Hen Egg White Lysozyme Aggregates Research Articles

Hexametaphosphate, a Common Food Additive, Aggregated the Hen Egg White Lysozyme.

Polyphosphate polymers are chains of phosphate monomers chemically bonded together via phosphoanhydride bonds. They are found in all prokaryotic and eukaryotic organisms and are among the earliest, most anionic, and most mysterious molecules known. They are everywhere, from small cellular components to additives in our food. There is a strong association between hyperphosphatemia and mortality. That is why it is crucial to assess how polyphosphates, as food additives, affect the quality of edible proteins. This study investigated the effect of inexpensive and widely used food additives (hexametaphosphate labeled as E452) on bakery items, meat products, fish, and soft drinks. Using various spectroscopic and microscopic techniques, we examined how hexametaphosphate affected the aggregation propensity, structure, and stability of a commonly used food protein: hen egg white lysozyme (HEWL). The solubility of HEWL is affected in a bimodal fashion by the concentration of hexametaphosphate. The bimodal concentration-dependent effect was also observed in the tertiary and secondary structural changes. Hexametaphosphate-induced HEWL aggregates were amorphous, as evidenced by ThT fluorescence, far-UV CD, and TEM imaging. This study showed that the food additive (hexametaphosphate) may denature and aggregate proteins and may lead to undesirable health issues.

Comparative scrutinize of BSA and HEWL in the vicinity of metallo-catanionic aggregates derived from single chain metallosurfactant and anionic surfactant

In recent times, protein-surfactant interactions have been extensively explored due to their stimulating applications in different fields. These systems have efficaciously addressed the economic, environmental, and ecological issues. Sensing their prominence, through this report, we investigate and compare the interactional behavior of Bovine Serum Albumin (BSA) and Hen Egg White Lysozyme (HEWL) with metallo-catanionic aggregate mixtures. Attempts have been made to synthesize the metallo-catanionic aggregates from single-chain metallosurfactant (FeCTAC I) and anionic surfactant (SDS) i.e., FeCTAC I:SDS. Before proceeding towards the interactional studies of metallo-catanionic aggregates with model proteins, the morphology of formed aggregates was elucidated. Here, out of different possible molar ratios of FeCTAC I:SDS, two ratios i.e 70:30 and 30:70 were opted. The aggregates were examined through conductivity, Dynamic Light Scattering (DLS), Field Emission Scanning Electron Microscopy (FESEM), Confocal Laser Scanning Microscopy (CLSM), Small Angle X-Ray Scattering (SAXS), Zeta Potential, and Vibrating Sample Magnetometer (VSM) techniques. Interestingly, the SAXS analysis demonstrated the micellar behavior of 70:30 and vesicular behavior of 30:70 aggregates mixture, due to the difference of overall charge on them. The interactional study of BSA and HEWL with FeCTAC I:SDS was carried out through SDS-PAGE, absorption, emission, and circular dichroism (CD) spectroscopy. The effect of FeCTAC I:SDS on the linear polypeptide chain of BSA and HEWL was observed to be different, however, the secondary and tertiary structures were affected similarly. To elucidate the exact locality of FeCTAC I:SDS aggregates on the BSA, a site marker experiment was carried out. The pyrene extrinsic fluorescence experiment was performed to verify the type of interactions among HEWL and metallo-catanionic aggregates. From, the comparative studies, we concluded that the overall system was controlled by the type of forces playing between the protein and aggregate mixture that emerged from the total charge on the protein as well as on the aggregates. These types of metallo-catanionic aggregates that are flexible to shape are considered as the new alternatives for nano-vehicle applications.

Structure Determination of Hen Egg-White Lysozyme Aggregates Adsorbed to Lipid/Water and Air/Water Interfaces.

We use vibrational sum-frequency generation (VSFG) spectroscopy to study the structure of hen egg-white lysozyme (HEWL) aggregates adsorbed to DOPG/D2O and air/D2O interfaces. We find that aggregates with a parallel and antiparallel β-sheet structure together with smaller unordered aggregates and a denaturated protein are adsorbed to both interfaces. We demonstrate that to retrieve this information, fitting of the VSFG spectra is essential. The number of bands contributing to the VSFG spectrum might be misinterpreted, due to interference between peaks with opposite orientation and a nonresonant background. Our study identified hydrophobicity as the main driving force for adsorption to the air/D2O interface. Adsorption to the DOPG/D2O interface is also influenced by hydrophobic interaction; however, electrostatic interaction between the charged protein's groups and the lipid's headgroups has the most significant effect on the adsorption. We find that the intensity of the VSFG spectrum at the DOPG/D2O interface is strongly enhanced by varying the pH of the solution. We show that this change is not due to a change of lysozyme's and its aggregates' charge but due to dipole reorientation at the DOPG/D2O interface. This finding suggests that extra care must be taken when interpreting the VSFG spectrum of proteins adsorbed at the lipid/water interface.

