Dissolved organic matter in water of Daugava river

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Efficiency of energy transfer,%Efficiency of energy transfer,%Interaction of Fibrillar Insulin and Globin with Liposomes: ResonanceEnergy Transfer StudyM.Romanova *1 , I.Maliyov 1 , A.Kastorna 1 , K.Vus 1 , V.Trusova 1 , J.Molotkovsky 2 , G.Gorbenko 11 V.N. Karazin Kharkov National University, Kharkov, Ukraine;2 Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russiae-mail: romanova-mari@mail.ruA wide range of proteins are capable of assembling into cross β-sheet structures (amyloidfibrils) under pathological or nonphysiological conditions. The toxicity of amyloid fibrilsand their precursors is supposed to be associated with their action on structural andfunctional state of biological membranes. One way of approaching this problem is basedon the use of model systems consisting of lipid vesicles and protein aggregates formed invitro.The present study was directed towards clarifying the molecular details of interactionbetween globin or insulin fibrils and liposomes of different composition. Fibrillaraggregates were obtained by protein incubation in glycine buffer (pH 2.2) at 60 o C during8 days. To assess the extent of fibril binding to liposomes, we measured energy transferefficiency from tryptophan residues of globin or insulin to anthrylvinyl (AV) fluorophorecovalently attached to phosphatidylcholine (PC). Lipid vesicles were prepared from PCand its mixtures with anionic lipid phosphatidylserine (PS) (10, 20, 40 mol% PS).Increasing the concentration of globin fibrils resulted in the raise of AV fluorescenceintensity for all model systems under study (Fig.1). It was found that the most effectiveenergy transfer occurs when globin interacts with neutral PC vesicles. In the case ofinsulin, conversely, the energy transfer from Trp to AV-PC was negligible. Thisobservation can be explained by the fact that insulin has negative charge under theemployed experimental conditions (pH 7.4), which prevents protein binding to negativelycharged PC/PS liposomes.4.03.5PCPS10PS20PS404,03,53.03,02.52,52.02,01.51.00.50.00 2 4 6 8 10 12Concentration of Globin, M1,51,00,50,0InsulinGlobinFig.1. Efficiency of energy transfer as afunction of fibrillar globin concentrationFig.2. Comparison of energy transfer fromTrp to AV-PC for insulin and globinFig.2 illustrates the difference between fibrillar insulin and globin in the energy transferefficiency from Trp to AV-PC in PC bilayers. The results obtained allowed us to supposethat the character of interaction between globin fibrils and liposomes is mostlyhydrophobic, while in the case of insulin the role of electrostatics may be predominant.This work was supported by the grant from Fundamental Research State Fund of Ukraine(project number F.41.4/014).- 30 -
Fluorescence Approaches to Detection of Protein AggregatesV.M.TrusovaV.N. Karazin Kharkov National University, Kharkov, Ukrainevaltrusova@yahoo.comFluorescence spectroscopy is one of the most powerful tools for characterization of amultitude of biological processes 1 . Of these, the phenomenon of protein oligomerizationattracts especial interest due to its crucial role in the formation of fibrillar proteinaggregates (amyloid fibrils) involved in ethiology of so-called protein misfoldingdiseases. It is becoming increasingly substantiated that protein fibrillization in vivo can beinitiated and modulated at membrane-water interface 2 . All steps of membrane-assistedfibrillogenesis, viz., protein adsorption onto lipid bilayer, structural transition ofpolypeptide chain into a highly aggregation-prone partially folded conformation, assemblyof oligomeric nucleus from membrane-bound monomeric species and fiber elongation canbe monitored with a mighty family of fluorescence-based techniques 3-5 . Little or nodamage to the examined matter, requirement of material micromolar concentration, rathersimple methodology, involvement of relatively inexpensive instrumentation, highsensitivity and specificity make fluorometry a broadly used research tool for studying theprotein-protein interactions. Various kinds of fluorescence technique, namely steady-stateand time-resolved fluorescence, fluorescence polarization and fluctuation spectroscopy,stopped-flow and laser-induced fluorescence provide the information about the structure,microenvironment and distribution of protein complexes 1 . The immense range ofparameters measured for both intrinsic and extrinsic protein fluorophores includesfluorescence intensity, quantum yield, anisotropy and quenching, lifetime, Försterresonance energy transfer efficiency (FRET) efficiency, diffusion coefficients, to namejust a few. In the present contribution the applications of fluorescence spectroscopy tomonitoring protein oligomerization in a membrane environment are exemplified and someproblems encountered in such kinds of studies are highlighted. More specifically, suchaspects as fluorescence probing of protein-membrane binding, fluorescence monitoring ofprotein structural changes, detection of protein aggregates with intrinsic and extrinsicfluorophores are discussed. In addition, optimal strategies for fluorescence analysis ofprotein aggregation are suggested.References:1. Lakowicz, J.R. Principles of Fluorescence Spectroscopy. NY: Springer, 2006.2. Gorbenko, G.P., Kinnunen, P.K.J. The Role of Protein-Lipid Interactions in Amyloid-Type Proteinfibril Formation. Chem. Phys. Lipids, 2006, vol. 141, p. 72-82.3. Zerovnik, E. Amyloid-fibril Formation. Proposed Mechanisms and Relevance to ConformationalDisease, Eur. J. Biochem., 2002, vol. 269, p. 3362-3371.4. Domanov, Ye.A., Kinnunen, P.K.J. Islet Amyloid Polypeptide Forms Rigid Lipid–Protein AmyloidFibrils on Supported Phospholipid Bilayers. J. Mol. Biol., 2008, vol. 376, p. 42-54.5. Trusova, V.M., Gorbenko, G.P., Sarkar, P. et al. Forster Resonance Energy Transfer Evidence forLysozyme Oligomerization in Lipid Environment. J. Phys. Chem. B, 2010, vol. 114, p. 16773-16782.- 31 -
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Page 34 and 35: ABinding of the Novel 4-tricyanovin
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Dissolved organic matter in water of Daugava river

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