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.     

Reduced global cooperativity is a common feature underlying the amyloidogenicity of pathogenic lysozyme mutations

One of the 20 or so human amyloid diseases is associated with the deposition in vital organs of full-length mutational variants of the antibacterial protein lysozyme. Here, we report experimental data that permit a detailed comparison to be made of the behaviour of two of these amyloidogenic variants, I56T and D67H, under identical conditions. Hydrogen/deuterium exchange experiments monitored by NMR and mass spectrometry reveal that, despite their different locations and the different effects of the two mutations on the structure of the native state of lysozyme, both mutations cause a cooperative destabilisation of a remarkably similar segment of the structure, comprising in both cases the beta-domain and the adjacent C-helix. As a result, both variant proteins populate transiently a closely similar, partially unstructured intermediate in which the beta-domain and the adjacent C-helix are substantially and simultaneously unfolded, whereas the three remaining alpha-helices that form the core of the alpha-domain still have their native-like structure. We show, in addition, that the binding of a camel antibody fragment, cAb-HuL6, which was raised against wild-type lysozyme, restores to both variant proteins the stability and cooperativity characteristic of the wild-type protein; as a consequence, it inhibits the formation of amyloid fibrils by both variants. These results indicate that the reduction in global cooperativity, and the associated ability to populate transiently a specific, partly unfolded intermediate state under physiologically relevant conditions, is a common feature underlying the behaviour of these two pathogenic mutations. The formation of intermolecular interactions between lysozyme molecules that are in this partially unfolded state is therefore likely to be the fundamental trigger of the aggregation process that ultimately leads to the formation and deposition in tissue of amyloid fibrils.     

Rationalising lysozyme amyloidosis: insights from the structure and solution dynamics of T70N lysozyme

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with those of the amyloidogenic variants of lysozyme is therefore important for understanding the determinants of amyloid disease. We report here the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45-51 and 68-75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5A. A marked decrease in the magnitudes of the generalised order parameter (S2) values of the amide nitrogen atom, for residues 70-74, shows that the T70N substitution increases the flexibility of the peptide backbone around the site of mutation. Hydrogen/deuterium exchange protection factors measured by NMR spectroscopy were calculated for the T70N variant and the wild-type protein. The protection factors for many of backbone amide groups in the beta-domain of the T70N variant are decreased relative to those in the wild-type protein, whereas those in the alpha-domain display wild-type-like values. In pulse-labelled hydrogen/deuterium exchange experiments monitored by mass spectrometry, transient but locally cooperative unfolding of the beta-domain of the T70N variant and the wild-type protein was observed, but at higher temperatures than for the amyloidogenic variants I56T and D67H. These findings reveal that such partial unfolding is an intrinsic property of the human lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs in vivo.     

Identification of a novel human islet amyloid polypeptide beta-sheet domain and factors influencing fibrillogenesis

Human islet amyloid polypeptide (hIAPP) accumulates as pancreatic amyloid in type 2 diabetes and readily forms fibrils in vitro. Investigations into the mechanism of hIAPP fibril formation have focused largely on residues 20 to 29, which are considered to comprise a primary amyloidogenic domain. In rodents, proline substitutions within this region and the subsequent beta-sheet disruption, prevents fibril formation. An additional amyloidogenic fragment within the C-terminal sequence, residues 30 to 37, has been identified recently. We have extended these observations by examining a series of overlapping peptide fragments from the human and rodent sequences. Using protein spectroscopy (CD/FTIR), electron microscopy and X-ray diffraction, a previously unrecognised amyloidogenic domain was localised within residues 8 to 20. Synthetic peptides corresponding to this region exhibited a transition from random coil to beta-sheet conformation and assembled into fibrils having a typical amyloid-like morphology. The comparable rat 8-20 sequence, which contains a single His18Arg substitution, was also capable of assembling into amyloid-like fibrils. Examination of peptide fragments corresponding to residues 1 to 13 revealed that the immediate N-terminal region is likely to have only a modulating influence on fibril formation or conformational conversion. The contributions of charged residues as they relate to the amyloid-forming 8-20 sequence were also investigated using IAPP fragments and by assessing the effects of pH and counterions. The identification of these principal amyloidogenic sequences and the effects of associated factors provide details on the IAPP aggregation pathway and structure of the peptide in its fibrillar state.     