Inhibition of lysozyme aggregation and cellular toxicity by organic acids at acidic and physiological pH conditions

The misfolding of proteins can lead to fibrillar and non-fibrillar deposits that are the hallmark of numerous human diseases. Inhibition of protein aggregation is considered as a promising strategy for the prevention of such diseases. Here we induced the fibrillar and non-fibrillar aggregates of hen egg white lysozyme (HEWL) at acidic (pH 3) and physiological (pH 7.4) environments. HEWL formed non-fibrillar aggregates rapidly at pH 7.4, whereas fibrillar HEWL aggregates were formed slowly at pH 3. Both fibrillar and non-fibrillar aggregates had cytotoxic effects on PC12 cells. Next, four organic acids, succinic acid, maleic acid, tartaric acid and citric acid, were tested for their inhibition potencies against fibrillar and non-fibrillar HEWL species. The four inhibitors were found to prevent the aggregation of HEWL at pH 7.4 with a reduction rate of over 95% as compared with the reduction rate of 42–58% for HEWL aggregation at pH 3. Other biophysical and computational analyses reveal that the candidate inhibitors have higher inhibition efficacy against HEWL monomers incubated at pH 7.4 than at pH 3. These results emphasize the importance of validating the newly identified aggregation drugs against different aggregate species, which would enhance the understanding of small molecules-induced protein aggregation inhibition.

Proline functionalized gold nanoparticles modulates lysozyme fibrillation.

Amyloid fibrils are the hallmarks of neurodegenerative diseases like Alzheimer's, Parkinson's and other proteopathies. Inhibition of fibrillation is a potential strategy to check the progress of amyloid associated diseases and further allied deterioration. In this study, we have synthesized proline functionalized gold nanoparticles (Pro-AuNPs) and scrutinized its antifibrillation property towards Hen Egg White Lysozyme (HEWL) aggregation. Pro-AuNPs were characterized using various biophysical methods like ultraviolet-visible spectroscopy, fourier transform infra-red spectroscopy, zeta potential measurement, dynamic light scattering and transmission electron microscopy. The effect of Pro-AuNPs on HEWL fibrillation was analyzed employing thioflavin T (ThT) and 8-Anilino-1-naphthalenesulfonic acid (ANS) assays. The kinetics of HEWL exhibited a typical sigmoidal nature of protein aggregation and was fitted to Boltzmann model. HEWL in the presence of bare gold nanoparticles (bAuNPs) exhibited similar aggregation kinetics as HEWL alone. However, HEWL fibrillation substantially reduced upon co-incubation with proline and Pro-AuNPs, and two slightly different intermediate species were formed with these two systems as predicted by CD spectroscopy. TEM images also supported the above observation displaying different morphological states of HEWL aggregates in the presence of proline and Pro-AuNPs. Using computational methods, the nature of interaction of HEWL and proline was found to be hydrogen bonding and hydrophobic interaction in multiple amyloidogenic regions. These interactions inhibited the formation of prefibrils (β-sheet rich intermediates) and also found to disintegrate fibrils. Furthermore, HEWL-Pro-AuNPs system resulted HEWL adsorption through hydrophobic patches, which blocked the intermolecular β-sheet formation. The present study successfully established Pro-AuNPs as a potential inhibitor of HEWL aggregation.

Protein Charge Transfer Absorption Spectra: An Intrinsic Probe to Monitor Structural and Oligomeric Transitions in Proteins

Protein Charge Transfer Spectra (ProCharTS) originate when charged amino/carboxylate groups in Lys/Glu, act as electronic charge acceptors/donors for photoinduced electron transfer either from/to the polypeptide backbone or to each other (Prasad et. al., 2017, DOI: 10.1039/c7sc00880e). The absorption band intensities in ProCharTS (250—800 nm) are dependent on 3D spatial proximity between charged functional groups across the protein. Intrinsically disordered proteins (IDPs) are rich in charged amino acids, but lack structure promoting intrinsic spectral probes like Tyr or Trp in their sequence, making their structural characterisation difficult. Here, we exploit, the richness of charged amino acid population among IDPs to sense structural transitions among multiple IDPs (like PEST fragment of human c-Myc, its mutant and Dehydrin from maize) using ProCharTS absorption spectra. Conformational changes induced in the IDP by altering pH/temperature of aqueous medium was monitored by ProCharTS and confirmed by CD spectra. Further, utility of ProCharTS to detect protein aggregation was examined using Hen Egg-White Lysozyme (HEWL) protein. Significant changes in ProCharTS spectrum was observed with changing pH in the range 3—11, which correlated with changes in secondary structure of PEST fragment. Importantly, absorbance at 280 nm, which is often employed as a measure of protein concentration, was profoundly altered by changes in ProCharTS intensity in response to changing pH in Dehydrin. ProCharTS intensity was sensitive to temperature induced changes in the secondary structure of PEST fragments between 25—85oC. Finally, increase in ProCharTS absorbance with time, in HEWL at pH 2, directly correlated with growth of HEWL aggregates and amyloid fibrils as confirmed by rising thioflavin T fluorescence. Taken together, our work highlights the utility of ProCharTS absorption as a novel label-free intrinsic probe to monitor changes in protein charge, structure and oligomeric state.

