Effects of dithiothreitol on the amyloid fibrillogenesis of hen egg-white lysozyme

At least 25 human proteins can fold abnormally to form pathological deposits that are associated with several degenerative diseases. Despite extensive investigation on amyloid fibrillation, the detailed molecular mechanisms remain rather elusive and there are currently no effective cures for treating these amyloid diseases. The present study examined the effects of dithiothreitol on the fibrillation of hen egg-white lysozyme (HEWL). Our results revealed that the fibrillation of hen lysozyme was significantly inhibited by reduced dithiothreitol (DTT^red) while oxidized dithiothreitol (DTT^ox) had no anti-aggregating activity. Effective inhibitory activity against hen lysozyme fibrillation was observed only when DTT^red was added within 8 days of incubation. Our results showed that the initial addition of DTT^red interacted with HEWL, leading to a loss in conformational stability. It was concluded from our findings that DTT^red-induced attenuation of HEWL fibrillation may be associated with disulfide disruption and extensive structural unfolding of HEWL. Our data may contribute to rational design of effective therapeutic strategies for amyloid diseases.     

The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two-state transition

Amyloid fibril depositions are associated with many neurodegenerative diseases as well as amyloidosis. The detailed molecular mechanism of fibrillation is still far from complete understanding. In our previous study of in vitro fibrillation of hen egg white lysozyme, an irreversible partially unfolded intermediate was characterized. A similarity of unfolding kinetics found for the secondary and tertiary structure of lysozyme using deep UV resonance Raman (DUVRR) and tryptophan fluorescence spectroscopy leads to a hypothesis that the unfolding might be a two-state transition. In this study, chemometric analysis, including abstract factor analysis (AFA), target factor analysis (TFA), evolving factor analysis (EFA), multivariate curve resolution-alternating least squares (ALS), and genetic algorithm, was employed to verify that only two principal components contribute to the DUVRR and fluorescence spectra of soluble fraction of lysozyme during the fibrillation process. However, a definite conclusion on the number of conformers cannot be made based solely on the above spectroscopic data although chemometric analysis suggested the existence of two principal components. Therefore, electrospray ionization mass spectrometry (ESI-MS) was also utilized to address the hypothesis. The protein ion charge state distribution (CSD) envelopes of the incubated lysozyme were well fitted with two principal components. Based on the above analysis, the partial unfolding of lysozyme during in vitro fibrillation was characterized quantitatively and proven to be a two-state transition. The combination of ESI-MS and Raman and fluorescence spectroscopies with advanced statistical analysis was demonstrated to be a powerful methodology for studying protein structural transformations.     

Thermal stability and enzymatic activity of a smaller lysozyme from silk moth (Bombyx mori)

Bombyx mori lysozyme is 10 amino acids shorter than hen egg-white lysozyme, which is a typical c-type lysozyme. It was expressed by using the methylotrophic yeast Pichia pastoris. The thermal stability and the enzymatic activity of the Bombyx mori lysozyme were estimated and compared with those of human and hen egg-white lysozymes. The denaturation temperature was 17-26°C lower than those of human and hen egg-white lysozymes. Further, the enthalpy change and the heat capacity change for unfolding were smaller than those of human lysozyme. It was also confirmed that the stability against guanidine hydrochloride was lower than those of the other two lysozymes. The enzymatic activity toward a simple synthetic substrate was measured and compared with those of human and hen egg-white lysozymes. The B-F binding mode was obviously dominant, although the A-E binding mode was preferred in human and hen egg-white lysozymes.     

Effect of ethanol on folding of hen egg-white lysozyme under acidic condition

The equilibrium and kinetics of folding of hen egg-white lysozyme were studied by means of CD spectroscopy in the presence of varying concentrations of ethanol under acidic condition. The equilibrium transition curves of guanidine hydrochloride-induced unfolding in 13 and 26% (v/v) ethanol have shown that the unfolding significantly deviates from a two-state mechanism. The kinetics of denaturant-induced refolding and unfolding of hen egg-white lysozyme were investigated by stopped-flow CD at three ethanol concentrations: 0, 13, and 26% (v/v). Immediately after dilution of the denaturant, the refolding curves showed a biphasic time course in the far-UV region, with a burst phase with a significant secondary structure and a slower observable phase. However, when monitored by the near-UV CD, the burst phase was not observed and all refolding kinetics were monophasic. To clarify the effect of nonnative secondary structure induced by the addition of ethanol on the folding/unfolding kinetics, the kinetic m values were estimated from the chevron plots obtained for the three ethanol concentrations. The data indicated that the folding/unfolding kinetics of hen lysozyme in the presence of varying concentrations of ethanol under acidic condition is explained by a model with both on-pathway and off-pathway intermediates of protein folding.     

