Bimodal Distribution and Fluorescence Response of Environment-Sensitive Probes in Lipid Bilayers (original) (raw)
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Excited State Proton Transfer and Solvent Relaxation of a 3-Hydroxyflavone Probe in Lipid Bilayers
The Journal of Physical Chemistry B, 2008
The photophysics of a ratiometric fluorescent probe, N-[[4′-N,N-diethylamino-3-hydroxy-6-flavonyl]methyl]-N-methyl-N-(3-sulfopropyl)-1-dodecanaminium, inner salt (F2N12S), incorporated into phospholipid unilamellar vesicles is presented. The reconstructed time-resolved emission spectra (TRES) unravels a unique feature in the photophysics of this probe. TRES exhibit signatures of both an excited-state intramolecular proton transfer (ESIPT) and a dynamic Stokes shift associated with solvent relaxation in the lipid bilayer. The ESIPT is fast, being characterized by a risetime of ∼30-40 ps that provides an equilibrium to be established between the excited normal (N*) and the ESIPT tautomer (T*) on a time scale of 100 ps. On the other hand, the solvent relaxation displays a bimodal decay kinetics with an average relaxation time of ∼1 ns. The observed slow solvent relaxation dynamics likely embodies a response of nonspecific dipolar solvation coupled with formation of probe-water H-bonds as well as the relocation of the fluorophore in the lipid bilayer. Taking into account that ESIPT and solvent relaxation are governed by different physicochemical properties of the probe microenvironment, the present study provides a physical background for the multiparametric sensing of lipid bilayers using ESIPT based probes.
Journal of Fluorescence, 2003
Time-resolved fluorescence of eight fluorescence probes were studied in EggPC bilayer membrane vesicles. Emission wavelength dependent fluorescence decays were analyzed in a model-independent way to obtain time resolved area normalized emission spectra (TRANES). The TRANES spectra of the probes studied were classified into four types: (1) spectra that are identical at all time (one emissive species), (2) spectra that show an isoemissive point (two emissive species), (3) spectra that shift continuously with time (slow solvation dynamics or multiple species), and (4) spectra that shift for a short time and thereafter one or two emissive species are indicated. The TRANES spectra of these eight probes, except RH421, belong to the type 1, 2, or 4. The continuous shift of the TRANES spectra that was observed for the probe RH421 is attributed to multiple ground state species and not due to slow solvation dynamics.
FEBS Letters, 2001
We report on dramatic differences in fluorescence spectra of 4P P-dimethylamino-3-hydroxyflavone (probe F) studied in phospholipid membranes of different charge (phosphatidyl glycerol, phosphatidylcholine (PC), their mixture and the mixture of PC with a cationic lipid). The effect consists in variations of relative intensities at two well-separated band maxima at 520 and 570 nm belonging to normal (N*) and tautomer (T*) excited states of flavone chromophore. Based on these studies we propose a new approach to measure electrostatic potential at the surface layer of phospholipid membranes, which is based on potential-dependent changes of bilayer hydration and involves very sensitive and convenient ratiometric measurements in fluorescence emission. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
Chemistry & Biology, 2002
Gebze-Kocaeli 41470 applied probes for fluorescent labeling. In addition to high chemical and photochemical stability and high fluo-Turkey 2 Department of Chemistry rescence quantum yield, they should provide strong change of color in response to different membrane per-Kyiv National Shevchenko University 01033 Kiev turbations. In this sense the common polarity-sensitive [5] and electrochromic [6] dyes have very limited capa-3 A.V. Palladin Institute of Biochemistry 9 Leontovicha Street bilities, as these probes commonly provide the response by shifting one broad band that is present in emission, 01030 Kiev Ukraine and the magnitude of the shift is usually smaller than the bandwidth. In order to be sensitive to the two-band 4 Laboratoire de Pharmacologie et Physicochimie des Interactions Cellulaires et Molé culaires ratiometric probe, the dye is required to exhibit an excited-state reaction: isomerization, electronic charge UMR 7034 du CNRS Faculté de Pharmacie transfer, or proton transfer [7]
Biophysical Journal, 1994
The effect of cholesterol on the gel, the liquid-crystalline, and mixed phospholipid phases has been studied using the fluorescence properties of 2-dimethylamino-6-lauroyinaphthalene (Laurdan). Laurdan sensitivity to the polarity and to the dynamics of its environment reveals that cholesterol affects phospholipid bilayers in the gel phase by expelling water and by increasing the amount of dipolar relaxation. In the liquid-crystalline phase, the effect of cholesterol is of a reduction of both water concentration and amount of dipolar relaxation. Detailed studies of Laurdan excitation and emission spectral contours as a function of cholesterol concentration show that there are some cholesterol concentrations at which Laurdan spectral properties changes discontinuously. These peculiar cholesterol concentrations are in agreement with recent observations of other workers showing the formation of local order in the liquid-crystalline phase of phospholipids upon addition of phospholipid derivatives of pyrene. A local organization of phospholipids around cholesterol molecule seems to be produced by the presence of peculiar concentrations of cholesterol itself. This local organization is stable enough to be observed during the excited state lifetime of Laurdan of approximately 5-6 ns.
