Наукові роботи. Факультет радіофізики, біомедичної електроніки та комп’ютерних систем
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Документ Chlorpromazine effect on lysozyme-lipid interactions(Харьковский Национальный Университет им. В.Н. Каразина, 2008) Trusova, V.M.Despite considerable research efforts, the molecular mechanisms of anaesthetic action still remain the matter of extensive debates. According to one viewpoint, anaesthetics alter the properties of lipid bilayer which, in turn, affects the functions of embedded membrane proteins. In contrast, protein-based theories of anaesthetic action postulate that the drugs modulate the functions of membrane proteins through direct association. To develop a unique conception of anaesthesia further in-depth investigations of drug-membrane interactions are strongly required. In the present work a well-known fluorescent probe pyrene has been employed to gain molecular insights into the interactions between amphipathic phenothiazine derivative chlorpromazine (CPZ) and model membranes composed of cationic globular protein lysozyme (Lz), and lipid vesicles prepared from zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin (CL) in the molar ratios 19:1, 9:1 and 4:1. To give unambiguous interpretation of the drug effect on protein-lipid interactions, we first analyzed the changes in pyrene excimerization due to the formation of either CPZ-lipid or Lz-lipid complexes. Pyrene excimer-to-monomer intensity ratio (E/M), a parameter which reflects the alterations in membrane free volume, was found to decrease upon Lz or CPZ binding to the lipid vesicles. Apparently, embedment of the protein and drug molecules into the hydrophobic region of lipid bilayer gives rise to the increase in lipid packing, decrease in the rate of trans-gauche isomerization of the lipid acyl chains and, consequently, reduction of membrane free volume. At the next step of the study, we analysed the changes in the rate of pyrene excimerization upon Lz addition to drug-lipid mixtures. In CL-containing liposomes the presence of CPZ does not modify the magnitude and sign of protein effect on membrane free volume. This implies that CPZ is incapable of perturbing Lz structure and exerted no influence on the protein interactions with this kind of liposomes. In contrast, in PC vesicles E/M ratio appeared to increase upon lysozyme binding to CPZ-modified model membranes. This finding may be explained in terms of two possibilities: (i) CPZ induces the formation of the new Lz conformer whose interactions with lipid bilayers are accompanied by the increase in membrane free volume; (ii) CPZ imparts the positive charge to the lipid bilayer thereby preventing Lz penetration into hydrophobic membrane region. Interfacially-located protein molecules are likely to generate structural defects coupled with the increased bilayer free volume. The results presented here clearly demonstrate that membrane composition can modulate the drug action on lipid-protein interactions. The recovered difference between CPZ effect on Lz-lipid binding in PC and CL-containing bilayers provide support to the idea that membrane environment can stabilize certain protein conformations differing in their responsiveness to drug action.Документ Hemoglobin binding to phospholipid membranes as revealed by pyrene fluorescence study(Харьковский Национальный Университет им. В.Н.Каразина, 2011) Kutsenko, O.K.; Gorbenko, G.P.; Trusova, V.M.In this work hemoglobin (Hb) association with lipid bilayers was investigated using fluorescent probe pyrene. Model membranes were prepared from zwitterionic lipid phosphatidylcholine (PC), anionic lipid phosphatidylglycerol (PG) and cholesterol (Chol). Hb-lipid binding was followed by the pyrene fluorescence quenching. Hb-induced decrease of pyrene monomer fluorescence was followed by the increase of relative intensities of vibronic bands. Presumably, Hb penetration into the bilayer increases the space between neighbouring lipids and promotes water penetration into the membrane core. Pyrene excimer emission quenching was interpreted in terms of resonance energy transfer. The greatest depth of Hb penetration into the lipid bilayer was observed in PC vesicles. In Chol-containing liposomes sterol condensing effect prevents deep protein penetration into the membrane. PG has an ability to stabilize lipid bilayers due to the ordered state of its lipid tails and H-bonding interactions between lipid molecules. This also can prevent Hb access to the inner membrane regions.Документ Influence of oligomeric and fibrillar lysozyme on physical properties of model membranes(Харьковский Национальный Университет им. В.Н.Каразина, 2011) Kastorna, A.P.; Trusova, V.M.; Gorbenko, G.P.A pathological hallmark of more than 20 human diseases including Alzheimer’s disease, Parkinson’s disease, type II diabetes is the deposition in organs and tissues of insoluble highly ordered protein aggregates, called amyloid fibrils. It is becoming widely recognized that toxicity of amyloid species is related to their interactions with cell membranes. In the present study we focused our efforts on the examination of the influence of amyloid fibrils and their precursors (oligomeric aggregates) of lysozyme on the structural and physical properties of the model membranes composed of phosphatidylcholine and its mixture with cholesterol. For evaluating the extent of lipid bilayer modifications, we used fluorescence spectroscopy technique. The results of pyrene excimerization measurements showed that amyloid protein reduces membrane fluidity. Analysis of Laurdan emission spectra revealed the ability of lysozyme aggregates to produce bilayer dehydration. The most pronounced membrane-modifying effects were observed in the case of oligomeric lysozyme. Significantly less influence of pathogenic protein aggregates on the physical properties of cholesterol-containing vesicles confirmed the hypothesis on the preventive role of cholesterol in amyloid-related diseases.Документ Lipid bilayer modification induced by fibrillar lysozyme: fluorescence spectroscopy study(Харьковский Национальный Университет им. В.Н.Каразина, 2010) Kastorna, A.P.; Trusova, V.M.; Gorbenko, G.P.The correlation between neurodegenerative diseases (Parkinson’s, Alzheimer’s diseases), type II diabetes, systemic amyloidosis, etc. and deposition of protein aggregates in brain and other tissues has long been established. A growing body of evidence has demonstrated that binding of amyloid proteins to the membrane may underlie their cytotoxic effect. It was shown that amyloid toxicity arises primarily from a soluble oligomeric form (pre-fibrillar aggregates) of the peptide rather than amyloid monomers or mature fibrils. The molecular basis of the amyloid protein toxicity is not sufficiently clear and requires further investigation. In view of this, the present study has been undertaken to ascertain the effect of fibrillar aggregates of lysozyme on the structural and physical properties of model membranes (liposomes) composed of zwitterionic lipid phosphatidylcholine and its mixture with cholesterol (30 mol%). To this end, two fluorescent probes with different properties and bilayer location, pyrene and Laurdan, have been employed. Pyrene spectra have characteristic vibronic structure in the region of 370-400 nm. Relative intensities of vibronic transitions exhibit dependence on solvent polarity. Excited species of pyrene can interact with non-excited ones thus forming excited state dimers – excimers. Excimer-to-monomer fluorescence intensity ratio reflects the extent of pyrene excimerization, which depends mainly on the rate of monomer lateral diffusion in lipid bilayer, being a function of the density of lipid molecular packing. Analysis of pyrene emission spectra revealed the absence of any influence of fibrillar lysozyme on the structural state of bilayer acyl chain region. Laurdan is an amphiphilic fluorescent probe, whose emission spectra are sensitive to the environmental polarity (hydration level). In the solvents of high polarity, Laurdan shows a considerable shift of its emission spectrum to longer wavelengths due to the dipolar relaxation processes. The changes in the emission spectrum of Laurdan were characterized by the generalized polarization value (GP). In all types of liposomes increasing concentration of fibrillar lysozyme resulted in the increment of GP, suggesting that amyloid fibrils cause the decrease in the lipid bilayer polarity.