The transition state in the folding-unfolding reaction of four species of three-disulfide variant of hen lysozyme: the role of each disulfide bridge

The effects of lacking a specific disulfide bridge on the transition state in folding were examined in order to explore the folding-unfolding mechanism of lysozyme. Four species of three-disulfide variant of hen lysozyme (3SS-lysozyme) were prepared by replacing two Cys residues with Ala or Ser: C6S/C127A, C30A/C115A, C64A/C80A and C76A/C94A. The recombinant hen lysozyme was studied as the standard reference containing four authentic disulfide bridges and the extra N-terminal Met: the recombinant hen lysozyme containing the extra N-terminal. Folding rates were measured by monitoring the change in fluorescence intensity associated with tri-N-acetyl-d-glucosamine binding to the active site of refolded lysozyme. It was confirmed that the folding rate of the recombinant hen lysozyme containing the extra N-terminal was the same as that of wild-type lysozyme, and that the folding rate was little affected by the presence of tri-N-acetyl-d-glucosamine (triNAG). The folding rate of C64A/C80A was found to be the fastest and almost the same as that of the recombinant hen lysozyme containing the extra N-terminal, and that of C30A/C115A the second, and that of C6S/C127A the third. The folding rate of C76A/C94A was particularly slow. On the other hand, the unfolding rates which were measured in the presence of triNAG showed the dependence on the concentration of triNAG. The intrinsic unfolding rate in the absence of triNAG was determined by extrapolation. Also in the unfolding rate, C76A/C94A was markedly slower than the others. It was found from the analysis of binding constants of triNAG to C64A/C80A during the unfolding process that the active site of C64A/C80A partly unfolds already prior to the unfolding transition. On the basis of these kinetic data, we suggest that C64A/C80A folding transition can occur with leaving the loop region around SS3 (C64-C80) flexible, while cross-linking by SS4 (C76-C94) is important for the promotion of folding, because it is an indispensable constraint on the way towards the folding transition state.     

A stopped-flow fluorescence study of the native and modified lysozyme

The protein folding kinetics of hen egg white lysozyme (HEWL) was studied using experimental and bioinformatics tools. The structure of the transition state in the unfolding pathway of lysozyme was determined with stopped-flow kinetics using intact HEWL and its chemically modified derivative, in which six lysine residues have been modified. The overall consistency of φ-value (φ ≈ 1) indicates that lysine side chains interactions are subject to breaking in the structure of the transition state. Following experimental evidences, multiple sequence alignment of lysozyme family in vertebrates and exact structural examination of lysozyme, showed that the α-helix in the structure of lysozyme has critical role in the unfolding kinetics.     

Effects of glutathione on amyloid fibrillation of hen egg-white lysozyme

This study examined the effects of glutathione on the fibrillation of hen egg-white lysozyme. We found that the fibrillation of lysozyme was considerably reduced by GSH while no anti-aggregating activity was detected with only GSSG. SDS-PAGE results also revealed that the addition of GSH led to an early occurrence of prominent lysozyme hydrolysis. Moreover, GSH was effective in inhibiting lysozyme fibrillation when GSH was added within 6 days of incubation. We conclude that the attenuation of lysozyme fibrillation is strongly dependent upon the redox environment. Our data may contribute to decipher the molecular mechanism of amyloid fibrillation.     

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions

The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.     