Perturbations to lipid bilayers by spectroscopic probes as determined by dielectric measurements
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1980
Dielectric measurements on lecithin/cholesterol bimolecular lipid membranes have indicated that the series of extrinsic fluorescent probe molecules, the n-(9anthroyloxy) fatty acids, cause significant perturbation to the bilayer structure at concentrations equivalent to those used in fluorescence experiments (0.1 mol%). Perturbations were observed in the capacitance and conductance of the electrically distinct substructural regions of the bilayer that were consistent with the putative location of the probe molecules. Inclusion of stearic acid decreased the thickness of the hydrocarbon region of the membrane, presumably by expanding the average surface area per unit membrane mass, and also significantly disrupted the surface regions. The attachment of the anthroyloxy moiety to position 2 of a fatty acid accentuated both these effects. Attachment at position 12 had the reverse effect by increasing the volume of the hydrocarbon region without further disturbance of the surface organisation. The 9-positioned probe had an intermediate effect. The degree of perturbation by the 2-positioned probe was dependent on the probe concentration within the range (probe:lipid) 1:1000 to 1:10000. The technique therefore detects perturbation of structure at probe levels which are lower than those commonly used in fluorescence-labelling experiments.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2004
The penetration of water into the hydrophobic interior leads to polarity and hydration profiles across lipid membranes which are fundamental in the maintenance of membrane architecture as well as in transport and insertion processes into the membrane. The present paper is an original attempt to evaluate simultaneously polarity and hydration properties of lipid bilayers by a fluorescence approach. We applied two 3-hydroxyflavone probes anchored in lipid bilayers at a relatively precise depth through their attached ammonium groups. They are present in two forms: either in H-bond-free form displaying a two-band emission due to an excited state intramolecular proton transfer reaction (ESIPT), or in H-bonded form displaying a single-band emission with no ESIPT. The individual emission profiles of these forms were obtained by deconvolution of the probes' fluorescence spectra. The polarity of the probe surrounding the bilayer was estimated from the two-band spectra of the H-bond-free form, while the local hydration was estimated from the relative contribution of the two forms. Our results confirm that by increasing the lipid order (phase transition from fluid to gel phase, addition of cholesterol or decrease in the lipid unsaturation), the polarity and to a lesser extent, the hydration of the bilayers decrease simultaneously. In contrast, when fluidity (i.e. lipid order) is kept invariant, increase of temperature and of bilayer curvature leads to a higher bilayer hydration with no effect on the polarity. Furthermore, no correlation was found between dipole potential and the hydration of the bilayers.
Flavonols - new fluorescent membrane probes for studying the interdigitation of lipid bilayers
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1998
Two flavonols, 3-hydroxy-4'-dimethylaminoflavone (FME) and 3-hydroxy-4'-(15-azacrown-5) flavone (FRC) have been investigated as new fluorescence probes for studying the formation of the interdigitated gel phase in lipid bilayers. The formation of the interdigitated gel phase in the saturated symmetrical phosphatidylcholines (PCs) and phosphatidylethanol (Peth) in the presence of ethanol has been well studied. The present study examines the behavior of these new probes in PC-ethanol and Peth-ethanol systems, as well as in PC-cholesterol and Peth-cholesterol vesicles. The present results demonstrate that both flavonols give distinctively different spectra in interdigitated lipids compared to non-interdigitated lipids, when examined in lipids in which the interdigitation behavior is known. This makes them useful for determinations of the structural state of unknown lipids, and for following the transitions between interdigitated and non-interdigitated phases. However, in the presence of cholesterol, only FCR gave appropriate indications of interdigitation. The results with FME in the presence of cholesterol were not consistent with the known behavior of the lipids examined; instead, FME appears to be located preferentially in the cholesterol-rich non-interdigitated regions of the bilayer.
Determination of the fluorescence labels location in lipid bilayer based on fluorescence quenching
Journal of Molecular Liquids, 2018
A comparison of Förster Resonance Energy Transfer (FRET) data of vesicles labeled with fluorescence labels on both inner and outer layers of the bilayer, with ones labeled only on the inner layer of bilayer to permit a determination of the location of fluorescence labels in relation to the bilayer center. The theoretical description of the FRET effect is based on the FRET model generalized and extended by Wolber and Hudson. A useful version of the FRET model implies location of acceptors with respect to a trigonal or tetragonal lattice of acceptors centered on donors located deeper than the acceptor layer. Several fluorescence labels were tested in order to find FRET pairs of labels for which the same quantity of quencher reduced label fluorescence to one half of the initial value before quencher treatment. 2,2,6,6tetramethylpiperidine-1-oxyl (TEMPO) was used as a fluorescence quencher of both labels located in outer layer of bilayer.