Curcumin and kaempferol prevent lysozyme fibril formation by modulating aggregation kinetic parameters

Interaction of small molecule inhibitors with protein aggregates has been studied extensively, but how these inhibitors modulate aggregation kinetic parameters is little understood. In this work, we investigated the ability of two potential aggregation inhibiting drugs, curcumin and kaempferol, to control the kinetic parameters of aggregation reaction. Using thioflavin T fluorescence and static light scattering, the kinetic parameters such as amplitude, elongation rate constant and lag time of guanidine hydrochloride-induced aggregation reactions of hen egg white lysozyme were studied. We observed a contrasting effect of inhibitors on the kinetic parameters when aggregation reactions were measured by these two probes. The interactions of these inhibitors with hen egg white lysozyme were investigated using fluorescence quench titration method and molecular dynamics simulations coupled with binding free energy calculations. We conclude that both the inhibitors prolong nucleation of amyloid aggregation through binding to region of the protein which is known to form the core of the protein fibril, but once the nucleus is formed the rate of elongation is not affected by the inhibitors. This work would provide insight into the mechanism of aggregation inhibition by these potential drug molecules.     

Involvement of water molecules in the association of monoclonal antibody HyHEL-5 with bobwhite quail lysozyme.

Fluorescence polarization spectroscopy and isothermal titration calorimetry were used to study the influence of osmolytes on the association of the anti-hen egg lysozyme (HEL) monoclonal antibody HyHEL-5 with bobwhite quail lysozyme (BWQL). BWQL is an avian species variant with an Arg-->Lys mutation in the HyHEL-5 epitope, as well as three other mutations outside the HyHEL-5 structural epitope. This mutation decreases the equilibrium association constant of HyHEL-5 for BWQL by over 1000-fold as compared to HEL. The three-dimensional structure of this complex has been obtained recently. Fluorescein-labeled BWQL, obtained by labeling at pH 7.5 and purified by hydrophobic interaction chromatograpy, bound HyHEL-5 with an equilibrium association constant close to that determined for unlabeled BWQL by isothermal titration calorimetry. Fluorescence titration, stopped-flow kinetics, and isothermal titration calorimetry experiments using various concentrations of the osmolytes glycerol, ethylene glycol, and betaine to perturb binding gave a lower limit of the uptake of approximately 6-12 water molecules upon formation of the HyHEL-5/BWQL complex.     

Sorbitol prevents the self-aggregation of unfolded lysozyme leading to and up to 13 degrees C stabilisation of the folded form

We present a calorimetric investigation of stabilisation of hen egg-white lysozyme with sorbitol in the pH range 3.8-10.5. Differential scanning calorimetry and steady-state fluorescence were used to determine the denaturation temperatures of lysozyme as a function of sorbitol concentration. The fluorescence data were collected in the presence of 2M urea to lower the melting point of the protein to an observable range of the instrument. The effect of sorbitol on the activation energy of unfolding was investigated by scanrate studies. The effect of sorbitol lysozyme interaction was investigated using isothermal titration calorimetry. The titration experiments were performed with folded as well as unfolded lysozyme to investigate in more detail the nature of the interaction. The data obtained in those experiments show a remarkable stabilisation effect of sorbitol. We observed a 4.0 degrees C increase in the Tm for 1 M sorbitol in the pH range 3.8-8.5 by scanning calorimetry. The effect increases dramatically at pH 9.5 where we observe a 9.5 degrees C stabilisation. An increase in the sorbitol concentration to 2 M stabilises lysozyme by 11.3-13.4 degrees C in the pH range 9.5-10.5. In the absence of urea, no significant effects of sorbitol were observed on the activation energy for unfolding for lysozyme at pH 4.5. This indicates together with the results from the titration experiments that sorbitol may stabilise the folded form of lysozyme by destabilising the unfolded form of lysozyme. At pH values at and above lysozyme's pI (approximately 9.3), the unfolding of the protein is accompanied with a substantial amount of self-aggregation seen in the calorimetry experiments in the ratio of DeltaH(cal)/DeltaH(vH). In the presence of sorbitol, the self-aggregation was counterbalanced by higher sorbitol concentrations. These results strongly suggest a negative influence of sorbitol on the unfolded form of lysozyme and thereby stabilising the native form.     

Acidic residue modifications restore chaperone activity of β-casein interacting with lysozyme

An important factor in medicine and related industries is the use of chaperones to reduce protein aggregation. Here we show that chaperone ability is induced in β-casein by modification of its acidic residues using Woodward's Reagent K (WRK). Lysozyme at pH 7.2 was used as a target protein to study β-casein chaperone activities. The mechanism for chaperone activity of the modified β-casein was determined using UV-vis absorbencies, fluorescence spectroscopy, differential scanning calorimetry and theoretical calculations. Our results indicated that the β-casein destabilizes the lysozyme and increases its aggregation rate. However, WRK-ring sulfonate anion modifications enhanced the hydrophobicity of β-casein resulting in its altered net negative charge upon interactions with lysozyme. The reversible stability of lysozyme increased in the presence of WRK-modified β-casein, and hence its aggregation rate decreased. These results demonstrate the enhanced chaperone activity of modified β-casein and its protective effects on lysozyme refolding.     

Protein Disulfide Isomerase Interacts with Tau Protein and Inhibits Its Fibrillization

Background

Tau protein is implicated in the pathogenesis of neurodegenerative disorders such as tauopathies including Alzheimer disease, and Tau fibrillization is thought to be related to neuronal toxicity. Physiological inhibitors of Tau fibrillization hold promise for developing new strategies for treatment of Alzheimer disease. Because protein disulfide isomerase (PDI) is both an enzyme and a chaperone, and implicated in neuroprotection against Alzheimer disease, we want to know whether PDI can prevent Tau fibrillization. In this study, we have investigated the interaction between PDI and Tau protein and the effect of PDI on Tau fibrillization.

Methodology/Principal Findings

As evidenced by co-immunoprecipitation and confocal laser scanning microscopy, human PDI interacts and co-locates with some endogenous human Tau on the endoplasmic reticulum of undifferentiated SH-SY5Y neuroblastoma cells. The results from isothermal titration calorimetry show that one full-length human PDI binds to one full-length human Tau (or human Tau fragment Tau_244–372) monomer with moderate, micromolar affinity at physiological pH and near physiological ionic strength. As revealed by thioflavin T binding assays, Sarkosyl-insoluble SDS-PAGE, and transmission electron microscopy, full-length human PDI remarkably inhibits both steps of nucleation and elongation of Tau_244–372 fibrillization in a concentration-dependent manner. Furthermore, we find that two molecules of the a-domain of human PDI interact with one Tau_244–372 molecule with sub-micromolar affinity, and inhibit both steps of nucleation and elongation of Tau_244–372 fibrillization more strongly than full-length human PDI.

Conclusions/Significance

We demonstrate for the first time that human PDI binds to Tau protein mainly through its thioredoxin-like catalytic domain a, forming a 1∶1 complex and preventing Tau misfolding. Our findings suggest that PDI could act as a physiological inhibitor of Tau fibrillization, and have applications for developing novel strategies for treatment and early diagnosis of Alzheimer disease.     

Interactions between l‐arginine/l‐arginine derivatives and lysozyme and implications to their inhibition effects on protein aggregation

l ‐Arginine (Arg), l ‐homoarginine (HArg), l ‐arginine ethylester (ArgEE), and l ‐arginine methylester (ArgME) were found effective in inhibiting protein aggregation, but the molecular mechanisms are not clear. Herein, stopped‐flow fluorescence spectroscopy, isothermal titration calorimetry, and mass spectroscopy were used to investigate the folding kinetics of lysozyme and the interactions of the additives with lysozyme. It was found that the interactions of ArgME and ArgEE with lysozyme were similar to that of guanidine hydrochloride and were much stronger than those of Arg and HArg. The binding forces were all mainly hydrogen bonding and cation‐π interaction from the guanidinium group, but their differences in molecular states led to the significantly different binding strengths. The additives formed molecular clusters in an increasing order of ArgEE, ArgME, HArg, and Arg. Arg and HArg mainly formed annular clusters with head‐to‐tail bonding, while ArgME and ArgEE formed linear clusters with guanidinium groups stacking. The interactions between the additives and lysozyme were positively related to the monomer contents. That is, the monomers were the primary species that participated in the direct interactions due to their intact guanidinium groups and small sizes, while the clusters performed as barriers to crowd out the protein–protein interactions for aggregation. Thus, it is concluded that the effects of Arg and its derivatives on protein aggregation stemmed from the direct interactions by the monomers and the crowding effects by the clusters. Interplay of the two effects led to the differences in their inhibition effects on protein aggregation. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1316–1324, 2013     

Strategies to increase the reproducibility of protein fibrillization in plate reader assays

There is great interest in developing reproducible high-throughput screens to identify small molecular inhibitors of protein fibrillization and aggregation for possible therapy against deposition diseases such as Alzheimer’s and Parkinson’s (PD). We have made a methodical analysis of factors increasing the reproducibility of the fibrillization of α-synuclein (αSN), a 140-amino-acid protein implicated in PD and notorious for its erratic fibrillization behavior. Salts and polyanionic polymers do not significantly improve the quality of the assay. However, an orbital agitation mode in the plate reader is a crucial first step toward reproducible αSN fibrillization. Higher reproducibility is achieved by the addition of glass beads, as evaluated by the percentage standard deviation of the nucleation and elongation rate constants and the end-stage fluorescence intensity of the fibril-binding dye thioflavin T (ThT). The highest reproducibility is obtained by either seeding the solution with preformed fibrils or by adding submicellar amounts of sodium dodecyl sulfate (SDS), where we obtain percentage standard deviations of 3-4% on the end ThT level. We conclude that there are multiple ways to achieve satisfactory levels of reproducibility, although the different conditions used to induce aggregation may lead to different fibrillization pathways.     

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

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

Fluorescence and thermotropic studies of the interactions of PEI-cholesterol based PEI-chol lipopolymers with dipalmitoyl phosphatidylcholine membranes

Numerous PEI derived polymers have been explored for their use in gene delivery. Nine PEI-chol lipopolymers based on cholesterol grafting on three polyethyleneimines (PEI) of different molecular weights have been synthesized. Firstly their aggregation behavior has been studied using transmission electron microscopy and then their interactions with l-α-dipalmitoyl phosphatidylcholine (DPPC) membranes have been examined using fluorescence anisotropy and differential scanning calorimetry (DSC). These PEI-chol lipopolymers are found to quench the chain motion of the acyl chains of DPPC, when incorporated in membranes, depending upon the cholesterol grafting on PEI. These PEI-chol lipopolymers act as filler molecules in membranes. Electron microscopy shows the different aggregation behavior of these new PEI-chol lipopolymers depending upon the molecular weight of PEI and percentage of cholesterol grafting. Detailed analysis of the fluorescence anisotropy and DSC data indicate that the nature of perturbation induced by PEI-chol lipopolymers is dependent upon the molecular weight of the PEI used and the % of cholesterol grafting on PEI. In general, PEI-chol lipopolymers rigidify the liquid-crystalline phase of the membranes without any noticeable effect on the gel phase unlike natural cholesterol, which is known to fluidize the gel phase of the membranes.     

Acridine derivatives inhibit lysozyme aggregation

We have screened a library of structurally distinct acridine derivatives (19 compounds) for their ability to inhibit lysozyme amyloid aggregation in vitro. Studied acridines were divided into three structurally different groups depending on the molecule planarity and type of the side chain-planar acridines, spiroacridines and tetrahydroacridines. Thioflavine T fluorescence assay and transmission electron microscopy were used for monitoring the inhibiting activity of acridines. We have found that both the structure of the acridine side chains and molecule planarity influence their antiamyloidogenic activity. The planar acridines inhibited lysozyme aggregation effectively. Spiroacridines and tetrahydroacridines had no significant effect on the prevention of lysozyme fibrillization, probably resulting from the presence of the heterocyclic 5-membered ring and non-planarity of molecule. Moreover, in the presence of some tetrahydroacridines the enhanced extent of aggregation was detected. We identified the most active acridine derivates from studied compound library characterized by low micromolar IC(50) values, which indicate their possible application for therapeutic purpose.     

Shape evolution and thermal stability of lysozyme crystals: effect of pH and temperature

The properties of crystalline protein materials are closely linked to crystal shape. However, the effective strategies for the shape control of protein crystals are lacking. The conventional sitting-drop vapor-diffusion method was employed to investigate the influence of pH and temperature on the crystal nucleation behavior of hen egg white lysozyme. Moreover, the size distributions of protein crystals grown at different conditions were analyzed. Differential scanning calorimetry was employed to evaluate the thermal stability of lysozyme crystals. The results indicated that pH and temperature will affect the supersaturation and electrostatic interactions among protein molecules in the nucleation process. In particular, the crystals with different aspect ratios can be selectively nucleated, depending upon the choice of pH and temperature. Therefore, this study provided a simple method for obtaining shape-controlled lysozyme crystals and supplied some information on thermal behaviors of lysozyme crystals grown at different pH values.     

Thermodynamic characterization of interactions between ornithine transcarbamylase leader peptide and phospholipid bilayer membranes

The interactions of the targeting sequence of the mitochondrial enzyme ornithine transcarbamylase with phospholipid bilayers of different molecular compositions have been studied by high-sensitivity heating and cooling differential scanning calorimetry, high-sensitivity isothermal titration calorimetry, fluorescence spectroscopy, and electron microscopy. These studies indicate that the leader peptide interacts strongly with dipalmitoylphosphatidylcholine (DPPC) bilayer membranes containing small mole percents of the anionic phospholipids dipalmitoylphosphatidylglycerol (DPPG) or brain phosphatidylserine (brain PS) but not with pure phosphatidylcholines. For the first time, the energetics of the leader peptide-membrane interaction have been measured directly by using calorimetric techniques. At 20 degrees C, the association of the peptide with the membrane is exothermic and characterized by an association constant of 2.3 X 10(6) M-1 in the case of phosphatidylglycerol-containing and 0.35 X 10(6) M-1 in the case of phosphatidylserine-containing phospholipid bilayers. In both cases, the enthalpy of association is -60 kcal/mol of peptide. Additional experiments using fluorescence techniques suggest that the peptide does not penetrate deeply into the hydrophobic core of the membrane. The addition of the leader peptide to DPPC/DPPG (5:1) or DPPC/brain PS (5:1) small sonicated vesicles results in vesicle fusion. The fusion process is dependent on peptide concentration and is maximal at the phase transition temperature of the vesicles and minimal at temperatures below the phase transition.     

Hsp104 targets multiple intermediates on the amyloid pathway and suppresses the seeding capacity of Abeta fibrils and protofibrils

The heat shock protein Hsp104 has been reported to possess the ability to modulate protein aggregation and toxicity and to “catalyze” the disaggregation and recovery of protein aggregates, including amyloid fibrils, in yeast, Escherichia coli, mammalian cell cultures, and animal models of Huntington's disease and Parkinson's disease. To provide mechanistic insight into the molecular mechanisms by which Hsp104 modulates aggregation and fibrillogenesis, the effect of Hsp104 on the fibrillogenesis of amyloid beta (Aβ) was investigated by characterizing its ability to interfere with oligomerization and fibrillogenesis of different species along the amyloid-formation pathway of Aβ. To probe the disaggregation activity of Hsp104, its ability to dissociate preformed protofibrillar and fibrillar aggregates of Aβ was assessed in the presence and in the absence of ATP. Our results show that Hsp104 inhibits the fibrillization of monomeric and protofibrillar forms of Aβ in a concentration-dependent but ATP-independent manner. Inhibition of Aβ fibrillization by Hsp104 is observable up to Hsp104/Aβ stoichiometric ratios of 1:1000, suggesting a preferential interaction of Hsp104 with aggregation intermediates (e.g., oligomers, protofibrils, small fibrils) on the pathway of Aβ amyloid formation. This hypothesis is consistent with our observations that Hsp104 (i) interacts with Aβ protofibrils, (ii) inhibits conversion of protofibrils into amyloid fibrils, (iii) arrests fibril elongation and reassembly, and (iv) abolishes the capacity of protofibrils and sonicated fibrils to seed the fibrillization of monomeric Aβ. Together, these findings suggest that the strong inhibition of Aβ fibrillization by Hsp104 is mediated by its ability to act at different stages and target multiple intermediates on the pathway to amyloid formation.     

Surface-modified magnetite nanoparticles affect lysozyme amyloid fibrillization

BackgroundThe surface of nanoparticles (NPs) is an important factor affecting the process of poly/peptides' amyloid aggregation. We have investigated the in vitro effect of trisodium citrate (TC), gum arabic (GA) and citric acid (CA) surface-modified magnetite nanoparticles (COAT-MNPs) on hen egg-white lysozyme (HEWL) amyloid fibrillization and mature HEWL fibrils.MethodsDynamic light scattering (DLS) was used to characterize the physico-chemical properties of studied COAT-MNPs and determine the adsorption potential of their surface towards HEWL. The anti-amyloid properties were studied using thioflavin T (ThT) and tryptophan (Trp) intrinsic fluorescence assays, and atomic force microscopy (AFM). The morphology of amyloid aggregates was analyzed using Gwyddion software. The cytotoxicity of COAT-MNPs was determined utilizing Trypan blue (TB) assay.ResultsAgents used for surface modification affect the COAT-MNPs physico-chemical properties and modulate their anti-amyloid potential. The results from ThT and intrinsic fluorescence showed that the inhibitory activities result from the more favorable interactions of COAT-MNPs with early pre-amyloid species, presumably reducing nuclei and oligomers formation necessary for amyloid fibrillization. COAT-MNPs also possess destroying potential, which is presumably caused by the interaction with hydrophobic residues of the fibrils, resulting in the interruption of an interface between β-sheets stabilizing the amyloid fibrils.ConclusionCOAT-MNPs were able to inhibit HEWL fibrillization and destroy mature fibrils with different efficacy depending on their properties, TC-MNPs being the most potent nanoparticles.General significanceThe study reports findings regarding the general impact of nanoparticles' surface modifications on the amyloid aggregation of proteins.     

Osmolytic Effect of Sucrose on Thermal Denaturation of Pea Seedling Copper Amine Oxidase

Protein stability is a subject of interest by many researchers. One of the common methods to increase the protein stability is using the osmolytes. Many studies and theories analyzed and explained osmolytic effect by equilibrium thermodynamic while most proteins undergo an irreversible denaturation. In current study we investigated the effect of sucrose as an osmolyte on the thermal denaturation of pea seedlings amine oxidase by the enzyme activity, fluorescence spectroscopy, circular dichroism, and differential scanning calorimetry. All experiments are in agreement that pea seedlings amine oxidase denaturation is controlled kinetically and its kinetic stability is increased in presence of sucrose. Differential scanning calorimetry experiments at different scanning rates showed that pea seedlings amine oxidase unfolding obeys two-state irreversible model. Fitting the differential scanning calorimetry data to two-state irreversible model showed that unfolding enthalpy and T ^*, temperature at which rate constant equals unit per minute, are increased while activation energy is not affected by increase in sucrose concentration. We concluded that osmolytes decrease the molecular oscillation of irreversible proteins which leads to decline in unfolding rate constant.     

Direct measurement of the energetics of association between myelin basic protein and phosphatidylserine vesicles

A newly designed high-sensitivity isothermal reaction calorimetry system has been used to investigate the thermodynamics of the association between myelin basic protein and phosphatidylserine vesicles. This instrument has allowed us to measure directly the energetics of the protein-lipid interaction under various conditions. Above the phospholipid phase transition temperature the enthalpy of association is highly exothermic amounting to -160 kcal/mol of protein. Below the phospholipid phase transition temperature the enthalpy of association is exothermic at protein/lipid ratios smaller than 1/50 and endothermic at higher protein/lipid ratios. These studies indicate that the association of myelin basic protein to phosphatidylserine vesicles consists of at least two stages involving different types of binding. The first stage, at low protein/lipid ratios, involves a strong exothermic association of the protein to the membrane and the second, at high protein/lipid ratios, a weaker association probably involving attachment of the protein to the membrane surface only. In the gel phase the second binding stage is endothermic and appears to be correlated with the formation of large vesicle aggregates. This vesicle aggregation is a reversible process dependent upon the physical state of the membrane. The isothermal titration studies have been complemented with high-sensitivity differential scanning calorimetry experiments. It is shown that the dependence of the phospholipid transition enthalpy on the protein/lipid molar ratio can be expressed in terms of the different protein-membrane association enthalpies in the gel and fluid phases of the membrane.     

Elucidating the mechanisms of protein antigen adsorption to the CAF/NAF liposomal vaccine adjuvant systems: Effect of charge,fluidity and antigen-to-lipid ratio

The reverse vaccinology approach has recently resulted in the identification of promising protein antigens, which in combination with appropriate adjuvants can stimulate customized, protective immune responses. Although antigen adsorption to adjuvants influences vaccine efficacy and safety, little is generally known about how antigens and adjuvants interact at the molecular level. The aim of this study was to elucidate the mechanisms of interactions between the equally sized, but oppositely charged model protein antigens α-lactalbumin and lysozyme, and i) the clinically tested cationic liposomal adjuvant CAF01 composed of cationic dimethyldioctadecylammonium (DDA) bromide and trehalose-6,6′-dibehenate (TDB) or ii) the neutral adjuvant formulation NAF01, where DDA was replaced with zwitterionic distearoylphosphatidylcholine (DSPC). The effect of liposome charge, bilayer rigidity, isoelectric point and antigen-to-lipid ratio was investigated using dynamic light scattering, transmission electron microscopy, differential scanning calorimetry, intrinsic fluorescence and Langmuir monolayers. The net anionic α-lactalbumin adsorbed onto the cationic liposomes, while there was no measureable attractive interaction with the zwitterionic liposomes. In contrast, the net cationic lysozyme showed very little interaction with either types of liposome. Adsorption of α-lactalbumin altered its tertiary structure, affected lipid membrane packing below and above the phase transition temperature, and neutralized the liposomal surface charge, resulting in reduced colloidal stability and liposome aggregation. Langmuir studies revealed that α-lactalbumin was not squeezed out of DDA monolayers upon compression, which suggests additional hydrophobic interactions.     

Cyanine dye N744 inhibits tau fibrillization by blocking filament extension: implications for the treatment of tauopathic neurodegenerative diseases

Tau fibrillization is a potential therapeutic target for Alzheimer's and other neurodegenerative diseases. Small molecules capable of both inhibiting aggregation and promoting filament disaggregation have been discovered, but knowledge of their mechanism of action and potential for testing in biological models is fragmentary. To clarify these issues, the interaction between a small-molecule inhibitor of tau fibrillization, 3,3'-bis(beta-hydroxyethyl)-9-ethyl-5,5'-dimethoxythiacarbocyanine iodide (N744), and full-length four-repeat tau protein was characterized in vitro using transmission electron microscopy and fluorescence spectroscopy. Analysis of reaction time courses performed in the presence of anionic fibrillization inducers revealed that increasing concentrations of N744 decreased the total filament length without modulating lag time, indicating that filament extension but not nucleation was affected by inhibitor under the conditions that were investigated. Critical concentration measurements confirmed that N744 shifted equilibria at filament ends away from the fibrillized state, resulting in endwise filament disaggregation when it was added to synthetic filaments. Both increasing bulk tau concentrations and filament stabilizing modifications such as pseudophosphorylation and glycation antagonized N744 activity. The results illustrate the importance of mechanism for the design and interpretation of pharmacological studies in biological models of tau aggregation.     

The impact of binding of macrocyclic metal complexes on amyloid fibrillization of insulin and lysozyme

Amyloid fibrils are insoluble protein aggregates whose accumulation in cells and tissues is connected with a range of pathological diseases. We studied the impact of 2 metal complexes (axially coordinated Hf phthalocyanine and iron (II) clathrochelate) on aggregation of insulin and lysozyme. For both proteins, the host‐guest interaction with these compounds changes the kinetics of fibrillization and affects the morphology of final aggregates. The Hf phthalocyanine is a very efficient inhibitor of insulin fibrillization; in its presence, only very low amounts of fibrils with the diameters of 0.8 to 5 nm and spherical aggregates were found. Effective concentration of fibrillization inhibition (IC₅₀) was estimated to be 0.11 ± 0.04 μM. The clathrochelate induced the formation of thin fibrils with the diameters of 0.8 to 2.5 nm; IC₅₀ was estimated as 20 ± 9 μM. The lysozyme fibrillization remained quite intensive in the presence of the studied compounds; they induced the formation of long filaments (the length up to 2.5 μm, the diameters of 1.5‐3.5 nm). These fibrils noticeably differed from those of free lysozyme short linear species (the diameters of 3‐5 nm, the length up to 0.6 μm). Thinning and elongation of fibrils suggest that the metal complexes bind mainly to the grooves of protofilaments; this hinders the stacking of early aggregates or protofilaments together but does not hinder their growth. The image of the fibril separated into 2 protofilaments allows suggesting that the fibril formation occurs via the growth of the parallel protofilaments with their subsequent twisting in the fibril. The changes of the lysozyme intrinsic fluorescence indicate that both metal complexes interact with the protein during the stage of the fibrillar seeds formation.     

Complexation of lysozyme with poly(sodium(sulfamate-carboxylate)isoprene)

The complexation between hen egg white lysozyme (HEWL) and a novel pH-sensitive and intrinsically hydrophobic polyelectrolyte poly(sodium(sulfamate-carboxylate)isoprene) (SCPI), was investigated by means of dynamic, static, and electrophoretic light scattering and isothermal titration calorimetry measurements. The complexation process was studied at both pH 7 and 3 (high and low charge density of the SCPI, respectively) and under low ionic strength conditions for two polyelectrolyte samples of different molecular weights. The solution behavior, structure, and effective charge of the formed complexes proved to be dependent on the pH, the [-]/[+] charge ratio, and the molecular weight of the polyelectrolyte. Increasing the ionic strength of the solution led to vast aggregation and eventually precipitation of the complexes. The interaction between HEWL and SCPI was found to be mainly electrostatic, associated with an exothermic enthalpy change. The structural investigation of the complexed protein by fluorescence, infrared, circular dichroism spectroscopic, and differential scanning calorimetric measurements revealed no signs of denaturation upon complexation.