson's disease (PD) and other neurodegenerative diseases, collectively referred to as synucleinopathies. For decades, light microscopy and electron microscopy studies of these inclusions have consistently shown that they are rich in filamentous structures that exhibit distinct distribution and organizational patterns depending on where they occur in the brain (e.g., classical brain-stem Lewy bodies (LBs) and cortical LBs) and the type of synucleinopathies. Although the identity of the protein that form these filaments was a subject of debate for decades, the discovery of PD-linked aSyn mutations, the demonstration that LBs are enriched in insoluble forms of aSyn, and the ability of aSyn to form fibrils of similar dimensions have led to convergence on the hypothesis that aSyn fibrils are key components of LBs. In a recent study, Shahmoradian et al used a combination of advanced electron microscopy and immunofluorescence based imaging techniques to investigate the structure, composition, and architecture of LBs from postmortem brain tissues of individuals with PD or other synucleinopathies (Shahmoradian et al., 2019). The paper's main conclusions suggest that "lipid membrane fragments and distorted organelles together with a non-fibrillar form of alpha Syn are the main structural building blocks for the formation of Lewy pathology". Their proposal that LBs are devoid of aSyn fibrils or that LB formation occurs independently of aSyn fibril formation casts doubts on a substantial body of work that forms the foundation of many of the current basic and translational research programs in academia and industry. In this article, I present a critical analysis of their data and claims in the context of the existing literature In addition, I examine the extent to which their findings and proposed models of the mechanisms of LB formation are consistent with existing data and are supported by other experimental evidence. The results from this analysis caution against overinterpretation of observations from a single report, especially given the limitations of the techniques and experimental approaches used by Shahmoradian et al and for more collaborative and systematic efforts to revisit and characterize LBs and other aSyn pathologies in the brain pathologies at the biochemical, morphological and structural level.

Aggregation and fibril formation of human alpha-Synuclein (αS) are neuropathological hallmarks of Parkinson's disease and other synucleinopathies. The molecular mechanisms of αS aggregation and fibrillogenesis are largely unknown. Several studies suggested a sequence of events from αS dimerization via oligomerization and pre-fibrillar aggregation to αS fibril formation. In contrast to αS, little evidence suggests that γS can form protein aggregates in the brain, and for βS its neurotoxic properties and aggregation propensities are controversially discussed. These apparent differences in aggregation behavior prompted us to investigate the first step in Synuclein aggregation, i.e. the formation of dimers or oligomers, by Bimolecular Fluorescence Complementation in cells. This assay showed some Synuclein-specific limitations, questioning its performance on a single cell level. Nevertheless, we unequivocally demonstrate that all Synucleins can interact with each other in a very similar way. Given the divergent aggregation properties of the three Synucleins this suggests that formation of dimers is not predictive for the aggregation of αS, βS or γS in the aged or diseased brain.

The aggregation of proteins with expanded polyglutamine (polyQ) tracts is directly relevant to the formation of neuronal intranuclear inclusions in Huntington’s disease. In vitro studies have uncovered the effects of flanking sequences as modulators of the driving forces and mechanisms of polyQ aggregation in sequence segments associated with HD. Specifically, a seventeen-residue amphipathic stretch (N17) that is directly N-terminal to the polyQ tract in huntingtin decreases the overall solubility, destabilizes nonfibrillar aggregates, and accelerates fibril formation. Published results from atomistic simulations showed that the N17 module reduces the frequency of intermolecular association. Our reanalysis of these simulation results demonstrates that the N17 module also reduces interchain entanglements between polyQ domains. These two effects, which are observed on the smallest lengthscales, are incorporated into phenomenological pair potentials and used in coarse-grained Brownian dynamics simulations to investigate their impact on large-scale aggregation. We analyze the results from Brownian dynamics simulations using the framework of diffusion-limited cluster aggregation. When entanglements prevail, which is true in the absence of N17, small spherical clusters and large linear aggregates form on distinct timescales, in accord with in vitro experiments. Conversely, when entanglements are quenched and a barrier to intermolecular associations is introduced, both of which are attributable to N17, the timescales for forming small species and large linear aggregates become similar. Therefore, the combination of a reduction of interchain entanglements through homopolymeric polyQ and barriers to intermolecular associations appears to be sufficient for providing a minimalist phenomenological rationalization of in vitro observations regarding the effects of N17 on polyQ aggregation.