Unfolding of hen egg lysozyme by molecular dynamics simulations at 300K: insight into the role of the interdomain interface

We present the results of two 1.2 ns molecular dynamics (MD) unfolding simulations on hen egg lysozyme in water at 300K, performed using a new procedure called PEDC (Path Exploration With Distance Constraints). This procedure allows exploration of low energy structures as a function of increasing RMSD from the native structure, and offers especially the possibility of extensive exploration of the conformational space during the initial unfolding stages. The two independent MD simulations gave similar chronology of unfolding events: disruption of the active site, kinking of helix C, partial unfolding of the three-stranded beta-sheet to a two-stranded sheet (during which the helices A, B, and D remain to a great extent native), and finally unfolding of the beta-domain and partial unfolding of the alpha-domain in which hydrophobic clusters persist. We show particularly that the loss of hydrophobic contacts between the beta-sheet turn residues Leu55 and Ile56 and the hydrobic patch of the alpha-domain destabilizes the beta-domain and leads to its unfolding, suggesting that the correct embedding of these residues in the alpha-beta interface may constitute the rate limiting step in folding. These results are in accord with experimental observations on the folding/unfolding behavior of hen egg lysozyme at room temperature. They would also explain the loss of stability and the tendency to aggregation observed for the mutant Leu55Thr, and the slow refolding kinetics observed in the analogous amyloidogenic variant of human lysozyme.     

Partially unfolded equilibrium state of hen lysozyme studied by circular dichroism spectroscopy

Equilibrium unfolding of hen egg white lysozyme as a function of GdnCl concentration at pH 0.9 was studied over a temperature range 268.2-303.2 K by means of CD spectroscopy. As monitored by far- and near-UV CD at 222 and 289 nm, the lack of coincidence between two unfolding transition curves was observed, which suggests the existence of a third conformational species in addition to native and unfolded states. The three-state model, in which a stable intermediate is populated, was employed to estimate the thermodynamic parameters for the GdnCl-induced unfolding. It was found that the transition from the native to intermediate states proceeds with significant changes in enthalpy and entropy due to an extremely cooperative process, while the transition from the intermediate to unfolded states shows a low cooperativity with small enthalpy and entropy changes. These results indicate that the highest energy barrier for the GdnCl-induced unfolding of hen lysozyme is located in the process from the native state to the intermediate state, and this process is largely responsible for the cooperativity of protein unfolding.     

Cysteine inhibits amyloid fibrillation of lysozyme and directs the formation of small worm‐like aggregates through non‐covalent interactions

In this article, we discuss the effects of amino acids on amyloid aggregation of lysozyme. l ‐cysteine (Cys) dramatically inhibited fibrillation of lysozyme, whereas other amino acids (including l ‐arginine) did not. In the presence of Cys, the aggregation pathway of lysozyme shifted from fibrillation to the formation of the small worm‐like aggregates with unfolding. The interaction between Cys and lysozyme was observed to be non‐covalent, suggesting that the thiophilic interaction between the thiol group on the side chain of Cys and the core sequence of lysozyme significantly contributes to the inhibition of amyloid aggregation. These findings provide a new basis for the design of a biocompatible additive to prevent amyloid fibrillation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:470–478, 2014     

Exploring the inhibitory activity of short-chain phospholipids against amyloid fibrillogenesis of hen egg-white lysozyme

Amyloid fibrillogenesis is an important pathological feature of a group of degenerative human diseases. The 129-residue enzyme hen egg-white lysozyme has been shown to form fibrils in vitro at pH 2.0 and 55°C. In this research, using various spectroscopic techniques, light scattering, and transmission electron microscopy, we first examined the influence of short-chain phospholipids on the amyloid fibrillogenesis and the structural changes derived from hen lysozyme in vitro. Both model short-chain phospholipids were observed to mitigate the fibrillogenesis of hen lysozyme. Also, urea-induced unfolding results suggested that the susceptibility of hen lysozyme to conformational changes elicited by the denaturant was observed to decrease upon addition of short-chain phospholipids. Moreover, our molecular dynamics simulations results demonstrated that the observed inhibitory action of short-chain phosoholipids against hen lysozyme fibrillogenesis might be attributable to the interference of β-strand extension by the binding of phospholipids to lysozyme's β-sheet-rich region. We believe that the outcome from this study may contribute to a better understanding the molecular factors affecting amyloid fibrillogenesis and the molecular mechanism(s) of the interactions between phospholipids/lipids and amyloid-forming proteins.     

