TRANES Spectra of Fluorescence Probes in Lipid Bilayer Membranes: An Assessment of Population Heterogeneity and Dynamics (original) (raw)
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Time-Resolved Fluorescence in Lipid Bilayers: Selected Applications and Advantages over Steady-State
2014
Fluorescence methods are versatile tools for obtaining dynamic and topological information about biomembranes as the molecular interactions taking place in lipid membranes frequently occur on the same time scale as fluorescence emission. The fluorescence intensity decay, in particular, is a powerful reporter of the molecular environment of a fluorophore. The fluorescence lifetime can be sensitive to the local polarity, hydration, viscosity and/or presence of fluorescence quenchers/energy acceptors within several nanometers of the vicinity of a fluorophore. Illustrative examples of how Time-Resolved (TR) fluorescence measurements can provide more valuable and detailed information about a system than the time-integrated (Steady-State) approach will be presented in this review: (a) determination of membrane polarity and mobility using time-dependent spectral shifts, (b) identification of submicroscopic domains by fluorescence lifetime imaging microscopy, (c) elucidation of membrane leakage mechanisms from dye self-quenching assays, and (d) evaluation of nanodomain sizes by TR Förster resonance energy transfer measurements.
Bimodal Distribution and Fluorescence Response of Environment-Sensitive Probes in Lipid Bilayers
Biophysical Journal, 2004
A remarkable heterogeneity is often observed in the spectroscopic properties of environment-sensitive fluorescence probes in phospholipid bilayers. To explain its origin, we provided a detailed investigation of the fluorescence excitation and emission spectra of 4#-dimethylamino-3-hydroxyflavone (probe F) in bilayer vesicles with the variations of fatty acid composition, polar heads, temperature, and cholesterol content. Probe F, due to excited-state intramolecular proton transfer, exhibits two bands in emission that are differently sensitive to intermolecular interactions-thereby allowing us to distinguish universal (dipole-dipole) and specific (H-bonding) interactions within the bilayer. Spectroscopic, quenching, and anisotropy data suggest the presence of two forms of probe F at different locations in the bilayer: an H-bond free form located below sn 1 -carbonyls and an H-bonded form located at the polar membrane interface. We provide a quantitative analysis of the distribution of the probe between these two locations as well as the polarity of these locations, and show that both the distribution and the polarity contribute to the probe response. Moreover, analysis of literature data on other environment-sensitive probes (Prodan, Laurdan, Nile Red, NBD lipids, etc.) in lipid bilayers allows us to suggest that the bimodal distribution in the lipid bilayer is probably a general feature of low-polar molecules with polar groups capable of H-bonding interactions.
Journal of Photochemistry and Photobiology A: Chemistry, 1995
Fluorescence energy transfer in lipid vesicles between N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)-labelled phosphatidylethanolamine (acting as donor) and N-(lissamine-rhodamine B)-labelled phosphatidylethanolamine (acting as acceptor) was studied by steady state and time-resolved fluorescence quenching analysis. Both fluorescent phospholipids were incorporated as minor components in four different types of lipid vesicle: dipalmitoylphosphatidylglycerol vesicles in their L~ gel phase at 20 °C and in their La liquid crystalline phase at 50 °C, and egg yolk phosphatidylethanolamine vesicles at 40 °C in their L,, liquid crystalline phase at pH 9.5 and in their Hn inverted hexagonal phase at pH 5.0. The quenching of the donor fluorescence by energy transfer is diffusion controlled in all cases, except in the L~ gel phase. The dimensionality and type of constraints imposed on diffusion are different in each case, with the most efficient diffusion-controlled quenching in the hexagonal phase.
Fluorescence decay of DPH in lipid membranes: Influence of the external refractive index
Biophysical Chemistry, 1993
The radiative decay rate of a fluorescent probe in an optically thin layer is known to depend on the orientation of the probe and on the refractive indices inside and outside the layer (W. Lukosz, Phys. Rev. B 22 (1980) 3030). Fluorescent probes in phospholipid bilayer membranes approximate such a system. The natural lifetime is expected to vary with the refractive index of the medium surrounding the bilayer. The lifetime variation with the refractive index depends on the orientation of the fluorescent probe. This can be used to retrieve the second-rank orientational order parameter, (Pz). The fluorescence decay of all-trans 1,6-diphenyl-1,3,5hexatriene in L-cY-dipalmitoyl-phosphatidylchohne large unilamellar vesicles (LUVs) was measured at a temperature well below that of the phase transition. The refractive index of the medium was varied by addition of glycerol or sucrose. The observed change of decay time with the refractive index followed the theoretical prediction. The value of the order parameter, (Pz), recovered is significantly lower than that obtained from fluorescence polarization data. Possible reasons for this disagreement are discussed.
