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Документ Intrinsic fluorescence of lysozyme in model protein-lipid systems(Харьковский Национальный Университет им. В.Н. Каразина, 2009) Trusova, V.M.Fluorescence spectroscopy is one of the most powerful tools providing new insights into the structural, dynamic and functional behavior of biological macromolecules, being particularly useful in investigating the molecular details of protein-lipid association. Complete and accurate information about the conformational dynamics of protein molecules can be obtained using tryptophan (Trp) residues as intrinsic fluorescence probes. The fluorescence of indole chromophore is extremely sensitive to environment making it an ideal choice for reporting protein conformational transitions upon membrane interactions. Hen egg white lysozyme (Lz) is a multi-tryptophan protein which is extensively used in elucidating fundamental aspects of protein-lipid interactions. The main emitters responsible for 80% of lysozyme fluorescence are Trp62 and Trp108. The present study was undertaken to ascertain the alterations in lysozyme structural state upon association with model membranes composed of zwitterionic lipid 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) and anionic lipid 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphoglycerol (POPG). Fluorescence lifetime measurements showed that intensity-averaged lifetime (<τ>) of Trp residues in lysozyme decreased upon the protein binding to model membranes. Furthermore, <τ> reduction from 1.94 to 1.74 ns was observed at decreasing lipid-to-protein molar ratio (L:P) from 1130 to 120. It was suggested that Trp specific interactions with certain amino acid residues in its surroundings is the main factor responsible for the recovered decrease in tryptophan lifetime and the observed contradictions between lifetime, quenching and steady-state experiments. Since Lz is a stable protein whose conformation is reported to change insignificantly upon the formation of protein-lipid contacts, it can be assumed that the processes behind the drop in <τ> involve Lz self-association in membrane-bound state. Trp62 and Trp108 are located in the protein active site which reportedly participates in Lz aggregation. Moreover, Cys76-Cys94 disulfide bridge capable of efficient quenching of Trp fluorescence and reducing the lifetime of protein fluorophores, also resides in the active site cleft. Thus, it may be supposed that interactions between Trp62 and Trp108 of one Lz monomeric molecule with disulfide bridge of another monomeric molecule during the protein aggregation result in reduction of <τ> values. The <τ> dependence on L:P can be explained by the fact that lysozyme self-association is apparently coverage-dependent process controlled by both electrostatic and hydrophobic protein-lipid interactions. Additional arguments in favor of the assumption on Lz aggregation come from the time- resolved anisotropy measurements. Lysozyme rotational correlation time which reflects the motion of the whole protein molecule, was found to exhibit twofold increase at increasing L:P values. The recovered membrane ability to modulate Lz aggregation behavior may largely determine the bactericidal and amyloidogenic propensities of this protein.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 Interaction of novel benzanthrone derivative with amyloid lysozyme(Харьковский Национальный Университет им. В.Н.Каразина, 2011) Vus, K.O.; Trusova, V.M.; Gorbenko, G.P.; Zhytniakivska, O.A.; Kirilova, E.; Kirilov, G.; Kalnina, I.A novel benzanthrone derivative AM18 was investigated with respect to its photophysical properties when bound to native, oligomeric and fibrillar hen egg white lysozyme. As shown by fluorimetric titration AM18 is more sensitive to pathogenic protein aggregates than Thioflavin T, however has no ability to differentiate between mature and immature lysozyme fibrils. The recovered affinity and fluorescence response of the novel probe to amyloid protein appeared to be similar to those of recently developed amyloid lysozyme-sensitive dyes like e. g. Nile Red and cyanine dye 7515. Despite the high increase of the probe emission in the presence of amyloid lysozyme compared to its fluorescence in buffer, the minimal amount that could be detected by 1 μM AM18 was 10 times lower for amyloid-native protein solutions due to high affinity of the dye for lysozyme monomers. In general, because of high quantum yields and “signal-to-noise” ratios in the presence of pathogenic protein aggregates AM18 appeared to be an effective tool for amyloid detection and characterization in vitro, being however unable to detect pathogenic protein aggregates in vivo like e.g. recently reported p-FTAA because of the sensitivity to lipids. Compared to previously reported AM3 a novel dye showed 2-fold lower “signal-to- noise” ratio in the presence of fibrillar lysozyme, and 2 fold lower blue shift of emission maximum. This tendency was explained in terms of decreased charge transfer from the donor to acceptor groupes of AM18 compared to AM3. Finally, as concluded from the comparison of AM18 and previously studied benzanthrone derivatives, the 5 nm – red edge excitation shift of AM18 is indicative of its possible binding to fibril “deep cavities”, containing no water. High anisotropy values of amyloid-bound dye led us to conclusion that the enhanced fluorescence of the probe is associated with the decrease of the rotational motion of the amino-substitute about the benzanthrone unit. This is a sign of AM18 behaviour as a molecular rotor.