The degree of collagen fibril formation was altered by varying the preservation conditions. The collagen substrate under atmosphere of nitrogen gas for 4 days exhibited well-developed collagen fibril network accompanied with the frequency of round-shaped chondrocyte cells (f(R)) of 0.62, the value of which was slightly lower than that on the reference substrate. The exposure to air and prolongation of preservation led to further degradation of collagen fibril networks accompanied with f(R) lower than 0.4. It was thus demonstrated that the preservation conditions of the collagen substrate influenced the chondrocyte cell morphology.

Aggregates of amyloid-β (Aβ) peptides have been implicated in the etiology of Alzheimer disease. Among the different forms of Aβ aggregates, low molecular weight species ranging between ~2- and 50-mers, also called "soluble oligomers," have emerged as the species responsible for early synaptic dysfunction and neuronal loss. Emerging evidence suggests that the neurotoxic oligomers need not be formed along the obligatory nucleation-dependant fibril formation pathway. In our earlier work, we reported the isolation of one such "off-pathway" 12-18-mer species of Aβ42 generated from fatty acids called large fatty acid-derived oligomers (LFAOs) (Kumar, A., Bullard, R. L., Patel, P., Paslay, L. C., Singh, D., Bienkiewicz, E. A., Morgan, S. E., and Rangachari, V. (2011) PLoS One 6, e18759). Here, we report the physiochemical aspects of LFAO-monomer interactions as well as LFAO-LFAO associations in the presence of interfaces. We discovered that LFAOs are a replicating strain of oligomers that recruit Aβ42 monomers and quantitatively convert them into LFAO assemblies at the expense of fibrils, a mechanism similar to prion propagation. We also found that in the presence of hexane-buffer or chloroform-buffer interfaces LFAOs are able to associate with themselves to form larger but non-fibrillar aggregates. These results further support the hypothesis that low molecular weight oligomers can be generated via non-fibril formation pathways. Furthermore, the unique replicating property of off-pathway oligomers may hold profound significance for Alzheimer disease pathology.

The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) is neurotrophic and prevents fibril formation of amyloid β protein (Aβ). It is a promising lead compound for the development of therapeutic drugs for Alzheimer's disease. To find even more effective drugs based on the structure of fisetin, we synthesized a series of fisetin analogues lacking the 7-hydroxyl group and compared their effects on Aβ fibril formation determined by the thioflavin T fluorescence assay. 3,3',4'-Trihydroxyflavone and 3',4'-dihydroxyflavone inhibited Aβ fibril formation more potently than fisetin or 3',4',7-trihydroxyflavone, suggesting that the 7-hydroxy group is not necessary for anti-amyloidogenic activity. 3,3',4',5'-Tetrahydroxyflavone and 3',4',5'-trihydroxyflavone inhibited Aβ fibril formation far more potently than 3,3',4'-trihydroxyflavone and 3',4'-dihydroxyflavone, suggesting that 3',4',5'-trihydroxyl group of the B ring is crucial for the anti-amyloidogenic activity of flavonoids. Based on the structure-activity relationship, we synthesized 3,3',4',5,5'-pentahydroxyflavone, and confirmed that this compound is the most potent inhibitor of Aβ fibril formation among fisetin analogues that have been tested. Cytotoxicity assay using rat hippocampal neuron cultures demonstrated that Aβ preincubated with 3,3',4',5,5'-pentahydroxyflavone was significantly less toxic than Aβ preincubated with vehicle. 3,3',4',5,5'-Pentahydroxyflavone could be a new therapeutic drug candidate for the treatment of Alzheimer's disease.

A hallmark of Alzheimer’s disease (AD) is the amyloid β (Aβ) peptide fibril formation and deposition. The murine Aβ displays high sequence identity to its human counterpart, while it remains obscure why the former is less likely to aggregate with age and even in the presence of metal ions. With an attempt to elucidate the mechanism underlying the distinct aggregation properties of murine and human Aβ, molecular dynamics (MD) technique was used to perform parallel simulations on murine and human Aβ(1–42), and three single-point mutants, i.e. , R5G, Y10F and H13R of human Aβ(1–42), respectively. Parallel MD simulations on murine and human Aβ(1–42) revealed that the conformational stability of murine Aβ(1–42) is comparable with that of human counterpart. The simulations on the three single-point mutants also indicated that the R5G, Y10F and H13R mutations affect little on the conformational stability of the peptide. Therefore, the non-occurrence of murine Aβ aggregation may not arise from the dynamic characters of the peptide. Although the result is negative, it advances our understanding on the pathogenesis of AD.

Mixtures of γ-oryzanol and β-sitosterol have been shown to structure pure edible oil phases. This paper reviews the phytosterol system, and compares it with other alternatives to structure edible oils. Furthermore, additional evidence based on small-angle X-ray scattering will be introduced to support the claim that structuring is based on fibril formation (diameter 7.2 nm). Finally, various aspects of the application of phytosterols structuring in water-in-oil emulsions were investigated.