Purification, amino acid sequence, and some properties of rabbit kidney lysozyme

The lysozyme (rabbit kidney lysozyme) from the homogenate of rabbit kidney (Japanese white) was purified by repeated cation-exchange chromatography on Bio-Rex 70. The amino acid sequence was determined by automated gas-phase Edman degradation of the peptides obtained from the digestion of reduced and S-carboxymethylated rabbit lysozyme with Achromobacter protease I (lysyl endopeptidase). The sequence thus determined was KIYERCELARTLKKLGLDGYKGVSLANWMCLAKWESSYNTRATNYNPGDKSTDYGIFQ INSRYWCNDGKTPRAVNACHIPCSDLLKDDITQAVACAKRVVSDPQGIRAWVAWRNHCQ NQDLTPYIRGCGV, indicating 25 amino acid substitutions from human lysozyme. The lytic activity of rabbit lysozyme against Micrococcus lysodeikticus at pH 7, ionic strength of 0.1, and 30 degrees C was found to be 190 and 60% of those of hen and human lysozymes, respectively. The lytic activity-pH profile of rabbit lysozyme was slightly different from those of hen and human lysozymes. While hen and human lysozymes had wide optimum activities at around pH 5.5-8.5, the optimum activity of rabbit lysozyme was at around pH 5.5-7.0. The high proline content (five residues per molecule compared with two prolines per molecule in hen or human lysozyme) is one of the interesting features of rabbit lysozyme. The transition temperatures for the unfolding of rabbit, human, and hen lysozymes in 3 M guanidine hydrochloride at pH 5.5 were 51.2, 45.5, and 45.4 degrees C, respectively, indicating that rabbit lysozyme is stabler than the other two lysozymes. The high proline content may be responsible for the increased stability of rabbit lysozyme.     

The stability curve of hen egg white lysozyme

The physiological stability curve--a plot of the free energy of unfolding versus temperature--is calculated for hen egg white lysozyme from a combination of extrapolated unfolding thermodynamic data from reversible conditions and isothermal titrations with guanidine hydrochloride. The shape of the curve suggests the existence of only one folded conformation.     

Structural studies of hen egg-white lysozyme dimer: comparison with monomer

A facile method for the formation of covalent bonds between protein molecules is zero length cross-linking. This method enables the formation of cross-links without use of any chemical reagents. Here, we report a cross-linking method for lysozyme and some structural studies as well as catalytic activity assay was performed on lysozyme dimer. The results showed that catalytic activity of lysozyme dimer was the same as monomer. Also, the GdnCl-induced equilibrium unfolding of hen egg-white lysozyme monomer and dimer at pH 2 was studied over a temperature range of 290.7-303.2 K by means of CD spectroscopy. The lack of coincidence between two unfolding curves at 222 and 289 nm in lysozyme dimer was observed, which suggested the existence of intermediate state in unfolding process, while lysozyme monomer showed a single cooperative transition. Thus, the thermodynamic parameters were estimated on the basis of two-state mechanism for lysozyme monomer and three-state one for lysozyme dimer. These results indicated that zero length cross-linking can stabilize the intermediate, so the population of intermediate increased. Our results offer a special opportunity to study the role of intermediates in protein folding mechanisms. In addition thermal unfolding of monomer and dimer in 222 nm was achieved.     

Heat-induced conversion of beta(2)-microglobulin and hen egg-white lysozyme into amyloid fibrils

Thermodynamic parameters characterizing protein stability can be obtained for a fully reversible folding/unfolding system directly by differential scanning calorimetry (DSC). However, the reversible DSC profile can be altered by an irreversible step causing aggregation. Here, to obtain insight into amyloid fibrils, ordered and fibrillar aggregates responsible for various amyloidoses, we studied the effects on human beta(2)-microglobulin and hen egg-white lysozyme of a combination of agitation and heating. Aggregates formed by mildly agitating protein solutions in the native state in the presence of NaCl were heated in the cell of the DSC instrument. For beta(2)-microglobulin, with an increase in the concentration of NaCl at neutral pH, the thermogram began to show an exothermic transition accompanied by a large decrease in heat capacity, followed by a kinetically controlled thermal response. Similarly, the aggregated lysozyme at a high concentration of NaCl revealed a similar distinct transition in the DSC thermogram over a wide pH range. Electron microscopy demonstrated the conformational change into amyloid fibrils. Taken together, the combined use of agitation and heating is a powerful way to generate amyloid fibrils from two proteins, beta(2)-microglobulin and hen egg-white lysozyme, and to evaluate the effects of heat on fibrillation, in which the heat capacity is crucial to characterizing the transition.     