Документ Spectral behavior of novel benzanthrone probe in model membranes(Харьковский Национальный Университет им. В.Н.Каразина, 2011) Zhytniakivska, O.A.; Kutsenko, O.K.; Trusova, V.M.; Gorbenko, G.P.; Kirilova, E.M.; Kirilov, G.K.; Kalnina, I.The present study was undertaken to evaluate the sensitivity of a newly synthesized benzanthrone dye to the changes in physicochemical properties of lipid bilayer. It was shown that the dye under study is non- emissive in buffer but exhibites strong fluorescence in lipid phase. Partitioning of AM15 into model membranes composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin and cholesterol was followed by significant increase of fluorescence quantum yield. Analysis of the partition coefficients showed that inclusion of cardiolipin and choleterol into phosphatidylcholine bilayer gives rise to the decrease of AM15 incorporation into lipid phase compared to the neat phosphatidylcholine membrane. It is assumed that AM15 resides in the hydrophobic bilater region, being oriented parallel to the lipid acyl chains.Документ Quantitative analysis of the benzanthrone aminoderivative binding to amyloid fibrils of lysozyme(Харьковский Национальный Университет им. В.Н.Каразина, 2010) Vus, K.O.; Trusova, V.M.; Gorbenko, G.P.; Kirilova, E.; Kirilov, G.; Kalnina, I.The accumulation of amyloid fibrils in different tissues is associated with a number of neurodegenerative diseases. Despite a huge variety of amyloid-specific probes, all of them suffer from many drawbacks, highlighting the necessity of searching for more preferable dyes. In the present work, the potential of new fluorescent probe AM3 for selective detection of fibrillar protein aggregates, formed from lysozyme, has been evaluated. To quantify the affinity of this dye for amyloid fibrils, the isotherms of dye binding to the fibrillar lysozyme have been derived from fluorimetric titration. Parameters of the dye-protein complexation: association constant, molar fluorescence and binding stoichiometry, calculated from the Langmuir adsorption model, revealed that AM3 interacts strongly with protein insoluble aggregates. High values of the binding parameters make AM3 an alternative to a widely-used amyloid-specific probe Thioflavin T. We also investigated the effects of polarity and viscosity on AM3 fluorescence properties. The binding of AM3 to the protein hydrophobic cavities has been followed by red shift of the dye emission spectra, which can be explained by H-bonding between proton-donating groups of the protein and carbonyl moiety of the probe. Long-wavelength shift of emission maximum was observed also upon increasing the excitation wavelength. This finding suggests that reorientation time of solvent molecules is higher, than the dye fluorescence lifetime. Fluorescence anisotropy studies revealed slow rotation diffusion of the probe, bound to amyloid fibrils being indicative of high viscosity of AM3 microenvironment. The observed photophysical properties of the new aminobenzanthrone derivative make AM3 a perspective probe for basic research and medical diagnostics.Документ Lipid-mediated lysozyme aggregation: furster resonance energy transfer study(Харьковский Национальный Университет им. В.Н.Каразина, 2010) Trusova, V.M.Aggregation of proteins into insoluble complexes is intimately linked to pathogenesis of several neurodegenerative diseases. Protein aggregation is commonly regarded as nonspecific coagulation of incompletely folded or partially denaturated polypeptides, driven by interaction between the exposed hydrophobic patches. Accumulating evidence indicates that protein self-association can be induced by protein-lipid interactions. The present study addresses a problem of aggregation behavior of lysozyme (Lz) bound to model membranes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) (8:2). The formation of Lz assemblies in lipid environment has been monitored by measuring steady-state resonance energy transfer (FRET). Several donor-acceptor pairs have been employed: tryptophan (Trp) – pyrene, pyrene – fluorescein 5’-isothiocyanate (FITC) and FITC – rhodamine-isothiocyanate (RITC). Fluorescence spectra of pyrene maleimide-labelled Lz (Lz-PM) recorded at different lipid-to-protein molar ratios (L:P) with excitation wavelength of 296 nm were featured by three bands corresponding to Trp, pyrene monomer and excimer emission. Analysis of the shape of emission spectra showed that the ratio of PM to Trp intensity rises with increasing Lz-PM concentration and decreasing L:P values from 379 to 77. This effect is most probably to arise from the enhanced FRET between Trp and pyrene. The finding that the magnitude of this effect depends on protein concentration suggests that FRET enhancement is caused by the formation of protein aggregates. The same result was obtained with Lz- attached pyrene as donor and Lz-attached FITC as acceptor – the efficiency of energy transfer increased with increasing total protein concentration and decreasing L:P. Notably, the most pronounced increase of energy transfer efficiency was observed at surface coverage ca. 38 lipids per one protein molecule suggesting that this L:P value is critical for formation of Lz self-associates. The assumption that Lz forms aggregates in membrane environment is also corroborated by the quantitative analysis of FRET between FITC and RITC. The distance between FITC and RITC was found to be ca. 8 nm which exceeds the dimensions of Lz molecule by 2-2.5 times, lending additional support to the idea about Lz self- association in lipid surroundings.