Wild-type hen egg white lysozyme aggregation in vitro can form self-seeding amyloid conformational variants

Misfolded β-sheet-rich protein aggregates termed amyloid, deposit in vivo leading to debilitating diseases such as Alzheimer's, prion and renal amyloidosis diseases etc. Strikingly, amyloid can induce conversion of their natively folded monomers into similarly aggregated conformation via ‘seeding’. The specificity of seeding is well documented in vivo for prions, where prion-variants arising from conformationally altered amyloids of the same protein, faithfully seed monomers into amyloid displaying the original variant's conformation. Thus far, amyloid variant formation is reported only for a few non-prion proteins like Alzheimer's Aβ42-peptide and β-2 microglobulin, however, their conformational cross-seeding capabilities are unexplored. While mutant human lysozyme causes renal amyloidosis, the hen egg white lysozyme (HEWL) has been extensively investigated in vitro as a model amyloid protein. Here we investigated if wild-type HEWL could form self-seeding amyloid variants to examine if variant formation is more wide-spread. We found that HEWL aggregates formed under quiescent versus agitated conditions, displayed different particle sizes, detergent stabilities & β-sheet content, and they only seeded monomeric HEWL under similar incubation conditions, but not under swapped incubation conditions thereby showing amyloid variant formation by HEWL analogous to prion variants. This may have implications to the amyloidosis caused by different mutants of human lysozyme.

Water‐Soluble Conjugated Polymers for the Detection and Inhibition of Protein Aggregation

Protein aggregation is etiologically associated with a wide range of devastating neurodegenerative diseases such as Alzheimer's and prion diseases, and therefore the new strategies for the detection and inhibition of protein aggregation are urgently desired. Herein, the capability of a cationic water‐soluble poly(phenylene vinylene) derivative (PPV‐NMe3+) to detect and inhibit the protein aggregation has been explored, using the hen egg white lysozyme (HEWL) as a model system whose aggregation is triggered by UV illumination. The PPV‐NMe3+ binds efficiently to the surface of the HEWL aggregates driven by the hydrophobic interactions, leading to the reduction of interchain contacts and therefore the enhancement of fluorescence intensity of the conjugated polymer. Importantly, the hydrophobic interactions between HEWL and PPV‐NMe3+ compete with the interaction that dominates the formation of the HEWL self‐assembly, and the electrostatic repulsion between the neighboring PPV‐NMe3+‐coated aggregates reduces and inhibits the further aggregation of HEWL. In this way, the water‐soluble conjugated polymer PPV‐NMe3+ serves as both the detector and inhibitor for the protein aggregation triggered by UV illumination, which promises advanced potential applications in the diagnose and prevention of diseases caused by protein aggregation.

Preventing Disulfide Bond Formation Weakens Non-Covalent Forces among Lysozyme Aggregates

Nonnative disulfide bonds have been observed among protein aggregates in several diseases like amyotrophic lateral sclerosis, cataract and so on. The molecular mechanism by which formation of such bonds promotes protein aggregation is poorly understood. Here in this work we employ previously well characterized aggregation of hen eggwhite lysozyme (HEWL) at alkaline pH to dissect the molecular role of nonnative disulfide bonds on growth of HEWL aggregates. We employed time-resolved fluorescence anisotropy, atomic force microscopy and single-molecule force spectroscopy to quantify the size, morphology and non-covalent interaction forces among the aggregates, respectively. These measurements were performed under conditions when disulfide bond formation was allowed (control) and alternatively when it was prevented by alkylation of free thiols using iodoacetamide. Blocking disulfide bond formation affected growth but not growth kinetics of aggregates which were ∼50% reduced in volume, flatter in vertical dimension and non-fibrillar in comparison to control. Interestingly, single-molecule force spectroscopy data revealed that preventing disulfide bond formation weakened the non-covalent interaction forces among monomers in the aggregate by at least ten fold, thereby stalling their growth and yielding smaller aggregates in comparison to control. We conclude that while constrained protein chain dynamics in correctly disulfide bonded amyloidogenic proteins may protect them from venturing into partial folded conformations that can trigger entry into aggregation pathways, aberrant disulfide bonds in non-amyloidogenic proteins (like HEWL) on the other hand, may strengthen non-covalent intermolecular forces among monomers and promote their aggregation.

