Design of Magnetic Fe3O4/CeO2 "Core/Shell"-Like Nanocomposites with Pronounced Antiamyloidogenic and Antioxidant Bioactivity.

"Core/shell" nanocomposites based on magnetic magnetite (Fe3O4) and redox-active cerium dioxide (CeO2) nanoparticles (NPs) are promising in the field of biomedical interests because they can combine the ability of magnetic NPs to heat up in an alternating magnetic field (AMF) with the pronounced antioxidant activity of CeO2 NPs. Thus, this report is devoted to Fe3O4/CeO2 nanocomposites (NCPs) synthesized by precipitation of the computed amount of "CeO2-shell" on the surface of prefabricated Fe3O4 NPs. The X-ray diffraction, X-ray photoelectron spectroscopy, and high-resolution transmission electron microscopy data validated the formation of Fe3O4/CeO2 "core/shell"-like NCPs, in which ultrafine CeO2 NPs with an average size of approximately 3-3.5 nm neatly surround Fe3O4 NPs. The presence of a CeO2 "shell" significantly increased the stability of Fe3O4/CeO2 NCPs in aqueous suspensions: Fe3O4/CeO2 NCPs with "shell thicknesses" of 5 and 7 nm formed highly stable magnetic fluids with ζ-potential values of >+30 mV. The magnetization values of Fe3O4/CeO2 NCPs decreased with a growing CeO2 "shell" around the magnetic NPs; however, the resulting composites retained the ability to heat efficiently in an AMF. The presence of a CeO2 "shell" generates a possibility to precisely regulate tuning of the maximum heating temperature of magnetic NCPs in the 42-50 °C range and stabilize it after a certain time of exposure to an AMF by changing the thickness of the "CeO2-shell". A great improvement was observed in both antioxidant and antiamyloidogenic activities. It was found that inhibition of insulin amyloid formation, expressed in IC50 concentration, using NCPs with a "shell thickness" of 7 nm was approximately 10 times lower compared to that of pure CeO2. For these NCPs, more than 2 times higher superoxide dismutase-like activity was observed. The coupling of both Fe3O4 and CeO2 results in higher bioactivity than either of them individually, probably due to a synergistic catalytic mechanism.

Biocompatible zeolite-dye composites with anti-amyloidogenic properties.

One of the most attractive approaches in biomedicine and pharmacy is the application of multifunctional materials. The mesoporous structure of clinoptilolite (CZ) absorbs various types of substances and can be used as a model for studying the carriers for targeted drug delivery with controlled release. CZ-dye composites are fabricated by incorporation into clinoptilolite pores commonly used dyes, aluminum phthalocyanine, zinc porphine, and hypericin. We examined and compared the effect of pure dyes and CZ-dye composites on insulin amyloidogenesis. The formation of insulin amyloid fibrils and the disassembly of preformed fibrils is significantly affected by any of the three compounds, however, the strongest effect is observed for aluminum phthalocyanine indicating a structurally-dependent anti-amyloidogenic activity of the dyes. The incorporation of dyes into CZ particles resulted in enhanced anti-amyloidogenic activity in comparison to pure CZ particles. The cell metabolic activity, biocompatibility and fluorescence biodistribution of the dyes entrapped in the composites were tested in vitro (U87 MG cells) and in vivo in the quail chorioallantoic membrane model. Considering the photoactive properties of the dyes used, we assume their applicability in photodiagnostics and photodynamic therapy. It can also be expected that their anti-amyloidogenic potential can be enhanced by photodynamic effect.

Surface-modified cerium dioxide nanoparticles with improved anti-amyloid and preserved nanozymatic activity.

Cerium dioxide nanoparticles (CeO2 NPs) are used increasingly in nanotechnology and particularly in biotechnology and bioresearch. Thus, CeO2 NPs have been successfully tested in vitro as a potential therapeutic agent for various pathologies associated with oxidative stress, including the formation of protein amyloid aggregates. In this study, to increase the anti-amyloidogenic efficiency and preserve the antioxidant potential, the surface of the synthesized CeO2 NPs is modified with a nonionic, sugar-based surfactant, dodecyl maltoside (DDM), which is known for its high anti-amyloidogenic activity and biocompatibility. Dynamic light scattering and Fourier transform infrared spectroscopy demonstrated successful modification by DDM. The apparent hydrodynamic diameters of CeO2 NPs and DDM-modified NPs (CeO2@DDM NPs) are found to be ⁓180 nm and ⁓260 nm, respectively. A positive zeta potential value of + 30.5 mV for CeO2 NPs and + 22.5 mV for CeO2 @DDM NPs suggest sufficient stability and good dispersion of NPs in an aqueous solution. A combination of Thioflavin T fluorescence analysis and atomic force microscopy is used to assess the effect of nanoparticles on the formation of insulin amyloid fibrils. Results show that the fibrillization of insulin is inhibited by both, naked and modified NPs in a dose-dependent manner. However, while the IC50 of naked NPs is found to be ∼270 ± 13 µg/mL, the surface-modified NPs are 50% more efficient with IC50 equaled to 135 ± 7 µg/mL. In addition, both, the naked CeO2 NPs and DDM-modified NPs displayed an antioxidant activity expressed as oxidase-, catalase- and SOD-like activity. Therefore, the resulting nanosized material is very well suited to prove or disprove the hypothesis that oxidative stress plays a role in the formation of amyloid fibrils.

Micelle Formation in Aqueous Solutions of the Cholesterol-Based Detergent Chobimalt Studied by Small-Angle Scattering.

The structure and interaction parameters of the water-soluble cholesterol-based surfactant, Chobimalt, are investigated by small-angle neutron and X-ray scattering techniques. The obtained data are analyzed by a model-independent approach applying the inverse Fourier transformation procedure as well as considering a model fitting procedure, using a core-shell form factor and hard-sphere structure factor. The analysis reveals the formation of the polydisperse spherical or moderately elongated ellipsoidal shapes of the Chobimalt micelles with the hard sphere interaction in the studied concentration range 0.17-6.88 mM. The aggregation numbers are estimated from the micelle geometry observed by small-angle scattering and are found to be in the range of 200-300. The low pH of the solution does not have a noticeable effect on the structure of the Chobimalt micelles. The critical micelle concentrations of the synthetic surfactant Chobimalt in water and in H2O-HCl solutions were obtained according to fluorescence measurements as ~3 µM and ~2.5 µM, respectively. In-depth knowledge of the basic structural properties of the detergent micelles is necessary for further applications in bioscience and biotechnology.

Cerium dioxide nanoparticles synthesized via precipitation at constant pH: Synthesis, physical-chemical and antioxidant properties.

Cerium oxide nanoparticles (CeO2 NPs) are well known for their application in various fields of industry, as well as in biology and medicine. Knowledge of synthesis schemes, physicochemical and morphological features of nanoscale CeO2 is important for assessing their antioxidant behavior and understanding the mechanism of oxidative stress and its consequences. The choice of the method of synthesis should be based on the possibility to choose the conditions and parameters for obtaining CeO2 with controlled dimensions and a ratio of Се3+/Се4+ on their surface. In this study, CeO2 NPs are synthesized by precipitation in mixed water-alcohol solutions at constant pH = 9. The properties of obtained NPs are studied using various methods of physical-chemical characterization such as X-ray diffraction, X-ray photoelectron spectroscopy, transmission electron microscopy, and dynamic light scattering. The size of CeO2 NPs varied from 14 to 4.2 nm with increasing alcohol concentration, while the effect of constant pH during synthesis on the morphology of the particles was insignificant. The synthesized nanoparticles form highly stable aqueous suspensions since their zeta-potential is higher than + 40 mV. It is found that the ability of CeO2 NPs to self-stabilize is associated with the presence of hydrated Ce4+ ions on their surface. In vitro biological studies have shown that, regardless of particle size, CeO2 NPs have antioxidant potential, but smaller NPs with a higher percentage of Ce3+ on the surface had a more effective antioxidant effect. In addition, the size-depended activity of CeO2 NPs to inhibit the amyloid formation of insulin is demonstrated.

The intriguing dose-dependent effect of selected amphiphilic compounds on insulin amyloid aggregation: Focus on a cholesterol-based detergent, Chobimalt.

The amyloidogenic self-assembly of many peptides and proteins largely depends on external conditions. Among amyloid-prone proteins, insulin attracts attention because of its physiological and therapeutic importance. In the present work, the amyloid aggregation of insulin is studied in the presence of cholesterol-based detergent, Chobimalt. The strategy to elucidate the Chobimalt-induced effect on insulin fibrillogenesis is based on performing the concentration- and time-dependent analysis using a combination of different experimental techniques, such as ThT fluorescence assay, CD, AFM, SANS, and SAXS. While at the lowest Chobimalt concentration (0.1 µM; insulin to Chobimalt molar ratio of 1:0.004) the formation of insulin fibrils was not affected, the gradual increase of Chobimalt concentration (up to 100 µM; molar ratio of 1:4) led to a significant increase in ThT fluorescence, and the maximal ThT fluorescence was 3-4-fold higher than the control insulin fibril's ThT fluorescence intensity. Kinetic studies confirm the dose-dependent experimental results. Depending on the concentration of Chobimalt, either (i) no effect is observed, or (ii) significantly, ∼10-times prolonged lag-phases accompanied by the substantial, ∼ 3-fold higher relative ThT fluorescence intensities at the steady-state phase are recorded. In addition, at certain concentrations of Chobimalt, changes in the elongation-phase are noticed. An increase in the Chobimalt concentrations also triggers the formation of insulin fibrils with sharply altered morphological appearance. The fibrils appear to be more flexible and wavy-like with a tendency to form circles. SANS and SAXS data also revealed the morphology changes of amyloid fibrils in the presence of Chobimalt. Amyloid aggregation requires the formation of unfolded intermediates, which subsequently generate amyloidogenic nuclei. We hypothesize that the different morphology of the formed insulin fibrils is the result of the gradual binding of Chobimalt to different binding sites on unfolded insulin. A similar explanation and the existence of such binding sites with different binding energies was shown previously for the nonionic detergent. Thus, the data also emphasize the importance of a protein partially-unfolded state which undergoes the process of fibrils formation; i.e., certain experimental conditions or the presence of additives may dramatically change not only kinetics but also the morphology of fibrillar aggregates.

Dual-Functional Antioxidant and Antiamyloid Cerium Oxide Nanoparticles Fabricated by Controlled Synthesis in Water-Alcohol Solutions.

Oxidative stress is known to be associated with a number of degenerative diseases. A better knowledge of the interplay between oxidative stress and amyloidogenesis is crucial for the understanding of both, aging and age-related neurodegenerative diseases. Cerium dioxide nanoparticles (CeO2 NPs, nanoceria) due to their remarkable properties are perspective nanomaterials in the study of the processes accompanying oxidative-stress-related diseases, including amyloid-related pathologies. In the present work, we analyze the effects of CeO2 NPs of different sizes and Ce4+/Ce3+ ratios on the fibrillogenesis of insulin, SOD-like enzymatic activity, oxidative stress, biocompatibility, and cell metabolic activity. CeO2 NPs (marked as Ce1-Ce5) with controlled physical-chemical parameters, such as different sizes and various Ce4+/Ce3+ ratios, are synthesized by precipitation in water-alcohol solutions. All synthesized NPs are monodispersed and exhibit good stability in aqueous suspensions. ThT and ANS fluorescence assays and AFM are applied to monitor the insulin amyloid aggregation and antiamyloid aggregation activity of CeO2 NPs. The analyzed Ce1-Ce5 nanoparticles strongly inhibit the formation of insulin amyloid aggregates in vitro. The bioactivity is analyzed using SOD and MTT assays, Western blot, fluorescence microscopy, and flow cytometry. The antioxidative effects and bioactivity of nanoparticles are size- or valence-dependent. CeO2 NPs show great potential benefits for studying the interplay between oxidative stress and amyloid-related diseases, and can be used for verification of the role of oxidative stress in amyloid-related diseases.

Monitoring the surface tension by the pendant drop technique for detection of insulin fibrillogenesis.

Monitoring the aggregation of amyloid-prone proteins is critical for understanding the mechanism of amyloid fibril formation. Insulin, when dissolved in low pH buffer, has a surface tension of 61-64 mN m-1, as measured by the pendant drop technique. Formation of insulin amyloid fibrils resulted in the increase of the surface tension values up to 71.2-73.5 mN m-1. The kinetics of fibril formation and fibril morphology were validated by ThT fluorescence and AFM, respectively. The results demonstrate that monitoring the surface tension by the pendant drop technique is a valuable tool for the detection of insulin amyloid aggregation.

Structural and physical-chemical characterization of redox active CeO2nanoparticles synthesized by precipitation in water-alcohol solutions.

A set of cerium dioxide nanoparticles (CeO2NPs) was synthesized by precipitation in water-alcohol solutions under conditions when the physical-chemical parameters of synthesized NPs were controlled by changing the ratio of the reaction components. The size of CeO2NPs is controlled largely by the dielectric constant of the reaction solution. An increase of the percentage of Ce3+ions at the surface was observed with a concomitant reduction of the NP sizes. All synthesized CeO2NPs possess relatively high positive values of zeta-potential (ζ > 40 mV) suggesting good stability in aqueous suspensions. Analysis of the valence- and size-dependent rate of hydrogen peroxide decomposition revealed that catalase/peroxidase-like activity of CeO2NPs is higher at a low percentage of Ce3+at the NP surface. In contrast, smaller CeO2NPs with a higher percentage of Ce3+at the NP surface display a higher oxidase-like activity.

Development of multifunctional nanocomposites for controlled drug delivery and hyperthermia.

Magnetic nano/micro-particles based on clinoptilolite-type of natural zeolite (CZ) were fabricated and were expected to act as carriers for controlled drug delivery/release, imaging and local heating in biological systems. Adsorption of rhodamine B, sulfonated aluminum phthalocyanine and hypericin by magnetic CZ nano/micro-particles was investigated, as was the release of hypericin. Using an alternating magnetic field, local temperature increase by 10 °C in animal tissue with injected magnetic CZ particles was demonstrated. In addition, the CZ-based particles have been found to exhibit an anti-amyloidogenic effect on the amyloid aggregation of insulin and lysozyme in a dose- and temperature-dependent manner. Therefore, the mesoporous structure of CZ particles provided a unique platform for preparation of multifunctional magnetic and optical probes suitable for optical imaging, MRI, thermo- and phototherapy and as effective containers for controlled drug delivery. We concluded that magnetic CZ nano/micro-particles could be evaluated for further application in cancer hyperthermia therapy and as anti-amyloidogenic agents.

Complementary experimental and computational analysis of the effects of non-ionic detergents and phospholipids on insulin amyloid aggregation.

Amphiphilic compounds, both detergents and lipids, are important tools for in vitro analysis of water-soluble and integral membrane proteins. A key question is whether these two groups of amphiphilic molecules use the same pathway to affect structural and functional integrity of proteins. In the present study, we tested the effect of non-ionic detergent dodecyl maltoside (DDM), two phospholipids, 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), and the detergent-phospholipid mixtures on insulin amyloidogenesis in vitro. Amyloidogenesis of insulin is significantly affected by DDM in a time-and dose-dependent manner, but only slightly affected by either of phospholipids. Addition of DHPC or DMPC to detergent does not alter the inhibiting pattern, suggesting that DDM preferably binds to insulin. The molecular modeling revealed that DDM and the phospholipids occupy equivalent binding sites. DDM, due to the presence of maltose with several oxygen atoms (hydroxylic, glycosidic and ring) is involved in more hydrogen bonds than DHPC or DMPC. Hydrophobic interactions are important factors to stabilize both, DDM and phospholipids in their binding sites. Our results indicate that certain detergents (applying DDM as an example) and selected phospholipids are not always interchangeable in their use to investigate the effect of amphiphilic compounds on the behavior of amyloid-prone proteins.

The Interplay among Subunit Composition, Cardiolipin Content, and Aggregation State of Bovine Heart Cytochrome c Oxidase.

Mitochondrial cytochrome c oxidase (CcO) is a multisubunit integral membrane complex consisting of 13 dissimilar subunits, as well as three to four tightly bound molecules of cardiolipin (CL). The monomeric unit of CcO is able to form a dimer and participate in the formation of supercomplexes in the inner mitochondrial membrane. The structural and functional integrity of the enzyme is crucially dependent on the full subunit complement and the presence of unperturbed bound CL. A direct consequence of subunit loss, CL removal, or its oxidative modification is the destabilization of the quaternary structure, loss of the activity, and the inability to dimerize. Thus, the intimate interplay between individual components of the complex is imperative for regulation of the CcO aggregation state. While it appears that the aggregation state of CcO might affect its conformational stability, the functional role of the aggregation remains unclear as both monomeric and dimeric forms of CcO seem to be fully active. Here, we review the current status of our knowledge with regard to the role of dimerization in the function and stability of CcO and factors, such as subunit composition, amphiphilic environment represented by phospholipids/detergents, and posttranslational modifications that play a role in the regulation of the CcO aggregation state.

Fullerenes as an Effective Amyloid Fibrils Disaggregating Nanomaterial.

Nowadays, determining the disassembly mechanism of amyloids under nanomaterials action is a crucial issue for their successful future use in therapy of neurodegenerative and overall amyloid-related diseases. In this study, the antiamyloid disassembly activity of fullerenes C60 and C70 dispersed in 1-methyl-2-pyrrolidinone (NMP) toward amyloid fibrils preformed from lysozyme and insulin was investigated using a combination of different experimental techniques. Thioflavin T fluorescence assay and atomic force microscopy were applied for monitoring of disaggregation activity of fullerenes. It was demonstrated that both types of fullerene-based complexes are very effective in disassembling preformed fibrils, and characterized by the low apparent half-maximal disaggregation concentration (DC50) in the range of ∼22-30 µg mL-1. Small-angle neutron scattering was employed to monitor the different stages of the disassembly process with respect to the size and morphology of the aggregates. Based on the obtained results, a possible disassembly mechanism for amyloid fibrils interacting with fullerene/NMP complexes was proposed. The study is a principal step in understanding of the fullerenes destruction mechanism of the protein amyloids, as well as providing valuable information on how macromolecules can be engineered to disassemble unwanted amyloid aggregates by different mechanisms.

Inhibition of amyloid fibril formation and disassembly of pre-formed fibrils by natural polyphenol rottlerin.

Natural polyphenols, curcumin, rottlerin and EGCG were selected for initial computational modeling of protein-ligand interaction patterns. The docking calculations demonstrated that these polyphenols can easily adjust their conformational shape to fit well into the binding sites of amyloidogenic proteins. The experimental part of the study focused on the effect of rottlerin on fibrillation of three distinct amyloidogenic proteins, namely insulin, lysozyme and Aß1-40 peptide. Different experimental protocols such as fluorescence spectroscopy, circular dichroism and atomic force microscopy, demonstrated that amyloid fibril formation of any of the three proteins is inhibited by low micromolar rottlerin concentrations. Most likely, the inhibition of amyloid formation proceeded via interaction of rottlerin with amyloidogenic regions of the studied proteins. Moreover, rottlerin was also effective in pre-formed fibrils disassembly, suggesting that interactions of rottlerin with fibrils were capable to interrupt the fibril-stabilizing bonds of ß-sheets. The apparent IC50 and DC50 values were calculated in the range of 1.3-36.4 µM and 15.6-25.8 µM, respectively. The strongest inhibiting/disassembling effect of rottlerin was observed on Aß1-40 peptide. The cytotoxicity assay performed on the Neuro 2a cells indicated time-dependent cell morphology changes but rottlerin affected the cell viability only at concentration above 50 µM. The results of this study suggest that chemical modifications on rottlerin could be tested in the future as a promising strategy for the modulation of amyloidogenic proteins aggregation.

Dual effect of non-ionic detergent Triton X-100 on insulin amyloid formation.

Atomic force microscopy, Thioflavin T (ThT) fluorescence assay, circular dichroism spectroscopy, differential scanning calorimetry, and molecular modeling techniques have been employed to investigate the amyloid aggregation of insulin in the presence of non-ionic detergent, Triton X-100 (TX-100). In contrast to recently described inhibition of lysozyme amyloid formation by non-ionic detergents (Siposova, 2017), the amyloid aggregation of insulin in the presence of sub-micellar TX-100 concentration exhibits two dissimilar phases. The first, inhibition phase, is observed at the protein to detergent molar ratio of 1:0.1 to 1:1. During this phase, the insulin amyloid fibril formation is inhibited by TX-100 up to ∼60%. The second, "morphological" phase, is observed at increasing detergent concentration, corresponding to protein:detergent molar ratio of ∼1:1 - 1:10. Under these conditions a significant increase of the steady-state ThT fluorescence intensities and a dramatically changed morphology of the insulin fibrils were observed. Increasing of the detergent concentration above the CMC led to complete inhibition of amyloidogenesis. Analysis of the experimental and molecular modeling results suggests an existence of up to six TX-100 binding sites within dimer of insulin with different binding energy. The physiological relevance of the results is discussed.

Advances in the Study of Cerium Oxide Nanoparticles: New Insights into Antiamyloidogenic Activity.

There seems to be general agreement that oxidative stress is involved in many pathological conditions including Parkinson's, Alzheimer's, and other neurodegenerative diseases, and overall aging. Cerium oxide nanoparticles, also known as nanoceria (CeO2-NPs), have shown promise as catalytic antioxidants, based on their ability to switch between Ce3+ and Ce4+ valence states. In the present work we have synthesized and characterized CeO2-NPs, examined the effect of CeO2-NPs on amyloidogenesis of insulin, and analyzed the impact of CeO2-NPs on oxidative stress and biocompatibility in vitro in three types of invasive cancer cells, and in vivo in the preclinical model of the chorioallantoic membrane (CAM) of quail embryos. The different experimental techniques revealed a high stability and homogeneity of the "naked" CeO2-NPs synthesized by precipitation from a reversal microemulsion. The CeO2-NPs were 5-6 nm in diameter (TEM) and monodispersed and have a ζ +46.9 mV ζ potential in Milli-Q water. We demonstrated for the first time that CeO2-NPs affect insulin fibrillation in a dose-dependent manner. The inhibiting, IC50, and disassembling, DC50, concentrations were calculated to be ∼100 ± 3.5 and ∼200 ± 5.5 µg/mL, respectively. Furthermore, CeO2-NPs demonstrated reliable biocompatibility and sufficient uptake by glioma and breast cancer cells. The presence of a high concentration of CeO2-NPs within the cells resulted only in local changes in metabolic activity and generation of oxidative stress at a low level. Moreover, high biocompatibility with CeO2-NPs was shown in vivo in the CAM.

Inhibition of lysozyme amyloidogenesis by phospholipids. Focus on long-chain dimyristoylphosphocholine.

BACKGROUND: Protein amyloid aggregation is an important pathological feature of a group of different degenerative human diseases called amyloidosis. We tested effect of two phospholipids, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) on amyloid aggregation of hen egg white (HEW) lysozyme in vitro. METHODS: Effect of phospholipids was investigated using spectroscopic techniques (fluorescence and CD spectroscopy), atomic force microscopy and image analysis. RESULTS: Phospholipids DMPC and DHPC are able dose-dependently inhibit lysozyme fibril formation. The length of the phospholipid tails and different structural arrangement of the phospholipid molecules affect inhibitory activity; long-chain DMPC inhibits fibrillization more efficiently. Interestingly, interference of DMPC with lysozyme amyloid fibrils has no effect on their morphology or amount. CONCLUSIONS: Phospholipid molecules have significant effect on lysozyme amyloid fibrillization. We suggest that inhibitory activity is due to the interference of phospholipids with lysozyme leading to the blocking of the intermolecular protein interactions important for formation of the cross-ß structure within the core of the fibrils. The higher inhibitory activity of DMPC is probably due to adsorption of protein molecules on the liposome surfaces which caused decrease of species needed for fibrillization. Interaction of the phospholipids with formed fibrils is not sufficient enough to interrupt the bonds in ß-sheets which are required for destroying of amyloid fibrils. GENERAL SIGNIFICANCE: The obtained results contribute to a better understanding of the effect of phospholipids on amyloid fibrillization of the lysozyme. The data suggest that DMPC and DHPC phospholipids represent agents able to modulate lysozyme amyloid aggregation.

Subunit analysis of mitochondrial cytochrome c oxidase and cytochrome bc1 by reversed-phase high-performance liquid chromatography.

A rapid separation of the ten nuclearly-encoded subunits of mitochondrial cytochrome c oxidase, and ten out of the eleven subunits of cytochrome bc1, was achieved using a short, 50 mm C18-reversed-phase column. The short column decreased the elution time 4-7 fold while maintaining the same resolution quality. Elution was similar to a previously published protocol, i.e., a water/acetonitrile elution gradient containing trifluoroacetic acid. Isolated subunits were identified by MALDI-TOF. The rapidity of the described method makes it extremely useful for determining the subunit composition of isolated mitochondrial complexes. The method can be used for both analytical and micro-preparative purposes.

Interaction of nonionic detergents with the specific sites of lysozyme amyloidogenic region - inhibition of amyloid fibrillization.

Two nonionic detergents, Triton X-100 (TX-100) and n-dodecyl-ß-d-maltoside (DDM) were tested for their ability to affect lysozyme amyloid aggregation. We have demonstrated that fibrillization of lysozyme is completely inhibited by low sub-micellar concentrations of both of these detergents. The apparent IC50 values were calculated to be 22µM and 26µM for TX-100 and DDM, respectively. The detergent/protein ratio is not the only parameter controlling inhibition. The precise timing of the detergent addition was found to be also crucial. It appears that the primary inhibitory activity of detergents resulted from inhibition of nuclei formation, in addition to inhibition of fibril polymerization at the early stage of protofibrils growth. The docking study revealed that Asn-59, Trp-63 and Ala-107, all present within the lysozyme amyloidogenic region, were involved in the interaction with both detergents. In addition, TX-100 also interacted with Gln-57 and Asp-103 within lysozyme. Moreover, based on our computational results, TX-100 bridges the Gln-57 and Ala-107 amino acids of the amyloidogenic segment of lysozyme and therefore inhibits more effectively the amyloid fibril formation. Along these lines, the knowledge gained from our study indicates that the detergents or their derivatives may be applicable as a promising strategy for the modulation of lysozyme protein aggregation.

Functional and structural evaluation of bovine heart cytochrome c oxidase incorporated into bicelles.

Bilayered long- and short-chain phospholipid assemblies, known as bicelles, have been widely used as model membranes in biological studies. However, to date, there has been no demonstration of structural or functional viability for the fundamental mitochondrial electron transport complexes reconstituted into or interacting with bicelles. In the present work, bicelles were formed from the mixture of long- and short-chain phospholipids, specifically 14:0 and 6:0 phosphatidylcholines (1,2-dimyristoyl-sn-glycero-3-phosphocholine, (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine, (DHPC)). Isolated from bovine heart, cytochrome c oxidase was successfully incorporated into bicelles. Bicelles and cytochrome c oxidase incorporated into bicelles ("proteobicelles") were characterized by absorption spectroscopy, dynamic light scattering, atomic force microscopy, sedimentation velocity and differential scanning calorimetry. It was demonstrated that at total concentration of phospholipids CL = 24 mM and the molar ratio (q) of long-chain DMPC over short-chain DHPC equal to 0.4, the diameter of bicelles formed at neutral pH is in the range of 30-60 nm with the thickness of bicelles of about 4 nm. Adding cytochrome c oxidase to bicelles unified the size of the resulting proteobicelles to about 160 nm. Cytochrome c oxidase in bicelles was fully reducible by artificial donors of electrons, exhibited "normal" reaction with external ligands, and was fully active. Both, sedimentation velocity analysis and temperature-induced denaturation indicated that enzyme in bicelles is monomeric. We concluded that cytochrome c oxidase in bicelles maintains its structural and functional integrity, and that bicelles can be used for more comprehensive investigation of cytochrome c oxidase and most likely other mitochondrial electron transfer complexes.