Characterization of the mechanisms by which proteins fold into their native conformations is important not only for protein structure prediction and design but also because protein misfolding intermediates may play critical roles in fibril formation that are commonplace in neurodegenerative disorders. In practice, the study of folding pathways is complicated by the fact that for the most part intermediates are low-populated and short-lived so that biophysical studies are difficult. Due to recent methodological advances, relaxation dispersion NMR spectroscopy has emerged as a particularly powerful tool to obtain high-resolution structural information about protein folding events on the millisecond timescale. Applications of the methodology to study the folding of SH3 domains have shown that folding proceeds via previously undetected on-pathway intermediates, sometimes stabilized by nonnative long-range interactions. The relaxation dispersion approach provides a detailed kinetic and thermodynamic description of the folding process as well as the promise of obtaining an atomic level structural description of intermediate states. We review the concerted application of a variety of recently developed NMR relaxation dispersion experiments to obtain a “high-resolution” picture of the folding pathway of the A39V/N53P/V55L Fyn SH3 domain.

We show that a series of peptides corresponding to individual beta-strands in native beta-lactoglobulin readily form amyloid aggregates and that such aggregates are capable of seeding fibril formation by a full-length form of beta-lactoglobulin in which the disulfide bonds are reduced. By contrast, preformed fibrils corresponding to only one of the beta-strands that we considered, betaA, were found to promote fibril formation by a full-length form of beta-lactoglobulin in which the disulfide bonds are intact. These results indicate that regions of high intrinsic aggregation propensity do not give rise to aggregation unless at least partial unfolding takes place. Furthermore, we found that the high aggregation propensity of one of the edge strands, betaI, promotes dimerisation of the native structure rather than misfolding and aggregation since the structure of betaI is stabilised by the presence of a disulfide bond. These findings demonstrate that the interactions that promote folding and native-state oligomerisation can also result in high intrinsic amyloidogenicity. However, we show that the presence of the remainder of the sequence dramatically reduces the net overall aggregation propensity by negative design principles that we suggest are very common in biological systems as a result of evolutionary processes.

The formation of collagen fibrils in poly(vinyl alcohol) (PVA) and in poly(vinyl pyrrolidone) (PVP) was investigated using Atomic Force Microscopy (AFM). The water solutions of PVA and PVP containing 1%, 3% and 5% of collagen were cast onto glass plate. After slow solvent evaporation thin polymeric films were obtained. AFM images showed the fibril formation in both, PVA and PVP films containing collagen. The amount of collagen in PVA and PVP matrix has an important effect on the structure and size of collagen fibril formed. The diameter of collagen fibrils in PVA films is bigger than the diameter of collagen fibrils formed in PVP films.

Transthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR, however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild type sea bream TTR (sbTTRWT) plus two mutants in which 6 (sbTTRM6) and 12 (sbTTRM12) N-terminal residues were removed. Ligand-binding studies revealed similar affinities for T3 (Kd=10.6+/-1.7nM) and T4 (Kd=9.8+/-0.97nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd=252.3+/-15.8nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine T. In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR.

Amyloid β proteins extracted from the amyloid cores of neuritic plaques are considerably racemized at their Asp residues. To assess the impact of d -Asp on amyloid β1–42 conformation and on initiation of amyloid fibril formation, we used wild-type amyloid β1–42 and analogs in which d -Asp was substituted for l -Asp at residues 1, 7, 23, and all combinations of these residues. Amyloid fibril formation was enhanced by d -Asp23; modulation of Asp chirality at N-terminal position 1 blocked this enhancement and modulation at position 7 augmented it. Knowledge of such chirality modifications may help to develop potent inhibitors of amyloid fibril formation.

The heat-induced denaturation and the molecular basis for aggregation in β -lactoglobulin has been investigated using a combination of molecular dynamics simulation and bioinformatics analysis. Molecular dynamics has been used to simulate the temperature induced unfolding of a single β -lactoglobulin molecule. Although the study is carried out at an elevated temperature to speed up the simulation, it confirms the experimental observation that the β -sheet structure in the protein is more stable to heat than the α-helical regions. We have also used bioinformatics analysis to search the β -lactoglobulin primary sequence for potential minimal sequences that may act as initiators for fibril formation in fine-stranded gels. Two potential candidate sequences were identified, and one GDLEIL was shown by molecular dynamics simulation to be able to form anti-parallel β -sheet with copies of itself. The potential significance of the minimal sequences to fine-stranded gel formation is discussed by way of analogy with the postulated mechanisms for amyloid fibril formation.