Local cooperativity in the unfolding of an amyloidogenic variant of human lysozyme

Hydrogen exchange experiments monitored by NMR and mass spectrometry reveal that the amyloidogenic D67H mutation in human lysozyme significantly reduces the stability of the beta-domain and the adjacent C-helix in the native structure. In addition, mass spectrometric data reveal that transient unfolding of these regions occurs with a high degree of cooperativity. This behavior results in the occasional population of a partially structured intermediate in which the three alpha-helices that form the core of the alpha-domain still have native-like structure, whereas the beta-domain and C-helix are simultaneously substantially unfolded. This finding suggests that the extensive intermolecular interactions that will be possible in such a species are likely to initiate the aggregation events that ultimately lead to the formation of the well-defined fibrillar structures observed in the tissues of patients carrying this mutation in the lysozyme gene.     

In vitro characterization of lactoferrin aggregation and amyloid formation

Lactoferrin has previously been identified in amyloid deposits in the cornea, seminal vesicles, and brain. We report in this paper a highly amyloidogenic region of lactoferrin (sequence of NAGDVAFV). This region was initially identified by sequence comparison with medin, a 5.5 kDa amyloidogenic fragment derived from lactadherin. Subsequent characterization revealed that this peptide forms amyloid fibrils at pH 7.4 when incubated at 37 degrees C. Furthermore, although full-length lactoferrin does not by itself form amyloid fibrils, the protein does bind to the peptide fibrils as revealed by an increase in thioflavin T fluorescence and the presence of enlarged fibrils by transmission electron microscopy and polarized light microscopy. The binding of lactoferrin is a selective interaction with the NAGDVAFV fibrils. Lactoferrin does not bind to insulin or lysozyme fibrils, and the NAGDVAFV fibrils do not bind to soluble insulin or lysozyme. The lactoferrin appears to coat the peptide fibril surface to form mixed peptide/protein fibrils, but again there is no evidence for the formation of lactoferrin-only fibrils. This interaction, therefore, seems to involve selective binding rather than conventional seeding of fibril formation. We suggest that such a process could be generally important in the formation of amyloid fibrils in vivo since the identification of both full-length protein and protein fragments is common in ex vivo amyloid deposits.     

Is polyproline II helix the killer conformation? A Raman optical activity study of the amyloidogenic prefibrillar intermediate of human lysozyme

The amyloidogenic prefibrillar partially denatured intermediate of human lysozyme, prepared by heating the native protein to 57 degrees C at pH 2.0, was studied using Raman optical activity (ROA). A positive band in the room temperature ROA spectrum of the native protein at approximately 1345 cm(-1), assigned to a hydrated form of alpha-helix, is not present in that of the prefibrillar intermediate, where a new strong positive band at approximately 1318 cm(-1) appears instead that is assigned to the poly(l-proline) II (PPII)-helical conformation. A sharp negative band at approximately 1241 cm(-1) in the native protein, assigned to beta-strand, shows little change in the ROA spectrum of the prefibrillar intermediate. The disappearance of a positive ROA band at approximately 1551 cm(-1) assigned to vibrations of tryptophan side-chains indicates that major conformational changes have occurred among the five tryptophan residues present in human lysozyme, four of which are located in the alpha-domain. The various ROA data suggest that a substantial loss of tertiary structure has occurred in the prefibrillar intermediate and that this is located more in the alpha-domain than in the beta-domain. There is no evidence for any increase in beta-structure. The ROA spectrum of hen lysozyme, which does not form amyloid fibrils so readily, remains much more native-like on heating to 57 degrees C at pH 2.0. The thermal behaviour of the alanine-rich alpha-helical peptide AK21 in aqueous solution was found to be similar to that of human lysozyme. Hydrated alpha-helix therefore appears to readily undergo a conformational change to PPII structure on heating, which may be a key step in the conversion of alpha-helix into beta-sheet in the formation of amyloid fibrils in human lysozyme. Since it is extended, flexible, lacks intrachain hydrogen bonds and is fully hydrated in aqueous solution, PPII helix has the appropriate characteristics to be implicated as a critical conformational element in many conformational diseases. Disorder of the PPII type may be a sine qua non for the formation of regular fibrils; whereas the more dynamic disorder of the random coil may lead only to amorphous aggregates.     