Correlation of structural differences in several bird lysozymes and their loop regions with immunological cross-reactivity

Goat antibodies that were specific, respectively, to hen egg white lysozyme, its loop region (residues 60 to 83) and to regions other than the loop, were reacted with the intact lysozyme or its loop region. The interference with this reaction by several bird lysozymes was tested. Bobwhite quail lysozyme was as efficient as hen lysozyme in the lysozyme-anti-lysozyme system, but much less reactive with anti-loop antibodies. Turkey lysozyme, on the other hand, was similar to hen lysozyme in its behaviour with anti-loop antibodies but different in its reactivity with anti-lysozyme. It is thus concluded that the loop region of hen lysozyme is far more reactive than that of bobwhite quail lysozyme with loop-specific goat antibodies. The large antigenic difference results from replacement of an arginine residue (at position 68) in the hen loop by a lysine residue in the quail loop. By contrast, the loop region of turkey lysozyme is antigenically similar to that of hen lysozyme. Yet the turkey loop also differs from the hen loop by a single lysine-for-arginine replacement (at position 73). To explain why the lysine substitution has a greater antigenic effect at position 68 than at position 73, two hypotheses are considered. First, as arginine 68 is the i + 2 residue of a β-bend (encompassing residues 66 to 69) and as the frequency of occurrence of lysine at the i + 2 position in β-bends is lower than that of arginine, the presence of lysine at position 68 may lower the stability of the β-bend and thereby cause a conformational change in the β-bend region of the loop. Alternatively, arginine 68 may be more exposed than is arginine 73 in hen lysozyme, and hence goat antibodies may more easily recognize the side-chain difference produced by the lysine substitution at position 68.     

Effect of curcumin on the amyloid fibrillogenesis of hen egg-white lysozyme

At least twenty human proteins can fold abnormally to form pathological deposits that are associated with several degenerative diseases. Despite extensive investigation on amyloid fibrillogenesis, its detailed molecular mechanisms remain unknown. This study is aimed at exploring the inhibitory activity of curcumin against the fibrillation of hen lysozyme. We found that the formation of amyloid fibrils at pH 2.0 in vitro was inhibited by curcumin in a dose-dependent manner. Moreover, quenching analysis confirmed the existence of an interaction between curcumin and lysozyme, and Van't Hoff analysis indicated that the curcumin–lysozyme interaction is predominantly governed by Van Der Waals force or hydrogen bonding. Curcumin was also found to acquire disaggregating ability on preformed lysozyme fibrils. Finally, we observed that curcumin pre-incubated at 25 °C for at least 7 days inhibited lysozyme fibrillogenesis better than untreated curcumin and the enhanced inhibition against HEWL fibrillation might be attributed to the presence of dimeric species.     

Nuclear magnetic resonance and ultraviolet difference spectral studies of the binding properties of turkey egg white lysozyme. Consequences of the replacement of Asp 101 by glycine

The nuclear magnetic resonance spectrum of the ¹⁹F nuclei in N-trifluoroacetylated chitotriose was studied in the presence of turkey lysozyme. In contrast to results previously obtained with hen lysozyme, the ¹⁹F nmr spectrum of the complex did not show any striking pH dependence. It was, in fact, very similar at all pH's to the spectrum of the trisaccharide complexed with hen lysozyme at low pH, where Asp 101 is protonated. The replacement of Asp 101 in turkey lysozyme by a glycine is thought to account for this difference and the results allow unequivocal assignment of a value of 4.2 to the pK_a of Asp 101 in hen lysozyme. The dissociation constant of the chitotriose-turkey lysozyme complex was measured at various pH's using uv difference methods and compared with that previously reported for the hen lysozyme-chitotriose complex. Again, the results could be attributed to the loss in binding energy due to the absence of Asp 101. In contrast to chitotriose, the binding of chitobiose and methyl-2-acetamido-2-deoxy-β-d-glucopyranoside as studied by both uv difference and nmr methods is the same within experimental error for turkey and hen lysozyme. The results obtained for binding of chitobiose suggest that Asp 101 does not contribute as much to the binding energy of the disaccharide as was previously thought. Finally, the specific activities of both of these lysozymes against Micrococcus lysodeikticus were found to be identical.     

Inactivation of Gram-Negative Bacteria by Lysozyme, Denatured Lysozyme, and Lysozyme-Derived Peptides under High Hydrostatic Pressure

We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except both Salmonella species showed higher inactivation in the presence of 100 μg of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coli and P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides.