Effects of salt concentration on association of the amyloid protofilaments of hen egg white lysozyme studied by time-resolved neutron scattering

Various proteins have been shown to form various aggregated structures including the filamentous aggregates known as amyloid fibrils depending on the solution conditions. Hen egg white lysozyme (HEWL) is one of the proteins that form the amyloid fibrils. To gain insight into the mechanism of this polymorphism of the aggregated structures, we employed a model system consisting of HEWL, pure water, and ethanol, and investigated the kinetic process of the fibril formation in various salt concentrations with time-resolved neutron scattering. It was shown that by addition of NaCl in a range between 0.3 mM and 1.0 mM to HEWL solution in 90% ethanol, gelation occurred, and this gelation proceeded through a two-step process: the lateral association of the protofilaments, followed by the cross-linking of these fibrils formed. Both the structures of the fibrils and the rate of the gelation depended on NaCl concentration. The average structures of the fibrils formed at 1.0 mM NaCl were characterized by the radius of gyration of their cross-section (45.9(+/-0.4)A) and the number of the protofilaments within the fibril (4.10(+/-0.12)), corresponding to the mature amyloid fibrils. A range of intermediate structures was formed below 1 mM NaCl. Above 2 mM NaCl, precipitation occurred because of the formation of amorphous aggregates. Here the branch point to the formation of the mature amyloid fibrils or to the amorphous aggregates was after the formation of the protofilaments. Sensitivity of the aggregated structures to salt concentration suggests that electrostatic interaction plays an essential role in the formation of these structures. The structural diversity both in the fibrils and the aggregated structures of the fibrils can be interpreted in terms of the difference in the degree of the electrostatic shielding at different salt concentrations.     

Lysozyme amyloidogenesis is accelerated by specific nicking and fragmentation but decelerated by intact protein binding and conversion

We have revisited the well-studied heat and acidic amyloid fibril formation pathway (pH 1.6, 65 degrees C) of hen egg-white lysozyme (HEWL) to map the barriers of the misfolding and amyloidogenesis pathways. A comprehensive kinetic mechanism is presented where all steps involving protein hydrolysis, fragmentation, assembly and conversion into amyloid fibrils are accounted for. Amyloid fibril formation of lysozyme has multiple kinetic barriers. First, HEWL unfolds within minutes, followed by irreversible steps of partial acid hydrolysis affording a large amount of nicked HEWL, the 49-101 amyloidogenic fragment and a variety of other species over 5-40 h. Fragmentation forming the 49-101 fragment is a requirement for efficient amyloid fibril formation, indicating that it forms the rate-determining nucleus. Nicked full-length HEWL is recruited efficiently into amyloid fibrils in the fibril growth phase or using mature fibrils as seeds, which abolished the lag phase completely. Mature amyloid fibrils of HEWL are composed mainly of nicked HEWL in the early equilibrium phase but go through a "fibril shaving" process, affording fibrils composed of the 49-101 fragment and 53-101 fragment during more extensive maturation (incubation for longer than ten days). Seeding of the amyloid fibril formation process using sonicated mature amyloid fibrils accelerates the fibril formation process efficiently; however, addition of intact full-length lysozyme at the end of the lag phase slows the rate of amyloidogenesis. The intact full-length protein, in contrast to nicked lysozyme, slows fibril formation due to its slow conversion into the amyloid fold, probably due to inclusion of the non-amyloidogenic 1-48/102-129 portion of HEWL in the fibrils, which can function as a "molecular bumper" stalling further growth.     

The protofilament substructure of amyloid fibrils

Tissue deposition of normally soluble proteins, or their fragments, as insoluble amyloid fibrils causes the usually fatal, acquired and hereditary systemic amyloidoses and is associated with the pathology of Alzheimer's disease, type 2 diabetes and the transmissible spongiform encephalopathies. Although each type of amyloidosis is characterised by a specific amyloid fibril protein, the deposits share pathognomonic histochemical properties and the structural morphology of all amyloid fibrils is very similar. We have previously demonstrated that transthyretin amyloid fibrils contain four constituent protofilaments packed in a square array. Here, we have used cross-correlation techniques to average electron microscopy images of multiple cross-sections in order to reconstruct the sub-structure of ex vivo amyloid fibrils composed of amyloid A protein, monoclonal immunoglobulin lambda light chain, Leu60Arg variant apolipoprotein AI, and Asp67His variant lysozyme, as well as synthetic fibrils derived from a ten-residue peptide corresponding to the A-strand of transthyretin. All the fibrils had an electron-lucent core but the packing arrangement comprised five or six protofilaments rather than four. The structural similarity that defines amyloid fibres thus exists principally at the level of beta-sheet folding of the polypeptides within the protofilament, while the different types vary in the supramolecular assembly of their protofilaments.     

Ultrastructural organization of amyloid fibrils by atomic force microscopy

Atomic force microscopy has been employed to investigate the structural organization of amyloid fibrils produced in vitro from three very different polypeptide sequences. The systems investigated are a 10-residue peptide derived from the sequence of transthyretin, the 90-residue SH3 domain of bovine phosphatidylinositol-3'-kinase, and human wild-type lysozyme, a 130-residue protein containing four disulfide bridges. The results demonstrate distinct similarities between the structures formed by the different classes of fibrils despite the contrasting nature of the polypeptide species involved. SH3 and lysozyme fibrils consist typically of four protofilaments, exhibiting a left-handed twist along the fibril axis. The substructure of TTR(10-19) fibrils is not resolved by atomic force microscopy and their uniform appearance is suggestive of a regular self-association of very thin filaments. We propose that the exact number and orientation of protofilaments within amyloid fibrils is dictated by packing of the regions of the polypeptide chains that are not directly involved in formation of the cross-beta core of the fibrils. The results obtained for these proteins, none of which is directly associated with any human disease, are closely similar to those of disease-related amyloid fibrils, supporting the concept that amyloid is a generic structure of polypeptide chains. The detailed architecture of an individual fibril, however, depends on the manner in which the protofilaments assemble into the fibrillar structure, which in turn is dependent on the sequence of the polypeptide and the conditions under which the fibril is formed.     

An atomic model for the pleated beta-sheet structure of Abeta amyloid protofilaments.

Synchrotron x-ray studies on amyloid fibrils have suggested that the stacked pleated beta-sheets are twisted so that a repeating unit of 24 beta-strands forms a helical turn around the fibril axis (. J. Mol. Biol. 273:729-739). Based on this morphological study, we have constructed an atomic model for the twisted pleated beta-sheet of human Abeta amyloid protofilament. In the model, 48 monomers of Abeta 12-42 stack (four per layer) to form a helical turn of beta-sheet. Each monomer is in an antiparallel beta-sheet conformation with a turn located at residues 25-28. Residues 17-21 and 31-36 form a hydrophobic core along the fibril axis. The hydrophobic core should play a critical role in initializing Abeta aggregation and in stabilizing the aggregates. The model was tested using molecular dynamics simulations in explicit aqueous solution, with the particle mesh Ewald (PME) method employed to accommodate long-range electrostatic forces. Based on the molecular dynamics simulations, we hypothesize that an isolated protofilament, if it exists, may not be twisted, as it appears to be when in the fibril environment. The twisted nature of the protofilaments in amyloid fibrils is likely the result of stabilizing packing interactions of the protofilaments. The model also provides a binding mode for Congo red on Abeta amyloid fibrils. The model may be useful for the design of Abeta aggregation inhibitors.     

Inhibition of amyloid fibrillation of lysozyme by indole derivatives--possible mechanism of action

Amyloid aggregation of polypeptides is related to a growing number of pathologic states known as amyloid disorders. There is a great deal of interest in developing small molecule inhibitors of the amyloidogenic processes. In the present article, the inhibitory effects of some indole derivatives on amyloid fibrillation of hen egg white lysozyme (HEWL) are reported. Acidic pH and high temperatures were used to drive HEWL towards amyloid formation. A variety of techniques, ranging from thioflavin T fluorescence and Congo red absorbance assays to far-UV CD and transmission electron microscopy, were employed to characterize the HEWL fibrillation process. Among the indole derivatives tested, indole 3-acetic acid, indole 3-carbinol and tryptophol had the most inhibitory effects on amyloid formation, indole and indole 3-propionic acid gave some inhibition, and indole aldehyde and tryptophan showed no significant inhibition. Although indoles did not protect the HEWL native state from conformational changes, they were effective in diminishing HEWL amyloid fibril formation, delaying both the nucleation and elongation phases. Disaggregation of previously formed HEWL amyloid fibrils was also enhanced by indole 3-acetic acid. Various medium conditions, such as the presence of different anions and alcoholic cosolvents, were explored to gain an insight into possible mechanisms. These observations, taken together, suggest that the indole ring is likely to play the main role in inhibition and that the side chain hydroxyl group may contribute positively, in contrast to the side chain carbonyl and intervening methylene groups.     

Cryo-electron microscopy structure of an SH3 amyloid fibril and model of the molecular packing

Amyloid fibrils are assemblies of misfolded proteins and are associated with pathological conditions such as Alzheimer's disease and the spongiform encephalopathies. In the amyloid diseases, a diverse group of normally soluble proteins self-assemble to form insoluble fibrils. X-ray fibre diffraction studies have shown that the protofilament cores of fibrils formed from the various proteins all contain a cross-beta-scaffold, with beta-strands perpendicular and beta-sheets parallel to the fibre axis. We have determined the threedimensional structure of an amyloid fibril, formed by the SH3 domain of phosphatidylinositol-3'-kinase, using cryo-electron microscopy and image processing at 25 A resolution. The structure is a double helix of two protofilament pairs wound around a hollow core, with a helical crossover repeat of approximately 600 A and an axial subunit repeat of approximately 27 A. The native SH3 domain is too compact to fit into the fibril density, and must unfold to adopt a longer, thinner shape in the amyloid form. The 20x40-A protofilaments can only accommodate one pair of flat beta-sheets stacked against each other, with very little inter-strand twist. We propose a model for the polypeptide packing as a basis for understanding the structure of amyloid fibrils in general.     

Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration

The calcium-binding equine lysozyme has been found to undergo conversion into amyloid fibrils during incubation in solution at acidic pH. At pH 4.5 and 57 degrees C, where equine lysozyme forms a partially unfolded molten globule state, the protein forms protofilaments with a width of ca. 2 nm. In the absence of Ca(2+) the protofilaments are present as annular structures with a diameter of 40-50 nm. In the presence of 10 mM CaCl(2) the protofilaments of equine lysozyme are straight or curved; they can assemble into thicker threads, but they do not appear to undergo circularisation. At pH 2.0, where the protein is more destabilised compared to pH 4.5, fibril formation occurs at 37 degrees C and 57 degrees C. At pH 2.0, both ring-shaped and linear protofilaments are formed, in which periodic repeats of ca 35 nm can be distinguished clearly. The rings constitute about 10% of all fibrillar species under these conditions and they are characterised by a larger diameter of 70-80 nm. All the structures bind Congo red and thioflavine T in a manner similar to fibrils associated with a variety of amyloid diseases. At pH 2.0, fibril formation is accompanied by some acidic hydrolysis, producing specific fragmentation of the protein, leading to the accumulation of two peptides in particular, consisting of residues 1-80 and 54-125. At the initial stages of incubation, however, full-length equine lysozyme represents the dominant species within the fibrils. We propose that the ring-shaped structures observed here, and in the case of disease-associated proteins such as alpha-synuclein, could be a second generic type of amyloid structure in addition to the more common linear fibrils.     