Identification of the core structure of lysozyme amyloid fibrils by proteolysis

Human lysozyme variants form amyloid fibrils in individuals suffering from a familial non-neuropathic systemic amyloidosis. In vitro, wild-type human and hen lysozyme, and the amyloidogenic mutants can be induced to form amyloid fibrils when incubated under appropriate conditions. In this study, fibrils of wild-type human lysozyme formed at low pH have been analyzed by a combination of limited proteolysis and Fourier-transform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates. After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26-123 and 32-108, although a significant minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive beta-sheet structure and a substantial element of non beta-sheet or random structure that is reduced significantly in the fibrils after digestion. The sequence 32-108 includes the beta-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme. The present proteolytic data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain. The majority of amyloid fibrils formed from lysozyme under the conditions used here contain a core structure involving some 50% of the polypeptide chain that is flanked by proteolytically accessible N and C-terminal regions.     

Distinct structural changes in wild-type and amyloidogenic chicken cystatin caused by disruption of C95–C115 disulfide bond

Human cystatin C (HCC) amyloid angiopathy (HCCAA) is characterized by tissue deposition of amyloid fibrils in blood vessels, which can lead to recurrent hemorrhagic stroke. Wild-type HCC forms part of the amyloid deposits in brain arteries of elderly people with amyloid angiopathy. A point mutation causing a glutamine to a leucine substitution at residue 68 in the HCC polypeptide chain greatly increases the amyloidogenic propensity of HCC and causes a more severe cerebral hemorrhage and premature death in young adults. In this study, we used molecular dynamics simulations to assess the importance of disulfide bridge formation upon the stability of chicken cystatin and how this may influence the propensity for amyloid formation. We found that disulfide bridge formation between Cys95 and Cys115 in human cystatin played a critical role in overall protein stability. Importantly, Cys95–Cys115 influenced cystatin structure in regions of the protein that play key roles in the protein-folding transitions that occur, which enable amyloid fibril formation. We hypothesized that correct disulfide bridge formation is a critical step in stabilizing cystatin toward its native conformation. Disrupting Cys95–Cys115 disulfide bridge formation within cystatin appears to significantly enhance the amyloidogenic properties of this protein. In addition, by combining in silico studies with our previous experimental results on Eps1, a molecular chaperone of the PDI family, we proposed that age-related HCCAA, may possess a different pathogenic mechanism compared with its amyloidogenic counterpart, the early onset amyloidogenic cystatin-related CAA.     

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.     

Insights into the conformational changes of several human lysozyme variants associated with hereditary systemic amyloidosis

In this study, various ethanol- and temperature-induced molecular dynamics simulations were conducted to investigate the conformational changes of several human lysozyme variants (I56T, D67H, and T70N) associated with hereditary systemic amyloidosis. The results show that these variants are all more sensitive to conditions affecting the structural integrity of this protein. The structural analyses of these variants reveal a high population of more unstable beta-domain and distorted hydrophobic core compared to the wild-type human lysozyme, particularly for the two natural amyloidogenic variants D67H and I56T. For the D67H variant, the distance between the mass centers of residues 54 and 67 was found to elongate as a result of the destruction of the hydrogen-bonding network stabilizing the two long loops in the beta-domain. It further accelerates the unfolding of this variant, starting from the hydrophobic core between the alpha- and beta-domains. For the I56T variant, the introduction of a hydrophilic residue in the hydrophobic core directly destroys the native contacts in the alpha-beta interface, leading to fast unfolding. The present results are consistent with the previous hypothesis suggesting that the distortion of the hydrophobic core at the alpha-beta interface putatively results in the formation of the initial "seed" for amyloid fibrils.     

In silico prediction of novel residues involved in amyloid primary nucleation of human I56T and D67H lysozyme

Background

Amyloidogenic proteins are most often associated with neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, but there are more than two dozen human proteins known to form amyloid fibrils associated with disease. Lysozyme is an antimicrobial protein that is used as a general model to study amyloid fibril formation. Studies aimed at elucidating the process of amyloid formation of lysozyme tend to focus on partial unfolding of the native state due to the relative instability of mutant amyloidogenic variants. While this is well supported, the data presented here suggest the native structure of the variants may also play a role in primary nucleation.

Results

Three-dimensional structural analysis identified lysozyme residues 21, 62, 104, and 122 as displaced in both amyloidogenic variants compared to wild type lysozyme. Residue interaction network (RIN) analysis found greater clustering of residues 112–117 in amyloidogenic variants of lysozyme compared to wild type. An analysis of the most energetically favored predicted dimers and trimers provided further evidence for a role for residues 21, 62, 104, 122, and 112–117 in amyloid formation.

Conclusions

This study used lysozyme as a model to demonstrate the utility of combining 3D structural analysis with RIN analysis for studying the general process of amyloidogenesis. Results indicated that binding of two or more amyloidogenic lysozyme mutants may be involved in amyloid nucleation by placing key residues (21, 62, 104, 122, and 112–117) in proximity before partial unfolding occurs. Identifying residues in the native state that may be involved in amyloid formation could provide novel drug targets to prevent a range of amyloidoses.

     

Mechanistic studies of the folding of human lysozyme and the origin of amyloidogenic behavior in its disease-related variants.

The unfolding and refolding properties of human lysozyme and two amyloidogenic variants (Ile56Thr and Asp67His) have been studied by stopped-flow fluorescence and hydrogen exchange pulse labeling coupled with mass spectrometry. The unfolding of each protein in 5.4 M guanidine hydrochloride (GuHCl) is well described as a two-state process, but the rates of unfolding of the Ile56Thr variant and the Asp67His variant in 5.4 M GuHCl are ca. 30 and 160 times greater, respectively, than that of the wild type. The refolding of all three proteins in 0.54 M GuHCl at pH 5.0 proceeds through persistent intermediates, revealed by multistep kinetics in fluorescence experiments and by the detection of well-defined populations in quenched-flow hydrogen exchange experiments. These findings are consistent with a predominant mechanism for refolding of human lysozyme in which one of the structural domains (the alpha-domain) is formed in two distinct steps and is followed by the folding of the other domain (the beta-domain) prior to the assembly of the two domains to form the native structure. The refolding kinetics of the Asp67His variant are closely similar to those of the wild-type protein, consistent with the location of this mutation in an outer loop of the beta-domain which gains native structure only toward the end of the refolding process. By contrast, the Ile56Thr mutation is located at the base of the beta-domain and is involved in the domain interface. The refolding of the alpha-domain is unaffected by this substitution, but the latter has the effect of dramatically slowing the folding of the beta-domain and the final assembly of the native structure. These studies suggest that the amyloidogenic nature of the lysozyme variants arises from a decrease in the stability of the native fold relative to partially folded intermediates. The origin of this instability is different in the two variants, being caused in one case primarily by a reduction in the folding rate and in the other by an increase in the unfolding rate. In both cases this results in a low population of soluble partially folded species that can aggregate in a slow and controlled manner to form amyloid fibrils.     

An Insight into the pathway of the amyloid fibril formation of hen egg white lysozyme obtained from a small-angle X-ray and neutron scattering study