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.
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]
2016
Nitrobenzoxadiazole (NBD)-labeled lipids are popular fluorescent membrane probes.However, the understanding of important aspects of the photophysics of NBD remains incomplete, including the observed shift in the emission spectrum of NBD-lipids to longer wavelengths following excitation at the red edge of the absorption spectrum (REES).REES of NBD-lipids in membrane environments has been previously interpreted as reflecting restricted mobility of solvent surrounding the fluorophore.However, this requires a large change in the dipole moment (Δμ) of NBD upon excitation.Previous calculations of the value of Δμ of NBD in the literature have been carried out using outdated semi-empirical methods, leading to conflicting values.Using up-to-date density functional theory methods, we recalculated the value of Δμ and verified that it is rather small (∼2D). Fluorescence measurements confirmed that the value of REES is ∼16nm for 1,2-dioleoyl-sn-glycero-3-phospho-l-serine-N-(NBD) (NBD-PS) in dioleoylphosphatidylcholine vesicles.However, the observed shift is independent of both the temperature and the presence of cholesterol and is therefore insensitive to the mobility and hydration of the membrane.Moreover, red-edge excitation leads to an increased contribution of the decay component with a shorter lifetime, whereas time-resolved emission spectra of NBD-PS displayed an atypical blue shift following excitation.This excludes restrictions to solvent relaxation as the cause of the measured REES and TRES of NBD, pointing instead to the heterogeneous transverse location of probes as the origin of these effects.The latter hypothesis was confirmed by molecular dynamics simulations, from which the calculated heterogeneity of the hydration and location of NBD correlated with the measured fluorescence lifetimes/REES.Globally, our combination of theoretical and experiment-based techniques has led to a considerably improved understanding of the photophysics of NBD...
Fluorescence spectroscopic studies on phase heterogeneity in lipid bilayer membranes
Journal of Fluorescence, 2001
There is a growing interest in functional membrane heterogeneity on the mesoscopic (several tens to hundreds of molecular dimensions) scale. However, the physical-chemical basis for this sort of heterogeneity in membranes is not entirely clear. Unambiguous methods to demonstrate that the cell plasma membrane and other cellular membranes are in fact heterogeneous on the mesoscopic level are also not generally available. Fluorescence techniques do, however, provide excellent tools for this purpose. In particular, the emerging techniques of scanning near-field optical microscopy and single-molecule fluorescence microscopy hold a great deal of promise for the near-future. All these methods require the use of fluorescent probes (lipids and/or proteins) and a clear definition of how these probes partition between domains of coexisting membrane phases. The development of the concept of membrane heterogeneity over the years since the first proposal of the "fluid mosaic" model is reviewed briefly. The use of lipid-binding proteins in experimental protocols for the labeling of membranes with fluorescent lipid amphiphiles as monomers in aqueous solutions at concentrations well above their critical aggregation concentrations is discussed. The methods of fluorescence spectroscopy available to the cell biologist for determining probe partition coefficients for partitioning between coexisting membrane phases are reviewed in some detail, as is the relevant theoretical and experimental work reported in the literature.
Spectrally constrained global analysis of fluorescence decays in biomembrane systems
Dynamic and steady-state fluorescence spectroscopic properties of a dye probe measured in a multicomponent biological system are often required to be separated into the spectra and lifetimes of individual spectroscopically distinct species. The conventional method of obtaining decay-associated spectra fails when the lifetimes of the fluorophore in the membrane phase and in the aqueous phase are very close to each other. This paper describes a global analysis method which takes advantage of the known spectrum and lifetime of the dye in the aqueous phase. This method is used to identify the spectra for two fluorescent species (lifetimes, 0.84 and 1.97 ns) of the dye DODCI in EggPC vesicle membranes by keeping the spectrum and lifetime (0.68 ns) of the dye in the aqueous phase as fixed parameters. The structural identity of the two membrane-bound dye species was established by the effect of refractive index and/or viscosity of the aqueous medium on the lifetimes.