Formation Of Amyloid-like Fibrils Research Articles

Hydration effects on the fibrillation process of a globular protein: the case of human serum albumin

In this work, we have studied the fibrillation process of human serum albumin (HSA) under different solution conditions. In particular, aggregation kinetics, fibril morphology, and composition structural changes were investigated under varying experimental conditions such as pH (2.0 and 7.4), temperature (at 25 and 65 °C), and solvent polarity (ethanol/water mixtures, 10–90% v/v). The characterization was carried out by means of static and dynamic light scattering (SLS and DLS), ThT fluorescence, circular dichroism (CD) and Fourier Transform Infrared (FT-IR) spectroscopy, and transmission electron microscopy (TEM). The aggregation process and the α-helix to β-sheet transitions were found to be favored by temperature and physiological pH. Also, pH was observed to influence both the fibrillation pathway and aggregation kinetics, changing from a classical fibrillation process with a lag phase under acidic conditions to a downhill polymerization process at physiological pH in the presence of the alcohol. Regarding protein structural composition, at room temperature and physiological pH ethanol was found to promote an α-helix to β-sheet conformational transition at intermediate alcohol concentrations, whereas at low and high ethanol contents α-helix prevailed as the predominant structure. Under acidic conditions, ethanol promotes an important fibrillation at high cosolvent concentrations due to screening of electric charges and a decrease in solvent polarity. On the other hand, important differences in the morphology of the resulting fibrils and aggregates are observed depending on the solution conditions. In particular, the formation of classical amyloid-like fibrils at physiological pH and high temperature is observed, in contrast to the usual curly morphology displayed by HSA fibrils under most of the solution conditions. Although the high temperature and pH are the main parameters influencing the protein structure destabilization and subsequent aggregation upon incubation, ethanol helps to regulate the hydrogen bonding, the attractive hydrophobic interactions, and the protein accessible surface area, thus, modifying the packing constraints and the resulting aggregate morphologies.

The impact of solubility and electrostatics on fibril formation by the H3 and H4 histones

The goal of this study was to examine fibril formation by the heterodimeric eukaryotic histones (H2A-H2B and H3-H4) and homodimeric archaeal histones (hMfB and hPyA1). The histone fold dimerization motif is an obligatorily domain-swapped structure comprised of two fused helix:β-loop:helix motifs. Domain swapping has been proposed as a mechanism for the evolution of protein oligomers as well as a means to form precursors in the formation of amyloid-like fibrils. Despite sharing a common fold, the eukaryotic histones of the core nucleosome and archaeal histones fold by kinetic mechanisms of differing complexity with transient population of partially folded monomeric and/or dimeric species. No relationship was apparent between fibrillation propensity and equilibrium stability or population of kinetic intermediates. Only H3 and H4, as isolated monomers and as a heterodimer, readily formed fibrils at room temperature, and this propensity correlates with the significantly lower solubility of these polypeptides. The fibrils were characterized by ThT fluorescence, FTIR, and far-UV CD spectroscopies and electron microscopy. The helical histone fold comprises the protease-resistant core of the fibrils, with little or no protease protection of the poorly structured N-terminal tails. The highly charged tails inhibit fibrillation through electrostatic repulsion. Kinetic studies indicate that H3 and H4 form a co-fibril, with simultaneous incorporation of both histones. The potential impact of H3 and H4 fibrillation on the cytotoxicity of extracellular histones and α-synuclein-mediated neurotoxicity and fibrillation is considered.

Aggregates with lysozyme and ovalbumin show features of amyloid-like fibrils

The interaction of egg-white lysozyme with N-ovalbumin, the native form of egg-white ovalbumin with the denaturation temperature, T(m), of 78 °C, was investigated by the inhibition of lysozyme muramidase activity, differential scanning calorimetry, and circular dichroism assay as indicators. Signals for the interaction were the most prominent when the mixture of lysozyme and N-ovalbumin was co-heated at 72 °C, slightly lower than the T(m) of N-ovalbumin. The interaction was also marked when unheated lysozyme was mixed with N-ovalbumin preheated at 72 °C. Moreover, the mixture rapidly formed fibrous precipitates, which were positive for thioflavin T fluorescent emission, a marker for the amyloid fibril formation. Also electron microscopic observation exhibited features of fibrils. The interaction potency of ovalbumin was ascribed to the tryptic fragment ILELPFASGT MSMLVLLPDE VSGLEQLESIINFEK (residues 229-263), derived from the 2B strands 2 and 3 of ovalbumin. From lysozyme, on the other hand, the chymotryptic peptide RNRCKGTDVQAW (residues 112-123), including cluster 6, and the chymotryptic/tryptic peptide GILQINSRW (residues 54-62), including cluster 3, were responsible for the interaction with N-ovalbumin. Interestingly, this nonamer peptide was found to have the ability to self-aggregate. To the authors knowledge, this may be the first report to document the possible involvement of dual proteins in the formation of amyloid-like fibrils.

Inhibition of Amyloid Aggregation by Formation of Helical Assemblies

The formation of amyloid aggregates is responsible for a wide range of diseases, including Alzheimer's and Parkinson's disease. Although the amyloid-forming proteins have different structures and sequences, all undergo a conformational change to form amyloid aggregates that have a characteristic cross-β-structure. The mechanistic details of this process are poorly understood, but different strategies for the development of inhibitors of amyloid formation have been proposed. In most cases, chemically diverse compounds bind to an elongated form of the protein in a β-strand conformation and thereby exert their therapeutic effect. However, this approach could favor the formation of prefibrillar oligomeric species, which are thought to be toxic. Herein, we report an alternative approach in which a helical coiled-coil-based inhibitor peptide has been designed to engage a coiled-coil-based amyloid-forming model peptide in a stable coiled-coil arrangement, thereby preventing rearrangement into a β-sheet conformation and the subsequent formation of amyloid-like fibrils. Moreover, we show that the helix-forming peptide is able to disassemble mature amyloid-like fibrils.

Amyloid-like Fibrils from a Domain-swapping Protein Feature a Parallel, in-Register Conformation without Native-like Interactions

The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain. Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. Intramolecular β-sheet contacts present in the monomeric state could constitute intermolecular β-sheets in the dimeric and fibrillar states. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded β-sheet and one α-helix. Under native conditions this mutant adopts a domain-swapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register β-sheet structure and a general loss of native conformation within the mature fibrils. This observation contrasts with predictions that native structure, and in particular intermolecular β-strand interactions seen in the dimeric state, may be preserved in "domain-swapping" fibrils. We discuss these observations in light of recent work on related amyloid-forming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.

Structural memory of natively unfolded tau protein detected by small‐angle X‐ray scattering

Small-angle X-ray scattering (SAXS) is a universal low-resolution method to study size and shape of globular proteins in solution but recent developments facilitate the quantitative characterization of the structure and structural transitions of metastable systems like partially or completely unfolded proteins. We present here a study of temperature induced transitions in tau, a natively unfolded protein involved in Alzheimer's disease. Previous studies on full length tau and several disease-related mutants provided information about the residual structure in different domains revealing a specific role and extended conformations of the so-called repeat domains, which are considered to be responsible for the formation of amyloid-like fibrils ("paired helical filaments"). Here, we employ SAXS to investigate the temperature dependent properties of tau. Slow heating/cooling of the full length protein from 10°C to 50°C did not lead to detectable changes in the overall size. Surprisingly, quick heating/cooling caused tau to adopt a significantly more compact conformation, which was stable over up to 3 h and represents a structural "memory" effect. This compaction is not observed for the shorter tau constructs containing largely the repeat domains. The structural and functional implications of the observed unusual behavior of tau under nonequilibrium conditions are discussed.

Species-Dependent Differences in Fibril Morphology and the Effects on Basal Forebrain Neurons of a Prion Protein Fragment (Residues 127-147)

Prion protein mis-folding is central to the transmission and pathobiology of transmissible spongiform encephalopathies. Unfortunately, the heterogeneous and aggregation-prone nature of the disease-associated mis-folded prion protein has complicated its study. Fragments of the prion protein, such as residues 106-126 and 127-147, exhibit amyloid-like fibril formation and neurotoxicity in cell culture. These prion protein fragments serve as simplified models for the study of the mis-folded prion protein and its role in disease. We have characterized a fibril-forming peptide (residues 127-147) from elk, hamster and bovine prion proteins. At neutral pH, the elk and hamster prion peptides form distinct fibril morphologies. The hamster fibrils are ∼20 nm in width, Thioflavin T-negative, and generally lack a repetitive helical structure. The elk fibrils are thinner (∼15 nm in width), Thioflavin T-positive, and have a repetitive helical structure. Notably, the elk fibrils appear more homogeneous in their morphology and helical periodicity, whereas the hamster fibrils appear more heterogeneous and non-periodic. As observed for the elk peptide, the bovine fibrils appear homogeneous in their morphology and periodicity. Three-dimensional reconstructions from negative-stain transmission electron microscopy images of representative elk, hamster, and bovine fibrils highlight their distinct morphologies. Interestingly, the elk prion peptide, but not the hamster peptide, significantly decreased whole cell currents in rat basal forebrain neurons, similar to previous studies of a human prion peptide (residues 106-126; Alier et al., Journal of Neuroscience Research, 2010).

Sonication-induced instant amyloid-like fibril formation and organogelation by a tripeptide

A terminally protected tripeptide Boc-Val(1)-Phe(2)-Phe(3)-OMe 1 having sequence similarity with Aβ18–20 (the central hydrophobic fragment 18–20 of the amyloid β-peptide Aβ42) forms sonication induced organogels. The peptide forms a very weak gel only in 1,2-dichlorobenzene after heating, cooling and ageing for 3 days. But ultrasound energy induces instant fibril formation and gelation in a wide range of organic solvents starting from hexane, cyclohexane, petrol, kerosene to benzene, toluene, xylene and ethanol. CD, FT-IR, NMR and wide angle X-ray scattering (WAXS) studies of the peptide 1 exhibit distinct structural changes before and after sonication. Atomic force microscopy (AFM) and field emission scanning electron microscopy (FE-SEM) of the xerogels reveal a nanofibrillar morphology, which is obtained by the sonication induced self-assembly of the gelator. These gels bind with Congo red, a physiological dye, and show a green-gold birefringence under polarized light, a characteristic feature of amyloid fibrils.

Use of yeast as a system to study amyloid toxicity

The formation of amyloid-like fibrils is a hallmark of several neurodegenerative diseases. How the assembly of amyloid-like fibrils contributes to cell death is a major unresolved question in the field. The budding yeast Saccharomyces cerevisiae is a powerful model organism to study basic mechanisms for how cellular pathways regulate amyloid assembly and proteotoxicity. For example, studies of the amyloidogenic yeast prion [RNQ+] have revealed novel roles by which molecular chaperones protect cells from the accumulation of cytotoxic protein species. In budding yeast there are a variety of cellular assays that can be employed to analyze the assembly of amyloid-like aggregates and mechanistically dissect how cellular pathways influence proteotoxicity. In this review, we describe several assays that are routinely used to investigate aggregation and toxicity of the [RNQ+] prion in yeast.

Formation and Characterization of Amyloid-like Fibrils from Soy β-Conglycinin and Glycinin

The fibrillar aggregation at pH 2.0 of soy β-conglycinin, glycinin, and the 1:1 mixture thereof, induced by heating at 80 °C for various periods of time, was investigated using Th T and Congo Red spectroscopic techniques. The morphology of the formed fibrillar aggregates was characterized using atomic force microscopy (AFM), whereas the conformational changes and the polypeptide hydrolysis of the proteins upon heating were also evaluated. Th T fluorescence analysis indicated that β-conglycinin had a much higher potential to form heat-induced amyloid-like aggregates than glycinin. AFM analyses showed that all of the soy globulins could form twisted screw-structure fibrils with heights of 1.4-2.2 nm, but the morphology of the amyloid-like fibrils considerably varied among various soy proteins. Significantly lower width at half-heights and higher coil periodicity values were observed for the β-conglycinin fibrils than the glycinin counterpart. Far-UV CD spectral analysis indicated that upon heating, the secondary conformations of the proteins changed considerably, especially during initial heating (e.g., <4 h), and the changes were much more distinct in the β-conglycinin case than in the glycinin case. Furthermore, reducing electrophoresis analyses indicated that progressive polypeptide hydrolysis occurred upon heating, but the polypeptide hydrolysis for the β-conglycinin was much more severe than that of glycinin. The data suggest that soy β-conglycinin exhibited a much higher potential to form thermally fibrillar aggregates than glycinin, and the differences seem to be mainly associated with the differences in their conformational changes and extent of polypeptide hydrolysis by the heating. The results would be of vital importance for the utilization of soy proteins to produce thermally induced fibrillar gels with excellent properties.

Amino Acid Sequence Requirements of Laminin β1 Chain Peptide B133 (DISTKYFQMSLE) for Amyloid-like Fibril Formation, Syndecan Binding, and Neurite Outgrowth Promotion

Peptide B133 (DSITKYFQMSLE), derived from mouse laminin beta1 chain (residues 1298-1309), promotes cell attachment, neurite outgrowth, and amyloid-like fibril formation. Previously, we showed that the N-terminal Asp-deleted peptide B133a (SITKYFQMSLE) promotes integrin alpha2beta1-mediated cell attachment and spreading but does not form amyloid-like fibrils, and that the C-terminal Glu-deleted peptide B133g (DSITKYFQMSL) attaches cells without cell spreading and forms amyloid-like fibrils. In this study, we further investigated the amino acid sequence requirements of B133 for biological function using a set of truncated and Ala-substituted peptides. Attachment of cells to B133g was inhibited by only heparin, and Congo Red analysis indicated that the amyloid-like fibril formation activity of B133g was stronger than that of B133. Alanine scan analysis for the B133g peptide indicated that Asp and Ile residues are essential for cell attachment. Additionally, the N-terminal Asp residue was required for neurite outgrowth. Further, amyloid-like fibril formation required Asp and Ile residues. These data suggest that the amyloid-like fibril formation of B133g is required for cell attachment activity. We also evaluated the attachment of cells to the peptides using syndecan- and glypican-overexpressing cells. B133g attached to syndecan-overexpressing cells but not to glypican-overexpressing cells, suggesting that the amyloidogenic peptides promote syndecan-mediated cell attachment. These findings were useful for clarifying the mechanism of amyloid-like fibril formation and biological functions. The B133 peptide promotes amyloid-like fibril formation, syndecan-mediated cell attachment, and neurite outgrowth and has the potential for use as a biomaterial for tissue engineering.

Inhibition by Flavonoids of Amyloid-like Fibril Formation by Plasmodium falciparum Merozoite Surface Protein 2

Merozoite surface protein 2 (MSP2) is a glycosylphosphatidylinositol (GPI)-anchored protein expressed abundantly on the surface of Plasmodium falciparum merozoites. The results of a phase 2 trial in Papua New Guinean children showed MSP2 to be a promising malaria vaccine candidate. MSP2 is intrinsically unstructured and forms amyloid-like fibrils under physiological conditions. Oligomers containing beta-strand interactions similar to those in amyloid fibrils may be a component of the fibrillar surface coat on P. falciparum merozoites. As the propensity of MSP2 to form fibrils in solution also has the potential to impede its development as a vaccine candidate, finding an inhibitor that specifically inhibits fibrillogenesis may enhance vaccine development. In this study, we tested the ability of three flavonoids, EGCG, baicalein, and resveratrol, to inhibit MSP2 fibrillogenesis and found marked inhibition with EGCG but not with the other two flavonoids. The inhibitory effect and the interactions of the flavonoids with MSP2 were characterized using NMR spectroscopy, thioflavin T fluorescence assays, electron microscopy, and other biophysical methods. EGCG stabilizes soluble oligomers and blocks fibrillogenesis by preventing the conformational transition of MSP2 from a random coil to an amyloidogenic beta-sheet structure. Structural comparison of the three flavonoids indicates an association between their propensity for autoxidation and their fibril inhibitory activity; the activity of EGCG can be attributed to the vicinal hydroxyl groups present in this flavonoid and their ability to form quinones. The molecular mechanism of fibril inhibition by EGCG appears to be complex and involves noncovalent binding followed by covalent modification of the protein. Although the addition of EGCG appears to be an effective means of stabilizing MSP2 in solution, the covalent modification of MSP2 would most likely not be acceptable in a vaccine formulation. However, these small molecule inhibitors of MSP2 fibril formation will be useful as mechanistic probes in studying oligomerization and fibril assembly of MSP2.

B133 (DSITKYFQMSLE), a laminin β1-derived peptide, contains distinct core sequences for both integrin α2β1-mediated cell adhesion and amyloid-like fibril formation

The B133 peptide (DSITKYFQMSLE, mouse laminin β1 chain 1319–1330) promotes cell attachment, and forms amyloid-like fibrils. Here, we evaluated the active core sequences using B133 deletion peptides. B133a, lacking the N-terminal Asp residue, promoted cell spreading via integrin α2β1, whereas B133g, lacking the C-terminal Glu residue, lost the activity. Congo red analysis using the truncated peptides determined that B133g forms amyloid-like fibrils but B133a did not. These results suggest that the N- and C-terminal amino acids contribute to integrin α2β1 binding and to fibril formation, respectively. Further analyses using the truncated peptides showed that the C-terminal eight residues (B133d: KYFQMSLE) are a minimum active sequence for integrin α2β1-mediated cell attachment and the N-terminal nine residues (B133i: DSITKYFQM) are critical for amyloid-like fibril formation. These results suggest that peptide B133 is multifunctional with two different active core sequences: integrin α2β1-mediated cell attachment and amyloid-like fibril formation. Moreover, alanine substitution analysis of B133a indicated that six amino acids, Ile, Thr, Tyr, Phe, Met, and Glu, are important for cell attachment activity. When the Ser residue at the 9th position of B133a was replaced with Ala, the cell attachment activity was enhanced. Further mutation analysis at the 9th position of B133a using various amino acids suggests that hydrophobic amino acids are effective for the integrin α2β1-mediated cell attachment activity. These findings define multifunctional and overlapping sites on the B133 peptide and are useful for designing multifunctional synthetic molecules.

L-Arginine reduces thioflavin T fluorescence but not fibrillation of bovine serum albumin

This work examines the effects of L-arginine (L-Arg) on the aggregation and amyloid fibrillation of bovine serum albumin (BSA). We demonstrate that L-Arg dose-dependently reduces thioflavin T (ThT) fluorescence of BSA within the L-Arg concentration range used (0-1.4 M). However, as revealed by electron microscopy, size exclusion chromatography, and dynamic light scattering results, L-Arg does not prevent amyloid-like fibril formation by BSA. We conclude that L-Arg competes against ThT for binding sites on BSA amyloid-like fibrils, leading to biased results in ThT fluorescence measurements. Moreover, the use of ThT fluorescence assay to screen for potential inhibitors against amyloid fibrillation can give misleading results.

Celastrol Inhibits Hsp90 Chaperoning of Steroid Receptors by Inducing Fibrillization of the Co-chaperone p23

Hsp90 is an ATP-dependent molecular chaperone. The best characterized inhibitors of Hsp90 target its ATP binding pocket, causing nonselective degradation of Hsp90 client proteins. Here, we show that the small molecule celastrol inhibits the Hsp90 chaperoning machinery by inactivating the co-chaperone p23, resulting in a more selective destabilization of steroid receptors compared with kinase clients. Our in vitro and in vivo results demonstrate that celastrol disrupts p23 function by altering its three-dimensional structure, leading to rapid formation of amyloid-like fibrils. This study reveals a unique inhibition mechanism of p23 by a small molecule that could be exploited in the dissection of protein fibrillization processes as well as in the therapeutics of steroid receptor-dependent diseases.

Formation of non-toxic amyloid-like fibrils in the presence of small heat shock protein

Formation of non-toxic amyloid-like fibrils in the presence of small heat shock protein

Design and activity of multifunctional fibrils using receptor-specific small peptides

We have designed multifunctional peptide fibrils using bioactive laminin-derived peptides and evaluated their potential as a biomedical material for tissue engineering. The Leu-Arg-Gly-Asp-Asn (LRGDN) peptide derived from laminin-111, which contains an RGD sequence bound to integrin αvβ3, was added to the N-terminus of the four amyloidogenic cell-adhesive laminin-derived peptides (A119: LSNIDYILIKAS, AG97: SAKVDAIGLEIV, B133: DISTKYFQMSLE, and B160: VILQQSAADIAR). The RGD-conjugated peptides were stained with Congo red and exhibited amyloid-like fibril formation in the electron microscopic. The RGD-conjugated peptides promoted human dermal fibroblasts spreading with well-organized actin stress fibers and focal contacts. Human dermal fibroblast attachment to the RGD-conjugated peptides was inhibited by anti-αv integrin antibody. Further, cell attachment to B133 was inhibited by anti-α2 and anti-β1 integrin antibodies, whereas attachment to RGD-B133 was inhibited by anti-αv and anti-β1 integrin antibodies. These results suggest that the RGD-conjugated peptides interact with integrin αvβ3 and that RGD-B133 interacts with both integrin αvβ3 and integrin β1. The RGD-conjugated peptide fibrils promoted neurite outgrowth in a peptide-dependent manner. These results support that biologically active sequence-conjugated peptide fibrils interact in a receptor-specific manner with cells and promote multifunctional activities. These fibrils may have use as biological supports for cell-specific tissue engineering.

Plasmodium falciparum merozoite surface protein 2 is unstructured and forms amyloid-like fibrils

Several merozoite surface proteins are being assessed as potential components of a vaccine against Plasmodium falciparum, the cause of the most serious form of human malaria. One of these proteins, merozoite surface protein 2 (MSP2), is unusually hydrophilic and contains tandem sequence repeats, characteristics of intrinsically unstructured proteins. A range of physicochemical studies has confirmed that recombinant forms of MSP2 are largely unstructured. Both dimorphic types of MSP2 (3D7 and FC27) are equivalently extended in solution and form amyloid-like fibrils although with different kinetics and structural characteristics. These fibrils have a regular underlying β-sheet structure and both fibril types stain with Congo Red, but only the FC27 fibrils stain with Thioflavin T. 3D7 MSP2 fibrils seeded the growth of fibrils from 3D7 or FC27 MSP2 monomer indicating the involvement of a conserved region of MSP2 in fibril formation. Consistent with this, digestion of fibrils with proteinase K generated resistant peptides, which included the N-terminal conserved region of MSP2. A monoclonal antibody that reacted preferentially with monomeric recombinant MSP2 did not react with the antigen in situ on the merozoite surface. Glutaraldehyde cross-linking of infected erythrocytes generated MSP2 oligomers similar to those formed by polymeric recombinant MSP2. We conclude that MSP2 oligomers containing intermolecular β-strand interactions similar to those in amyloid fibrils may be a component of the fibrillar surface coat on P. falciparum merozoites.

Molecular Dynamics Simulations to Investigate the Aggregation Behaviors of the Aß(17–42) Oligomers

The amyloid β-peptides (Aßs) are the main protein components of amyloid deposits in Alzheimer's disease (AD). Detailed knowledge of the structure and assembly dynamics of Aß is important for the development of properly targeted AD therapeutics. So far, the process of the monomeric Aß assembling into oligomeric fibrils and the mechanism underlying the aggregation process remain unclear. In this study, several molecular dynamics simulations were conducted to investigate the aggregation behaviors of the Aß(17–42) oligomers associated with various numbers of monomers (dimer, trimer, tetramer, and pentamer). Our results showed that the structural stability of the Aß(17–42) oligomers increases with increasing the number of monomer. We further demonstrated that the native hydrophobic contacts are positive correlated with the ß-sheet contents, indicating that hydrophobic interaction plays an important role in maintaining the structural stability of the Aß(17–42) oligomers, particularly for those associated with more monomers. Our results also showed that the stability of the C-terminal hydrophobic segment 2 (residues 30–42) is higher than that of the N-terminal hydrophobic segment 1 (residues 17–21), suggesting that hydrophobic segment 2 may act as the nucleation site for aggregation. We further identified that Met35 residue initiates the hydrophobic interactions and that the intermolecular contact pairs, Gly33-Gly33 and Gly37-Gly37, form a stable “molecular notch”, which may mediate the packing of the ß-sheet involving many other hydrophobic residues during the early stage of amyloid-like fibril formation.

Tertiary Butanol Induced Amyloidogenesis of Hen Egg White Lysozyme (HEWL) Is Facilitated by Aggregation-Prone Alkali-Induced Molten Globule Like Conformational State

Proteins may form undesirable aggregates during the process of folding. Increasing evidence suggests that amyloid fibrils may arise from partially folded precursor molecules. We have previously demonstrated that hen egg white lysozyme [HEWL] exists as molten globule at pH 12.7. Here, we report that lysozyme at pH 7.0 and 11.0 are nearly stable to the addition of up to 45% t-butanol, but treatment of the alkali-induced molten globule form of HEWL [AMGL] with 20% t-butanol caused the formation of amyloid-like fibrils as evidenced by enhanced Thioflavin T binding and DLS measurements.