It is known that hen egg white lysozyme (HEWL) forms amyloid fibrils. Since HEWL is one of the proteins that have been studied most extensively and is closely related to human lysozyme, the variants of which form the amyloid fibrils that are related to hereditary systemic amyloidosis, this protein is an ideal model to study the mechanism of amyloid fibril formation. In order to gain an insight into the mechanism of amyloid fibril formation, systematic and detailed studies to detect and characterize various structural states of HEWL were conducted. Since HEWL forms amyloid fibrils in highly concentrated ethanol solutions, solutions of various concentrations of HEWL in various concentrations of ethanol were prepared, and the structures of HEWL in these solutions were investigated by small-angle X-ray and neutron scattering. It was shown that the structural states of HEWL were distinguished as the monomer state, the state of the dimer formation, the state of the protofilament formation, the protofilament state, and the state towards the formation of amyloid fibrils. A phase diagram of these structural states was obtained as a function of protein, water and ethanol concentrations. It was found that under the monomer state the structural changes of HEWL were not gross changes in shape but local conformational changes, and the dimers, formed by the association at the end of the long axis of HEWL, had an elongated shape. Circular dichroism measurements showed that the large changes in the secondary structures of HEWL occurred during dimer formation. The protofilaments were formed by stacking of the dimers with their long axis (nearly) perpendicular to and rotated around the protofilament axis to form a helical structure. These protofilaments were characterized by their radius of gyration of the cross-section of 2.4nm and the mass per unit length of 16,000(+/-2300)Da/nm. It was shown that the changes of the structural states towards the amyloid fibril formation occurred via lateral association of the protofilaments. A pathway of the amyloid fibril formation of HEWL was proposed from these results.     

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.     

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.     

Trehalose and Magnesium Chloride Exert a Common Anti-amyloidogenic Effect Towards Hen Egg White Lysozyme

Many degenerative disorder such as Parkinsons, Alzheimers, Huntingtons disease, etc are caused due to the deposition of amyloid fibrils, formed due to the ordered aggregation of misfolded/unfolded proteins. Misfolded or unfolded proteins aggregate mostly through hydrophobic interactions which are unexposed in native state, but become exposed upon unfolding. To counteract amyloid related diseases, inhibition of the protein self assembly into fibril is a potential therapeutic strategy. The study aims at investigating the effect of selected compounds, namely trehalose and magnesium chloride hexahydrate towards inhibition and disaggregation of amyloid fibrils using Hen Egg White Lysozyme as a model. We further attempted to understand the mechanism of action with the help of various biophysical, microscopic as well as computational studies. A common mechanism of action was identified where the selected compounds exert their anti-amyloidogenic effects by altering HEWL conformations characterized by reduction in the beta sheet content and decrease in exposed hydrophobic surfaces. The altered conformation seems to have lesser amyloidogenic propensity leading to inhibition as well as disaggregation of amyloids.     

Formation of amyloid fibrils from fully reduced hen egg white lysozyme

The fully reduced hen egg white lysozyme (HEWL), which is a good model of random coil structure, has been converted to highly organized amyloid fibrils at low pH by adding ethanol. In the presence of 90% (v/v) ethanol, the fully reduced HEWL adopts beta-sheet secondary structure at pH 4.5 and 5.0, and an alpha-to-beta transition is observed at pH 4.0. A red shift of the Congo red absorption spectrum caused by the precipitation of the fully reduced HEWL in the presence of 90% (v/v) ethanol is typical of the presence of amyloid aggregation. EM reveals unbranched fibrils with a diameter of 2-5 nm and as long as 1-2 microm. The pH dependence of the initial structure of the fully reduced HEWL in the presence of 90% (v/v) ethanol suggests that Asp and His residues may play an important role.     

Conjugated polyelectrolytes: conformation-sensitive optical probes for detection of amyloid fibril formation

The in vivo deposition of amyloid fibrils is a hallmark of many devastating diseases known as the amyloidoses. Amyloid formation in vitro may also complicate production of proteins in the biotechnology industry. Simple, sensitive, and versatile tools that detect the fibrillar conformation of amyloidogenic proteins are thus of great importance. We have developed a negatively charged conjugated polyelectrolyte that displays different characteristic optical changes, detected visually or by absorption and emission, depending on whether the protein with which it forms a complex is in its native state or amyloid fibril conformation. This simple, rapid, and novel methodology was applied here to two amyloidogenic proteins, insulin and lysozyme, and its validity for detection of their fibrillar conformation was verified by currently used methods such as circular dichroism, transmission electron microscopy, and Congo red absorption.     

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.