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

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

Interactions of lysozyme with phospholipid vesicles: effects of vesicle biophysical features on protein misfolding and aggregation

Hen egg-white lysozyme aggregates into amyloid-like assemblies when incubated at pH 2 and 60 °C for some days or under physiological conditions in the presence of lipid membranes. This makes the protein an ideal model to study the effect of membranes on the unfolding and aggregation of pathologically relevant peptides. In this study, by choosing appropriate ratios of phosphatidylserine and phosphatidylcholine, we modulate the surface density of negative charge to investigate the effects of lipid vesicles' composition on hen egg-white lysozyme aggregation. Aggregation was investigated with dynamic light scattering and zeta potential measurements; aggregate morphology was characterized by transmission electron microscopy. We found that negatively charged lipids are needed to allow protein–vesicle interaction; in addition, protein aggregation occurs only after a threshold of the liposomal surface charge density. The spectroscopic properties of two amyloid-specific fluorescent probes, Thioflavine T and Congo Red, provided information on the presence of an amyloid-like signature in the aggregates, and together with the determination of residual enzymatic activity, confirmed the importance of surface clusters of negative charge for lysozyme aggregation. Finally, the cytotoxicity data confirmed that a high content of acidic phospholipids in the vesicles is needed to induce growth of toxic aggregates.

Decrease in Size of Hen Egg white Lysozyme Aggregates with Decrease in Monomer Concentration from Micro to Nanomolar in Alkaline pH

Earlier work from our laboratory has demonstrated that hen egg white lysozyme (HEWL) is an excellent model protein for investigating protein aggregation at an alkaline pH of 12.2. Exposure of HEWL to this pH at monomer concentrations from 0.3 to 120 uM, yielded amyloid fibrils as shown previously. Here we present the following concentration dependent changes observed with HEWL aggregates subsequent to incubation at pH 12.2 at room temperature from 0 to 24 hours. A) a two-fold increase in exposure of trp residues in HEWL to water at 0.3 uM compared to 120 uM as revealed by fluorescence quenching experiments with iodide B) a several fold increase in ANS fluorescence intensity accompanied by significantly blue-shifted emission on binding to HEWL moving from 0.3 to 120 uM. C) a marginal increase in dansyl probe fluorescence intensity accompanied by significant blue-shifted dansyl emission in dansyl conjugated HEWL moving from 0.3 to 120 uM. D) a gradual increase in steady state fluorescence anisotropy of dansyl probe in dansyl conjugated HEWL moving from 3 to 120 uM, although the dansyl mean fluorescence lifetime remained concentration independent between 3 and 120 uM. E) presence of all cys in -SH form with absence of any change in free [-SH] between 20 & 120 uM, as revealed by DTNP assays. In addition, FCS measurements with rhodamine conjugated HEWL demonstrated facile aggregation of HEWL in the concentration range 25 nM to 120 uM. The above results imply that, the size of HEWL aggregates, are clearly dependent on the initial monomer concentration, with lower concentrations like 300 nM favoring small aggregates that possess solvent exposed trp, diminished binding towards ANS and shorter mean fluorescence lifetime for dansyl probe.

Oligomerisation, Fibrillation and Activity of Hen Lysozyme in Alkaline Medium: A Concentration Dependent Investigation

Oligomerisation and fibrillation are hallmarks of protein misfolding diseases like Alzheimer's, Prion, Parkinson's, systemic amyloidosis and so on. Hen eggwhite lysozyme (HEWL) which spontaneously aggregates at pH 12.2 under room temperature as shown by us previously is an excellent model protein for aggregation studies. The present work was focussed on investigating the role of protein monomer concentration on the size and morphology of aggregates. For this purpose we employed HEWL concentrations ranging from 120 μM to 300 nM. Our findings reveal that A) FRET efficiency between dansyl labelled HEWL and dabcyl labelled HEWL in the aggregate, monitored over 12 hours was inversely proportional to HEWL monomer concentration B) Size exclusion chromatography after 12 hours showed that while all aggregates of 50 & 120 uM HEWL eluted much later, a small population of aggregates from 10 uM HEWL eluted early through the void volume C) Binding of ANS revealed fluorescence spectra that were gradually blue shifted and more intense, over 12h, in the following order with aggregates of 120 > 50 > 3 uM, while for 300 nM, these spectra were fairly invariant in emission wavelength and intensity over 12 hours. D) HEWL enzymatic activity decreased almost uniformly with time in alkaline pH for all concentrations suggesting a concentration independent early unfolding step E) AFM images showed extensive fibrils in 3 & 0.3 μM HEWL within 12 hours but predominantly large globular aggregates with 120 μM and 50 μM HEWL samples in the same time period. The above results clearly suggest that size and morphology of HEWL aggregates at alkaline pH are critically dependent on the initial monomer concentration.