Amyloid protofilament formation of hen egg lysozyme in highly concentrated ethanol solution

Mutant human lysozymes (Ile56Thr & Asp67His) have been reported to form amyloid deposits in the viscera. From the standpoint of understanding the mechanism of amyloid formation, we searched for conditions of amyloid formation in vitro using hen egg lysozyme, which has been extensively studied from a physicochemical standpoint. It was found that the circular dichroism spectra in the far-ultraviolet region of the hen egg lysozyme changed to those characteristic of a beta-structure from the native alpha-helix rich spectrum in 90% ethanol solution. When the concentration of protein was increased to 10 mg/mL, the protein solution formed a gel in the presence of 90% ethanol, and precipitated on further addition of 10 mM NaCl. The precipitates were examined by electron microscopy, their ability to bind Congo red, and X-ray diffraction to determine whether amyloid fibrils were formed in the precipitates. Electron micrographs displayed unbranched protofilament with a diameter of approximately 70 A. The peak point of the difference spectrum for the Congo red binding assay was 541 nm, which is characteristic of amyloid fibrils. The X-ray diffraction pattern showed a sharp and intense diffraction ring at 4.7 A, a reflection that arises from the interstrand spacing in beta-sheets. These results indicate that the precipitates of hen egg lysozyme are amyloid protofilament, and that the amyloid protofilament formation of hen egg lysozyme closely follows upon the destruction of the helical and tertiary structures.     

Sodium louroyl sarcosinate (sarkosyl) modulate amyloid fibril formation in hen egg white lysozyme (HEWL) at alkaline pH: a molecular insight study

Amyloid fibril formation is responsible for several neurodegenerative diseases and are formed when native proteins misfold and stick together with different interactive forces. In the present study, we have determined the mode of interaction of the anionic surfactant sarkosyl with hen egg white lysozyme (HEWL) [EC No. 3.2.1.17] at two pHs (9.0 and 13.0) and investigated its impact on fibrillogenesis. Our data suggested that sarkosyl is promoting amyloid fibril formation in HEWL at the concentration range between 0.9 and 3.0 mM and no amyloid fibril formation was observed in the concentration range of 3.0–20.0 mM at pH 9.0. The results were confirmed by several biophysical and computational techniques, such as turbidity measurement, dynamic light scattering, Raleigh scattering, ThT fluorescence, intrinsic fluorescence, far-UV CD and atomic force microscopy. Sarkosyl was unable to induce aggregation in HEWL at pH 13.0 as confirmed by turbidity and RLS measurements. HEWL forms larger amyloid fibrils in the presence of 1.6 mM of sarkosyl. The spectroscopic, microscopic and molecular docking data suggest that the negatively charged carboxylate group and 12-carbon hydrophobic tail of sarkosyl stimulate amyloid fibril formation in HEWL via electrostatic and hydrophobic interaction. This study leads to new insight into the process of suppression of fibrillogenesis in HEWL which can be prevented by designing ligands that can retard the electrostatic and hydrophobic interaction between sarkosyl and HEWL.     

Formation and seeding of amyloid fibrils from wild-type hen lysozyme and a peptide fragment from the beta-domain

Wild-type hen lysozyme has been converted from its soluble native state into highly organized amyloid fibrils. In order to achieve this conversion, conditions were chosen to promote partial unfolding of the native globular fold and included heating of low-pH solutions and addition of organic solvents. Two peptides derived from the beta-sheet region of hen lysozyme were also found to form fibrils very readily. The properties and morphologies of the amyloid fibrils formed by incubation either of the protein or the peptides are similar to those produced from the group of proteins associated with clinical amyloidoses. Fibril formation by hen lysozyme was substantially accelerated when aliquots of solutions in which fibrils of either one of the peptides or the full-length protein had previously formed were added to fresh solutions of the protein, revealing the importance of seeding in the kinetics of fibril formation. These findings support the proposition that the beta-domain is of particular significance in the formation of fibrils from the full-length protein and suggest similarities between the species giving rise to fibril formation and the intermediates formed during protein folding.     

Analysis of Core Region from Egg White Lysozyme Forming Amyloid Fibrils

Some of the lysozyme mutants in humans cause systemic amyloidosis. Hen egg white lysozyme (HEWL) has been well studied as a model protein of amyloid fibrils formation. We previously identified an amyloid core region consisting of nine amino acids (designated as the K peptide), which is present at 54-62 in HEWL. The K peptide, with tryptophan at its C- terminus, has the ability of self-aggregation. In the present work we focused on its structural properties in relation to the formation of fibrils. The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C. A substantial number of fibrils were generated under this pH condition and incubation period. Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils. Tryptophan 62 in lysozyme was suggested to be especially crucial to forming amyloid fibrils. We also show that amyloid fibrils formation of the K peptide requires not only tryptophan 62 but also a certain length containing hydrophobic amino acids. A core region is involved in the significant formation of amyloid fibrils of lysozyme.     