The aggregation and deposition onto neuronal cells of amyloid beta-peptide (Abeta) is central to the pathogenesis of Alzheimer's disease. Accumulating evidence suggests that membranes play a catalytic role in the aggregation of Abeta. This article summarizes the structures and properties of Abeta in solution and the physicochemical interaction of Abeta with lipid bilayers of various compositions. Reasons for discrepancies between results by different research groups are discussed. The importance of ganglioside clusters in the aggregation of Abeta is emphasized. Finally, a hypothetical physicochemical cascade in the pathogenesis of the disease is proposed.

Abeta40 and Abeta42 are the predominant Abeta species in the human body. Toxic Abeta42 oligomers and fibrils are believed to play a key role in causing Alzheimer's disease (AD). However, the role of Abeta40 in AD pathogenesis is not well established. Emerging evidence indicates a protective role for Abeta40 in AD pathogenesis. Although Abeta40 is known to inhibit Abeta42 fibril formation, it is not clear whether the inhibition acts on the non-toxic monomer or acts on the toxic Abeta42 oligomers. In contrast to conventional methods that detect the appearance of fibrils, in our study Abeta42 aggregation was monitored by the decreasing NMR signals from Abeta42 monomers. In addition, differential NMR isotope labelling enabled the selective observation of Abeta42 aggregation in a mixture of Abeta42 and Abeta40. We found Abeta40 monomers inhibit the aggregation of non-toxic Abeta42 monomers, in an Abeta42/Abeta40 ratio-dependent manner. NMR titration revealed that Abeta40 monomers bind to Abeta42 aggregates with higher affinity than Abeta42 monomers. Abeta40 can also release Abeta42 monomers from Abeta42 aggregates. Thus, Abeta40 likely protects Abeta42 monomers by competing for the binding sites on pre-existing Abeta42 aggregates. Combining our data with growing evidence from transgenic mice and human genetics, we propose that Abeta40 plays a critical, protective role in Alzheimer's by inhibiting the aggregation of Abeta42 monomer. Abeta40 itself, a peptide already present in the human body, may therefore be useful for AD prevention and therapy.

Early events in aggregation of proteins are not easily accessible by experiments. In this work, we perform a 5-ns molecular dynamics simulation of an ensemble of 27 copies of beta(2)-microglobulin in explicit solvent. During the simulation, the formation of intermolecular contacts is observed. The simulation highlights the importance of apical residues and, in particular, of those at the N-terminus end of the molecule. The most frequently found pattern of interaction involves a head-to-head contact arrangement of molecules. Hydrophobic contacts appear to be important for the establishment of long-lived (on the simulation timescale) contacts. Although early events on the pathway to aggregation and fibril formation are not directly related to the end-state of the process, which is reached on a much longer timescale, simulation results are consistent with experimental data and in general with a parallel arrangement of intermolecular beta-strand pairs.

—This review examines how protein misfolding pathways can be used to make self-assembling rod-like nanofibers. The development of these fibers and their potential application as protein-based, hybrid and functional materials is then discussed. Comparisons are also drawn between protein fibers and carbon nanotubes.

The aggregation (fibril formation) of amyloid beta-protein (Abeta) is considered to be a crucial step in the etiology of Alzheimer's disease (AD). The inhibition of Abeta aggregation and/or decomposition of fibrils formed in aqueous solution by small compounds have been studied extensively for the prevention and treatment of AD. However, recent studies suggest that Abeta aggregation also occurs in lipid rafts mediated by a cluster of monosialoganglioside GM1. This study examined the effects of representative compounds on Abeta aggregation and fibril destabilization in the presence of GM1-containing raft-like liposomes. Among the compounds tested, nordihydroguaiaretic acid (NDGA), rifampicin (RIF), tannic acid (TA), and quercetin (QUE) showed strong fibrillization inhibitory activity. NDGA and RIF inhibited the binding of Abeta to GM1 liposomes by competitively binding to the membranes and/or direct interaction with Abeta in solution, thus at least partly preventing fibrils from forming. Coincubation of Abeta with NDGA, RIF, and QUE in the presence of GM1 liposomes resulted in elongate particles, whereas the presence of TA yielded protofibrillar structures. TA and RIF also destabilized fibrils. The most potent NDGA prevented Abeta-induced toxicity in PC12 cells by inhibiting Abeta accumulation. Furthermore, a comparison of the inhibitory effects of various compounds between aqueous-phase and GM1-mediated aggregation of Abeta suggested that the two aggregation processes are not identical.

