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.     

Branching in Amyloid Fibril Growth

Using the peptide hormone glucagon and Aβ(1-40) as model systems, we have sought to elucidate the mechanisms by which fibrils grow and multiply. We here present real-time observations of growing fibrils at a single-fibril level. Growing from preformed seeds, glucagon fibrils were able to generate new fibril ends by continuously branching into new fibrils. To our knowledge, this is the first time amyloid fibril branching has been observed in real-time. Glucagon fibrils formed by branching always grew in the forward direction of the parent fibril with a preferred angle of 35-40°. Furthermore, branching never occurred at the tip of the parent fibril. In contrast, in a previous study by some of us, Aβ(1-40) fibrils grew exclusively by elongation of preformed seeds. Fibrillation kinetics in bulk solution were characterized by light scattering. A growth process with branching, or other processes that generate new ends from existing fibrils, should theoretically give rise to different fibrillation kinetics than growth without such a process. We show that the effect of adding seeds should be particularly different in the two cases. Our light-scattering data on glucagon and Aβ(1-40) confirm this theoretical prediction, demonstrating the central role of fibril-dependent nucleation in amyloid fibril growth     

The changing face of glucagon fibrillation: structural polymorphism and conformational imprinting

We have established a time-resolved fluorescence assay to study fibrillation of the 29 residue peptide hormone glucagon under a variety of different conditions in a high-throughput format. Fibrils formed at pH 2.5 differ in fibrillation kinetics, morphology, thioflavin T staining and FTIR/CD spectra depending on salts, glucagon concentration and fibrillation temperature. Apparent fibrillar stability correlates with spectral and kinetic properties; generally, fibrils formed under conditions favourable for rapid fibrillation (ambient temperatures, high glucagon concentration or high salt concentration) appear less thermostable than those formed under more challenging conditions (high temperatures, low glucagon or low salt concentrations). Properties of preformed fibrils used for seeding are inherited in a prion-like manner. Thus, we conclude that the structure of fibrils formed by glucagon is not the result of the global energy minimization, but rather kinetically controlled by solvent conditions and seed-imprinting. Fibrillar polymorphism, which is being reported for an increasing number of proteins, probably reflects that fibrils have not been under evolutionary constraints to retain a single active conformation. Our results highlight the complexity of the fibrillation mechanism of glucagon, since even subtle changes in fibrillation conditions can alter the type of fibrils formed, or result in formation of mixtures of several types of fibrils.     

Amyloid fibril formation by peptide LYS (11-36) in aqueous trifluoroethanol

Peptide LYS (11-36), derived from the beta-sheet region of T4 lysozyme, forms an amyloid fibril in aqueous trifluoroethanol (TFE) at elevated temperature. The peptide has a moderate alpha-helix content in 20 and 50% (v/v) TFE solution; large quantities of fibrils were formed after incubation at 55 degrees C for 2 weeks as monitored by a thioflavin T fluorescence assay. No fibrils were observed when the peptide initially existed predominantly as a random coil or as a complete alpha helix. Our results suggest that a moderate amount of alpha helix and random coil present in the peptide initially facilitates the fibril-formation process, but a high alpha-helix content inhibits fibril formation. Transmission electron microscopy revealed several types of fibril morphologies at different TFE concentrations. The fibrils were highly twisted and consisted of interleaved protofilaments in 50% TFE, while smooth and flat ribbonlike fibrils were found in 20% TFE. In 50% TFE, the fibril growth rate of LYS (11-36) was found to depend strongly on peptide concentration and seeding but was insensitive to solution pH and ionic strength.     

Visualization of polymorphism in apolipoprotein C-II amyloid fibrils

The misfolding and self-assembly of proteins into amyloid fibrils, which occur in several debilitating and age-related diseases, are affected by common components of amyloid deposits, notably lipids and lipid complexes. Previously, the effects of phospholipids on amyloid fibril formation by apolipoprotein (apo) C-II have been examined, where low concentrations of micellar phospholipids and lipid bilayers induce a new, straight rod-like morphology for apoC-II fibrils. This fibril appearance is distinct from the twisted-ribbon morphology observed when apoC-II fibrils are formed in the absence of lipids. We used total internal reflection fluorescence microscopy (TIRFM) to visualize the described polymorphism of apoC-II amyloid fibrils. The spontaneous assembly of apoC-II into either twisted-ribbon fibrils in the absence of lipids or into fibrils of straight rod-like morphology when lipids are present was captured by TIRFM. The latter was found to be better suited for visualization using TIRFM. The difference between seeding of apoC-II straight fibrils on microscopic quartz slide and in test tube suggested a role for the effects of incubation surface on fibril formation. Seed-dependent growth of apoC-II straight fibrils was probed further by using a dual-labelling construct, giving insights into the straight fibril growth pattern.     

Shear flow induced changes in apolipoprotein C-II conformation and amyloid fibril formation

The misfolding and self-assembly of proteins into amyloid fibrils that occur in several debilitating diseases are affected by a variety of environmental factors, including mechanical factors associated with shear flow. We examined the effects of shear flow on amyloid fibril formation by human apolipoprotein C-II (apoC-II). Shear fields (150, 300, and 500 s(-1)) accelerated the rate of apoC-II fibril formation (1 mg/mL) approximately 5-10-fold. Fibrils produced at shear rates of 150 and 300 s(-1) were similar to the twisted ribbon fibrils formed in the absence of shear, while at 500 s(-1), tangled ropelike structures were observed. The mechanism of the shear-induced acceleration of amyloid fibril formation was investigated at low apoC-II concentrations (50 μg/mL) where fibril formation does not occur. Circular dichroism and tryptophan fluorescence indicated that shear induced an irreversible change in apoC-II secondary structure. Fluorescence resonance energy transfer experiments using the single tryptophan residue in apoC-II as the donor and covalently attached acceptors showed that shear flow increased the distance between the donor and acceptor molecules. Shear-induced higher-order oligomeric species were identified by sedimentation velocity experiments using fluorescence detection, while fibril seeding experiments showed that species formed during shear flow are on the fibril formation pathway. These studies suggest that physiological shear flow conditions and conditions experienced during protein manufacturing can exert significant effects on protein conformation, leading to protein misfolding, aggregation, and amyloid fibril formation.     

Early stages of amyloid fibril formation studied by liquid-state NMR: the peptide hormone glucagon

The 29-residue peptide hormone glucagon forms amyloid fibrils within a few hours at low pH. In this study, we use glucagon as a model system to investigate fibril formation by liquid-state ¹H-NMR spectroscopy One-dimensional, correlation, and diffusion experiments monitoring the fibril formation process provide insight into the early stages of the pathway on which the molecules aggregate to fibrils. In conjunction with these techniques, exchange experiments give information about the end-state conformation. Within the limits of detection, there are no signs of larger oligomeric intermediates in the course of the fibril formation process. Kinetic information is extracted from the time course of the residual free glucagon signal decay. This suggests that glucagon amyloids form by a nucleated growth mechanism in which trimers (rather than monomers) of glucagon interact directly with the growing fibrils rather than with each other. The results of proton/deuterium exchange experiments on mature fibrils with subsequent dissolution show that the N-terminal of glucagon is the least amenable to exchange, which indicates that this part is strongly involved in the intermolecular bonds of the fibrils.     

Environmental conditions affect the kinetics of nucleation of amyloid fibrils and determine their morphology

To understand and tackle amyloid-related diseases, it is crucial to investigate the factors that modulate amyloid formation of proteins. Our previous studies proved that the N47A mutant of the α-spectrin SH3 (Spc-SH3) domain forms amyloid fibrils quickly under mildly acidic conditions. Here, we analyze how experimental conditions influence the kinetics of assembly and the final morphology of the fibrils. Early formation of curly fibrils occurs after a considerable conformational change of the protein and the concomitant formation of small oligomers. These processes are strongly accelerated by an increase in salt concentration and temperature, and to a lesser extent by a reduction in pH. The rate-limiting step in these events has a high activation enthalpy, which is significantly reduced by an increase in NaCl concentration. At low-to-moderate NaCl concentrations, the curly fibrils convert to straight and twisted amyloid fibrils after long incubation times, but only in the presence of soluble species in the mixture, which suggests that the curly fibrils and the twisted amyloid fibrils are diverging assembly pathways. The results suggest that the influence of environmental variables on protein solvation is crucial in determining the nucleation kinetics, the pathway of assembly, and the final fibril morphology.     

Suppression of apolipoprotein C-II amyloid formation by the extracellular chaperone, clusterin.

The effect of the extracellular chaperone, clusterin, on amyloid fibril formation by lipid-free human apolipoprotein C-II (apoC-II) was investigated. Sub-stoichiometric levels of clusterin, derived from either plasma or semen, potently inhibit amyloid formation by apoC-II. Inhibition is dependent on apoC-II concentration, with more effective inhibition by clusterin observed at lower concentrations of apoC-II. The average sedimentation coefficient of apoC-II fibrils formed from apoC-II (0.3 mg.mL-1) is reduced by coincubation with clusterin (10 microg x mL(-1)). In contrast, addition of clusterin (0.1 mg x mL(-1)) to preformed apoC-II amyloid fibrils (0.3 mg x mL(-1)) does not affect the size distribution after 2 days. This sedimentation velocity data suggests that clusterin inhibits fibril growth but does not promote fibril dissociation. Electron micrographs indicate similar morphologies for amyloid fibrils formed in the presence or absence of clusterin. The substoichiometric nature of the inhibition suggests that clusterin interacts with transient amyloid nuclei leading to dissociation of the monomeric subunits. We propose a general role for clusterin in suppressing the growth of extracellular amyloid.     

Heterogeneous amylin fibril growth mechanisms imaged by total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy has been used to visualize the fibrillization of amylin, a hormone which in aggregated forms plays a role in type 2 diabetes pathology. Data were obtained at acidic pH where fibrillization is hindered by the charging of histidine 18 and at slightly basic pH where the loss of charge on the histidine promotes aggregation. The experiments show three types of aggregate growth processes. In the earliest steps globular seeds are formed with some expanding radially during the course of the reaction. The dimensions of the globular seeds as well as their staining with the amyloid-specific dye thioflavin T indicate that they are plaques of short fibrils. The next species observed are fibrils that invariably grow from large globular seeds or smaller punctate granules. Fibril elongation appears to be unidirectional, although in some cases multiple fibrils radiate from a single seed or granule. After fibrils are formed, some show an increase in fluorescence intensity that we attribute to the growth of new fibrils alongside those previously formed. All three aggregation processes are suggestive of secondary (heterogeneous) nucleation mechanisms in which nucleation occurs on preformed fibrils. Consistently, electron micrographs show changes in fibril morphology well after fibrils are first formed, and the growth processes observed by fluorescence microscopy occur after the corresponding solution reactions have reached an initial apparent plateau. Taken together, the results highlight the importance of secondary nucleation in the fibrillization of amylin, as this could provide a pathway to continue fibril growth once an initial population of fibrils is established.     

Phospholipid interaction induces molecular-level polymorphism in apolipoprotein C-II amyloid fibrils via alternative assembly pathways

A common feature of many of the most important and prominent amyloid-forming proteins is their ability to bind lipids and lipid complexes. Lipids are ubiquitous components of disease-associated amyloid plaques and deposits in humans, yet the specific roles of lipid in the process of amyloid fibril formation are poorly understood. This study investigated the effect of phospholipids on amyloid fibril formation by human apolipoprotein (apo) C-II using phosphatidylcholine derivatives comprising acyl chains of up to 14 carbon atoms. Submicellar concentrations of short-chain phospholipids increase the rate of apoC-II fibril formation in an acyl-chain-length- and concentration-dependent fashion, while high micellar concentrations of phospholipids completely inhibited amyloid formation. At lower concentrations of soluble phospholipid complexes, fibril formation by apoC-II was only partially inhibited, and under these conditions, aggregation followed a two-phase process. Electron microscopy showed that the fibrils resulting from the second phase of aggregation were straight, cablelike, and about 13 nm wide, in contrast to the homogeneous twisted-ribbon morphology of apoC-II fibrils formed under lipid-free conditions. Seeding experiments showed that this alternative fibril structure could be templated both in the presence and in the absence of lipid complex, suggesting that the two morphologies result from distinct assembly pathways. Circular dichroism spectroscopy studies indicated that the secondary structural conformation within the straight-type and ribbon-type fibrils were distinct, further suggesting divergent assembly pathways. These studies show that phospholipid complexes can change the structural architecture of mature fibrils and generate new fibril morphologies with the potential to alter the in vivo behaviour of amyloid. Such lipid interactions may play a role in defining the structural features of fibrils formed by diverse amyloidogenic proteins.     

Reversible heat-induced dissociation of β2-microglobulin amyloid fibrils

Recent progress in the field of amyloid research indicates that the classical view of amyloid fibrils, being irreversibly formed highly stable structures resistant to perturbating conditions and proteolytic digestion, is getting more complex. We studied the thermal stability and heat-induced depolymerization of amyloid fibrils of β(2)-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis. We found that freshly polymerized β2m fibrils at 0.1-0.3 mg/mL concentration completely dissociated to monomers upon 10 min incubation at 99 °C. Fibril depolymerization was followed by thioflavin-T fluorescence and circular dichroism spectroscopy at various temperatures. Dissociation of β2m fibrils was found to be a reversible and dynamic process reaching equilibrium between fibrils and monomers within minutes. Repolymerization experiments revealed that the number of extendable fibril ends increased significantly upon incubation at elevated temperatures suggesting that the mechanism of fibril unfolding involves two distinct processes: (1) dissociation of monomers from the fibril ends and (2) the breakage of fibrils. The breakage of fibrils may be an important in vivo factor multiplying the number of fibril nuclei and thus affecting the onset and progress of disease. We investigated the effects of some additives and different factors on the stability of amyloid fibrils. Sample aging increased the thermal stability of β2m fibril solution. 0.5 mM SDS completely prevented β2m fibrils from dissociation up to the applied highest temperature of 99 °C. The generality of our findings was proved on fibrils of K3 peptide and α-synuclein. Our simple method may also be beneficial for screening and developing amyloid-active compounds for therapeutic purposes.     

Thermodynamic characteristics of collagen fibrils reconstructed in vitro at different temperatures and concentrations

The results of a calorimetric study of type I collagen fibrillogenesis were analyzed. The dependence of the half-width of the temperature transition of a collagen solution on the concentration and temperature of collagen formation was studied. It was demonstrated that, by varying temperature and collagen concentration, one can regulate the density of packing and dimensions of cooperative fibril blocks. At temperatures below the physiological level (25 degrees C and 30 degrees C), and a relatively low concentration of collagen (0.3 mg/ml), fibrils with the lowest density of packing are formed. The degree of order does not change as the collagen concentration increases twofold but grows as the concentration increases fourfold. It was shown that, at the physiological temperature (35 degrees C), fibrils with a dense packing of molecules are formed at all collagen concentrations studied. The value of fibril formation enthalpy is minimal at a temperature of 35 degrees C, pH 7.2, an ionic strength of 0.17 M and a concentration of 1.2 mg/ml. Based on the results obtained, a conclusion was made that the packing density of fibrils formed at physiological temperature does not depend on collagen concentration over the concentration range of 0.3 - 1.2 mg/ml.     

Sensitive detection of glucagon aggregation using amyloid fibril-specific antibodies

Sensitive detection of protein aggregates is important for evaluating the quality of biopharmaceuticals and detecting misfolded proteins in several neurodegenerative diseases. However, it is challenging to detect extremely low concentrations (<10 ppm) of aggregated protein in the presence of high concentrations of soluble protein. Glucagon, a peptide hormone used in the treatment of extreme hypoglycemia, is aggregation-prone and forms amyloid fibrils. Detection of glucagon fibrils using conformation-specific antibodies is an attractive approach for identifying such aggregates during process and formulation development. Therefore, we have used yeast surface display and magnetic-activated cell sorting to sort single-chain antibody libraries to identify antibody variants with high conformational specificity for glucagon fibrils. Notably, we find several high-affinity antibodies that display excellent selectivity for glucagon fibrils, and we have integrated these antibodies into a sensitive immunoassay. Surprisingly, the sensitivity of our assay—which involves direct (nonantibody mediated) glucagon immobilization in microtiter plates—can be significantly enhanced by pretreating the microtiter plates with various types of globular proteins before glucagon immobilization. Moreover, increased total concentrations of glucagon peptide also significantly improve the sensitivity of our assay, which appears to be due to the strong seeding activity of immobilized fibrils at high glucagon concentrations. Our final assay is highly sensitive (fibril detection limit of ~0.5–1 ppm) and is >20 times more sensitive than detection using a conventional, amyloid-specific fluorescent dye (Thioflavin T). We expect that this type of sensitive immunoassay can be readily integrated into the drug development process to improve the generation of safe and potent peptide therapeutics.     

Amyloid-like fibril formation of co-chaperonin GroES: nucleation and extension prefer different degrees of molecular compactness

The molecular chaperone GroES, together with GroEL from Escherichia coli, is the best characterized protein of the molecular chaperone family. Here, we report on the in vitro formation of GroES amyloid-like fibrils and the mechanism of formation. When incubated for several weeks at neutral pH in the presence of the denaturant guanidine hydrochloride, GroES formed a typical amyloid fibril; unbranched, twisted, and extended filaments stainable by thioflavin T and Congo red. GroES fibril formation was accelerated by the addition of preformed fibril seeds, in accordance with a nucleation-extension mechanism. Interestingly, whereas the spontaneous formation of GroES fibrils was favored in the structural transition region of GroES dissociation/unfolding, the extension of fibrils from preformed fibril seeds was favored in the region corresponding to an expanded molecular state. We concluded that the two stages of GroES fibril formation prefer different molecular states of the same protein. The significance of this preference is discussed.     

Formation of collagen type I fibrils in vitro

The assembly of collagen fibrils as a function of temperature and collagen concentration was studied. It was shown that temperature increases from 25 to 35 degrees C, the degree of ordering of collagen fibrils increases 1.5-fold at collagen concentration above 1 mg/ml and 2-fold at low collagen concentration. A maximum ordering of fibril structure occurs under conditions close to physiological (T approximately 35 degrees C and collagen concentration 1.2 mg/ml). As temperature is elevated from 30 to 35 degrees C, the packing of collagen molecules in fibrils becomes more ordered: the values of enthalpy and entropy of the transition of fibrils from the native to a disordered state decrease at all collagen concentrations used. At high collagen concentration, the dimensions of cooperative blocks in fibrils formed at 25 and 30 degrees C coincide with those of cooperative blocks of monomeric collagen in solution. Upon increasing the temperature to 35 degrees C, the dimensions of cooperative blocks increase.     

Guanidine hydrochloride can induce amyloid fibril formation from hen egg-white lysozyme

The formation of amyloid fibrils is an intractable problem in which normally soluble protein polymerizes and forms insoluble ordered aggregates. Such aggregates can range from being a nuisance in vitro to being toxic in vivo. The latter is true for lysozyme, which has been shown to form toxic deposits in humans. In the present study, the effects of partial denaturation of hen egg-white lysozyme via incubation in a concentrated solution of the denaturant guanidine hydrochloride are investigated. Results show that when lysozyme is incubated under moderate guanidine hydrochloride concentrations (i.e., 2-5 M), where lysozyme is partially unfolded, fibrils form rapidly. Thioflavin T, Congo red, X-ray diffraction, transmission electron microscopy, atomic force microscopy, and circular dichroism spectroscopy are all used to verify the production of fibrils under these conditions. Incubation at very low or very high guanidine hydrochloride concentrations fails to produce fibrils. At very low denaturant concentrations, the structure of lysozyme is fully native and very stable. On the other hand, at very high denaturant concentrations, guanidine hydrochloride is capable of dissolving and dis-aggregating fibrils that are formed. Raising the temperature and/or concentration of lysozyme accelerates fibril formation by further adding to the concentration of partially unfolded species. The addition of preformed fibrils also accelerates fibril formation but only under partially unfolding conditions. The results presented here provide further evidence that partial unfolding is a prerequisite to fibril formation. Partial denaturation can accelerate fibril formation in much the same way that mutations have been shown to accelerate fibril formation.     

Investigation of a peptide responsible for amyloid fibril formation of beta 2-microglobulin by achromobacter protease I.

To obtain insight into the mechanism of amyloid fibril formation from beta(2)-microglobulin (beta2-m), we prepared a series of peptide fragments using a lysine-specific protease from Achromobacter lyticus and examined their ability to form amyloid fibrils at pH 2.5. Among the nine peptides prepared by the digestion, the peptide Ser(20)-Lys(41) (K3) spontaneously formed amyloid fibrils, confirmed by thioflavin T binding and electron microscopy. The fibrils composed of K3 peptide induced fibril formation of intact beta2-m with a lag phase, distinct from the extension reaction without a lag phase observed for intact beta2-m seeds. Fibril formation of K3 peptide with intact beta2-m seeds also exhibited a lag phase. On the other hand, the extension reaction of K3 peptide with the K3 seeds occurred without a lag phase. At neutral pH, the fibrils composed of either intact beta2-m or K3 peptide spontaneously depolymerized. Intriguingly, the depolymerization of K3 fibrils was faster than that of intact beta2-m fibrils. These results indicated that, although K3 peptide can form fibrils by itself more readily than intact beta2-m, the K3 fibrils are less stable than the intact beta2-m fibrils, suggesting a close relation between the free energy barrier of amyloid fibril formation and its stability.     

pH-dependent amyloid and protofibril formation by the ABri peptide of familial British dementia

The ABri is a 34 residue peptide that is the major component of amyloid deposits in familial British dementia. In the amyloid deposits, the ABri peptide adopts aggregated beta-pleated sheet structures, similar to those formed by the Abeta peptide of Alzheimer's disease and other amyloid forming proteins. As a first step toward elucidating the molecular mechanisms of the beta-amyloidosis, we explored the ability of the environmental variables (pH and peptide concentration) to promote beta-sheet fibril structures for synthetic ABri peptides. The secondary structures and fibril morphology were characterized in parallel using circular dichroism, atomic force microscopy, negative stain electron microscopy, Congo red, and thioflavin-T fluorescence spectroscopic techniques. As seen with other amyloid proteins, the ABri fibrils had characteristic binding with Congo red and thioflavin-T, and the relative amounts of beta-sheet and amyloid fibril-like structures are influenced strongly by pH. In the acidic pH range 3.1-4.3, the ABri peptide adopts almost exclusively random structure and a predominantly monomeric aggregation state, on the basis of analytical ultracentrifugation measurements. At neutral pH, 7.1-7.3, the ABri peptide had limited solubility and produced spherical and amorphous aggregates with predominantly beta-sheet secondary structure, whereas at slightly acidic pH, 4.9, spherical aggregates, intermediate-sized protofibrils, and larger-sized mature amyloid fibrils were detected by atomic force microscopy. With aging at pH 4.9, the protofibrils underwent further association and eventually formed mature fibrils. The presence of small amounts of aggregated peptide material or seeds encourage fibril formation at neutral pH, suggesting that generation of such seeds in vivo could promote amyloid formation. At slightly basic pH, 9.0, scrambling of the Cys5-Cys22 disulfide bond occurred, which could lead to the formation of covalently linked aggregates. The presence of the protofibrils and the enhanced aggregation at slightly acidic pH is consistent with the behavior of other amyloid-forming proteins, which supports the premise that a common mechanism may be involved in protein misfolding and beta-amyloidosis.