Organization of designed nanofibrils assembled from alpha-helical peptides as determined by electron microscopy.

Self-assembling peptides present attractive platforms for engineering materials with controlled nanostructures. Recently, an alpha-helical fibril forming peptide (alphaFFP) was designed that self-assembles into nanofibrils at acid pH. Circular dichroism spectroscopy, electron-microscopy and x-ray fibre diffraction data showed that the most likely structure of alphaFFP fibrils is a five-stranded coiled coil rope. In the present study, scanning transmission electron microscopy (STEM) was used to improve our understanding of the alphaFFP fibril structure. The measurements of fibril mass per length suggest that there are ten alpha-helices in transverse sections of the fibrils. Based on the known data, it is proposed that a predominant fibrillar structure of alphaFFP is a dimer of alpha-helical five stranded protofilaments wrapped around a common axis. It is shown that these structures have an axial dimension of 58 +/- 16 nm and a width of 4 +/- 1 nm. A small number of thin fibrils is also observed in the negative stained preparation and STEM images. The thin fibrils may correspond to the single protofilament.     

Higher-order molecular packing in amyloid-like fibrils constructed with linear arrangements of hydrophobic and hydrogen-bonding side-chains

Various mutants of the protein fragment, barnase module-1 (1-24) were investigated in order to reveal the structural principle of amyloid-like fibrils. By means of circular dichroism spectroscopy, X-ray diffraction, electron microscopy, and thioflavin T binding assay, we found that the molecules containing two beta-strands and an intervening turn structure are assembled to form a cross-beta structure. Stabilization by both the hydrophobic interactions and hydrogen bonding between the respective paired side-chains on the coupled beta-strands was essential for fibril formation. These two types of interaction can also arrange the corresponding residues in lines on both sheet surfaces of protofilaments with a cross-beta structure. This leads to the most probable fibril structure constructed with the line-matching interactions between protofilaments. Consideration of the geometrical symmetry resulted in our finding that a limited number of essential models for molecular packing in fibril structure are stable, which would rationally explain the occurrence of two or three morphologies from an identical molecular species. The ribbon-like fibrils exhibited striped texture along the axis, which was assigned to a stacked two-sheet repeat as a structural unit. The comprehensively proposed structural model, that is, the sheet-sheet interaction between left-handed cross-beta structures, results in a slightly right-handed twist of beta-sheet stacking, which reasonably elucidates the intrinsic sizes of the fibril width and its helical period along the fibril axis, as the bias in the orientation of the hydrogen-bonded beta-strand pair at the lateral edge is larger than that at the central protofilament.     

Structural features of the Abeta amyloid fibril elucidated by limited proteolysis

Although the gross morphology of amyloid fibrils is fairly well understood, very little is known about how the constituent polypeptides fold within the amyloid folding motif. In the experiments reported here, we used trypsin and chymotrypsin to conduct limited proteolysis studies on synthetic amyloid fibrils composed of the Alzheimer's disease peptide Abeta(1-40). In both reactions, the extreme N-terminal proteolytic fragment is released from fibrils as rapidly as it is from the Abeta monomer, while other proteolytic fragments are generated much more slowly. Furthermore, aggregated material isolated by centrifugation of intermediate digestion time points from both proteases contains, in addition to full-length material, peptides that possess mature C-termini but truncated N-termini. These data strongly suggest that the N-terminal region of Abeta is not involved in the beta-sheet network of the amyloid fibril, while the C-terminus is essentially completely engaged in protective-presumably beta-sheet-structure. In both digests, release of the extreme N-terminal fragments of Abeta(1-40) reaches plateau values corresponding to about 80% of the total available Abeta. This suggests that there are two classes of peptides in the fibril: while the majority of Abeta molecules have an exposed N-terminus, about 20% of the peptides have an N-terminus that is protected from proteolysis within the fibril structure. The most likely cause of this heterogeneity is the lateral association of protofilaments into the fibril structure, which would be expected to generate a unique environment for those Abeta N-termini located at protofilament packing interfaces and/or in the interior core region between the packed protofilaments. This suggests that the N-terminal region of Abeta, while not directly involved in the beta-sheet network of the fibril, may contribute to fibril stability by participating in protofilament packing.     