More than 20 different human proteins can fold abnormally resulting in the formation of pathological deposits and several lethal degenerative diseases. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained far from complete. In this work, utilizing hen egg-white lysozymes as a model system, two objectives were pursued: (1) to search for suitable conditions for producing amyloid fibrils and (2) to investigate inhibitory activities of two potential molecules against lysozyme fibril formation. Via numerous spectroscopic analyses and electron microscopy, our results showed that the formation of lysozyme amyloid fibrils at pH 2.0 was considerably increased by the addition of salt. Moreover, the inhibition of lysozyme amyloid formation by either p-benzoquinone or melatonin followed a concentration-dependent fashion. Furthermore, p-benzoquinone, in comparison with melatonin, served as a more effective inhibitor against amyloid fibril formation of lysozyme. We believe that a better understanding of how hen egg-white lysozymes aggregate will not only aid in deciphering the molecular mechanism of amyloid fibrillogenesis, but also shed light on a rational design of effective therapeutics for amyloidogenic diseases.

The pathological hallmarks of Alzheimer's disease (AD) consist of P-amyloid plaques and neurofibrillary tangles in affected brain areas. The processes, which drive this host reaction are unknown. To determine whether an analogous host reaction to that occurring in AD could be induced by infectious agents, we exposed mammalian glial and neuronal cells in vitro to Borrelia burgdorferi spirochetes and to the inflammatory bacterial lipopolysaccharide (LPS). Morphological changes analogous to the amyloid deposits of AD brain were observed following 2-8 weeks of exposure to the spirochetes. Increased levels of beta-amyloid presursor protein (A beta PP) and hyperphosphorylated tau were also detected by Western blots of extracts of cultured cells that had been treated with spirochetes or LPS. These observations indicate that, by exposure to bacteria or to their toxic products, host responses similar in nature to those observed in AD may be induced. (C) 2005 Elsevier Inc. All rights reserved.

Alpha-synuclein has been linked to amyloidogenesis in Parkinson's disease and other neurodegenerative disorders. We have previously shown that a peptide comprising residues 68-78 of alpha-synuclein is the minimum fragment that, like alpha-synuclein itself, forms amyloid fibrils and exhibits toxicity towards cells in culture. Hughes et al. [J. Biol. Chem. 275 (2000) 25109] showed that an N-methylated derivative of Abeta(25-35) inhibited the formation of fibrils by Abeta(25-35) and reduced its toxicity. We have now extended this concept to an amyloidogenic alpha-synuclein-based peptide. Alpha-synuclein(68-78), N-methylated at G1y73, was compared to non-methylated peptide. Whereas alpha-synuclein(68-78) formed fibrils and was toxic to cells, the N-methylated analogue had neither of these properties. Moreover, an equimolar mixture of the non-methylated and methylated peptides formed very few fibrils and toxicity was markedly reduced.

α-Synuclein (α-Syn) is a small intrinsically disordered 140 amino acids (14 kDa) protein expressed abundantly in human brain, regulating the release of neurotransmitters, vesicle recycling, and synaptic integrity. Adequate α-Syn monomers in solution undergo a nucleation-dependent mechanism growing into oligomers, which are able to elongate to form profibers through monomers addition and finally mature fibers. It is well acknowledged that the abnormal aggregation process of α-Syn monomers is associated with pathophysiology of Parkinson's disease. Identifying different oligomer types and tracking their aggregation dynamic process in lipid free or lipid co-incubated system, which not only present the significant step during fibrillation but also play a key role in pathogenesis of Parkinson's disease, is hard to know due to the inherent challenges of studying heterogeneous and transient system by using traditional biotechnology. In this study, we use solid-state nanopores to study the aggregation progress of α-Syn only sample and lipid-small unilamellar vesicles co-incubated α-Syn sample in a single-molecule approach. A tween 20 surface coating method was developed to enable continuous and smooth α-Syn translocation through SiN nanopores. Through analysis of translocation events for α-Syn incubated at different time, we identified four types of oligomers forming during aggregation process. These four types oligomers inferred as intermediates show a time-dependent quantity fraction change, suggesting that the formation of large oligomers is based on the consumption of small oligomers. Furthermore, we investigated the effect of lipid small unilamellar vesicles on α-Syn aggregation process. The experimental results indicate that it is the presence of 20% 1,2-dioleoyl-sn- glycero-3- [phospho-L-serine] (DOPS) dramatically enhances the aggregation rate of α-Syn, while the aggregation pathway is similar to α-Syn only samples.