Role of different regions of alpha-synuclein in the assembly of fibrils

Elucidating the details of the assembly of amyloid fibrils is a key step to understanding the mechanism of amyloid deposition diseases including Parkinson's disease. Although several models have been proposed, based on analyses of polypeptides and short peptides, a detailed understanding of the structure and mechanism of alpha-synuclein fibrillation remains elusive. In this study, we used trypsin and endoproteinase GluC to digest intact alpha-synuclein fibrils and to analyze the detailed morphology of the resultant fibrils/remnants. We also created three mutants of alpha-synuclein, in which the N-terminal and C-terminal regions were removed, both individually and in combination, and investigated the detailed morphology of the fibrils from these mutants. Our results indicate that the assembly of mature alpha-synuclein fibrils is hierarchical: protofilaments --> protofibrils --> mature fibrils. There is a core region of approximately 70 amino acids, from residues approximately 32 to 102, which comprises the beta-rich core of the protofilaments and fibrils. In contrast, the two terminal regions show no evidence of participating in the assembly of the protofilament core but play a key role in the interactions between the protofilaments, which is necessary for the fibril maturation.     

Conformational Stability of Fibrillar Amyloid-Beta Oligomers via Protofilament Pair Formation – A Systematic Computational Study

Amyloid- (A) oligomers play a crucial role in Alzheimer’s disease due to their neurotoxic aggregation properties. Fibrillar A oligomerization can lead to protofilaments and protofilament pairs via oligomer elongation and oligomer association, respectively. Small fibrillar oligomers adopt the protofilament topology, whereas fibrils contain at least protofilament pairs. To date, the underlying growth mechanism from oligomers to the mature fibril still remains to be elucidated. Here, we performed all-atom molecular dynamics simulations in explicit solvent on single layer-like protofilaments and fibril-like protofilament pairs of different size ranging from the tetramer to the 48-mer. We found that the initial U-shaped topology per monomer is maintained over time in all oligomers. The observed deviations of protofilaments from the starting structure increase significantly with size due to the twisting of the in-register parallel -sheets. This twist causes long protofilaments to be unstable and leads to a breakage. Protofilament pairs, which are stabilized by a hydrophobic interface, exhibit more fibril-like properties such as the overall structure and the twist angle. Thus, they can act as stable conformational templates for further fibril growth. Key properties like the twist angle, shape complementarity, and energetics show a size-dependent behavior so that small oligomers favor the protofilament topology, whereas large oligomers favor the protofilament pair topology. The region for this conformational transition is at the size of approximately twelve A monomers. From that, we propose the following growth mechanism from A oligomers to fibrils: (1) elongation of short protofilaments; (2) breakage of large protofilaments; (3) formation of short protofilament pairs; and (4) elongation of protofilament pairs.     

Amyloid fibril formation of alpha-synuclein is accelerated by preformed amyloid seeds of other proteins: implications for the mechanism of transmissible conformational diseases

Alpha-synuclein is one of the causative proteins of familial Parkinson disease, which is characterized by neuronal inclusions named Lewy bodies. Lewy bodies include not only alpha-synuclein but also aggregates of other proteins. This fact raises a question as to whether the formation of alpha-synuclein amyloid fibrils in Lewy bodies may occur via interaction with fibrils derived from different proteins. To probe this hypothesis, we investigated in vitro fibril formation of human alpha-synuclein in the presence of preformed fibril seeds of various different proteins. We used three proteins, Escherichia coli chaperonin GroES, hen lysozyme, and bovine insulin, all of which have been shown to form amyloid fibrils. Very surprisingly, the formation of alpha-synuclein amyloid fibril was accelerated markedly in the presence of preformed seeds of GroES, lysozyme, and insulin fibrils. The structural characteristics of the natively unfolded state of alpha-synuclein may allow binding to various protein particles, which in turn triggers the formation (extension) of alpha-synuclein amyloid fibrils. This finding is very important for understanding the molecular mechanism of Parkinson disease and also provides interesting implications into the mechanism of transmissible conformational diseases.     

On the Characterization of Intermediates in the Isodesmic Aggregation Pathway of Hen Lysozyme at Alkaline pH

Protein aggregation leading to formation of amyloid fibrils is a symptom of several diseases like Alzheimer’s, type 2 diabetes and so on. Elucidating the poorly understood mechanism of such phenomena entails the difficult task of characterizing the species involved at each of the multiple steps in the aggregation pathway. It was previously shown by us that spontaneous aggregation of hen-eggwhite lysozyme (HEWL) at room temperature in pH 12.2 is a good model to study aggregation. Here in this paper we investigate the growth kinetics, structure, function and dynamics of multiple intermediate species populating the aggregation pathway of HEWL at pH 12.2. The different intermediates were isolated by varying the HEWL monomer concentration in the 300 nM—0.12 mM range. The intermediates were characterized using techniques like steady-state and nanosecond time-resolved fluorescence, atomic force microscopy and dynamic light scattering. Growth kinetics of non-fibrillar HEWL aggregates were fitted to the von Bertalanffy equation to yield a HEWL concentration independent rate constant (k = (6.6±0.6)×10⁻⁵ s⁻¹). Our results reveal stepwise changes in size, molecular packing and enzymatic activity among growing HEWL aggregates consistent with an isodesmic aggregation model. Formation of disulphide bonds that crosslink the monomers in the aggregate appear as a unique feature of this aggregation. AFM images of multiple amyloid fibrils emanating radially from amorphous aggregates directly confirmed that on-pathway fibril formation was feasible under isodesmic polymerization. The isolated HEWL aggregates are revealed as polycationic protein nanoparticles that are robust at neutral pH with ability to take up non-polar molecules like ANS.     

Glucagon Fibril Polymorphism Reflects Differences in Protofilament Backbone Structure

Amyloid fibrils formed by the 29-residue peptide hormone glucagon at different concentrations have strikingly different morphologies when observed by transmission electron microscopy. Fibrils formed at low concentration (0.25 mg/mL) consist of two or more protofilaments with a regular twist, while fibrils at high concentration (8 mg/mL) consist of two straight protofilaments. Here, we explore the structural differences underlying glucagon polymorphism using proteolytic degradation, linear and circular dichroism, Fourier transform infrared spectroscopy (FTIR), and X-ray fiber diffraction. Morphological differences are perpetuated at all structural levels, indicating that the two fibril classes differ in terms of protofilament backbone regions, secondary structure, chromophore alignment along the fibril axis, and fibril superstructure. Straight fibrils show a conventional β-sheet-rich far-UV circular dichroism spectrum whereas that of twisted fibrils is dominated by contributions from β-turns. Fourier transform infrared spectroscopy confirms this and also indicates a more dense backbone with weaker hydrogen bonding for the twisted morphology. According to linear dichroism, the secondary structural elements and the aromatic side chains in the straight fibrils are more highly ordered with respect to the alignment axis than the twisted fibrils. A series of highly periodical reflections in the diffractogram of the straight fibrils can be fitted to the diffraction pattern expected from a cylinder. Thus, the highly integrated structural organization in the straight fibril leads to a compact and highly uniform fibril with a well-defined edge. Prolonged proteolytic digestion confirmed that the straight fibrils are very compact and stable, while parts of the twisted fibril backbone are much more readily degraded. Differences in the digest patterns of the two morphologies correlate with predictions from two algorithms, suggesting that the polymorphism is inherent in the glucagon sequence. Glucagon provides a striking illustration of how the same short sequence can be folded into two remarkably different fibrillar structures.