Electric dipole moments of the fluorescent probes Prodan and Laurdan: experimental and theoretical evaluations

Several experimental and theoretical approaches can be used for a comprehensive understanding of solvent effects on the electronic structure of solutes. In this review, we revisit the influence of solvents on the electronic structure of the fluorescent probes Prodan and Laurdan, focusing on their electric dipole moments. These biologically used probes were synthesized to be sensitive to the environment polarity. However, their solvent-dependent electronic structures are still a matter of discussion in the literature. The absorption and emission spectra of Prodan and Laurdan in different solvents indicate that the two probes have very similar electronic structures in both the ground and excited states. Theoretical calculations confirm that their electronic ground states are very much alike. In this review, we discuss the electric dipole moments of the ground and excited states calculated using the widely applied Lippert–Mataga equation, using both spherical and spheroid prolate cavities for the solute. The dimensions of the cavity were found to be crucial for the calculated dipole moments. These values are compared to those obtained by quantum mechanics calculations, considering Prodan in vacuum, in a polarizable continuum solvent, and using a hybrid quantum mechanics–molecular mechanics methodology. Based on the theoretical approaches it is evident that the Prodan dipole moment can change even in the absence of solute–solvent-specific interactions, which is not taken into consideration with the experimental Lippert–Mataga method. Moreover, in water, for electric dipole moment calculations, it is fundamental to consider hydrogen-bonded molecules.     

Theory of lipid bilayer phase transitions as detected by fluorescent probes

Summary We present a quantitative theory that relates the fluorescence intensityvs. temperature (I vs. T) profile of a fluorescent-labeled two-component lipid bilayer to the phase diagram of the bilayer and the partition coefficientK of the fluorophore between fluid and solid phases of the bilayer. We show how the theory can be used to evaluateK from experimentalI vs. T profiles and the appropriate phase diagrams as well as to understand the different shapes ofI vs. T profiles obtained with particular fluorophores and phase diagrams. Using calculatedI vs. T graphs, we discuss the meaning of parameters, such as midpoint of the phase transition and onset and termination of a transition, which are often used to characterize phase transitions on the basis of fluorescence intensityvs. temperature profiles.     

Broad lipid phase transitions in mammalian cell membranes measured by Laurdan fluorescence spectroscopy

Employing fluorescence spectroscopy and the membrane-embedded dye Laurdan we experimentally show that linear changes of cell membrane order in the physiological temperature regime are part of broad order-disorder-phase transitions which extend over a much broader temperature range. Even though these extreme temperatures are usually not object of live science research due to failure of cellular functions, our findings help to understand and predict cell membrane properties under physiological conditions as they explain the underlying physics of a broad order-disorder phase transition. Therefore, we analyzed the membranes of various cell lines, red blood cell ghosts and lipid vesicles by spectral decomposition in a custom-made setup in a temperature range from ?40 °C to +90 °C. While the generalized polarization as a measure for membrane order of artificial lipid membranes like phosphatidylcholine show sharp transitions as known from calorimetry measurements, living cells in a physiological temperature range do only show linear changes. However, extending the temperature range shows the existence of broad transitions and their sensitivity to cholesterol content, pH and anaesthetic. Moreover, adaptation to culture conditions like decreased temperature and morphological changes like detachment of adherent cells or dendrite growth are accompanied by changes in membrane order as well. The observed changes of the generalized polarization are equivalent to temperature changes dT in the range of +12 K < dT < -6 K.     

Anion binding to lipid bilayers: a study using fluorescent lipid probes

Anion-induced fluorescence quenching of lipid probes incorporated into the liposomal membrane was used to study the binding of anions to the lipid membrane. Lipid derivatives bearing nonpolar fluorophore located either in the proximity of the polar headgroups (anthrylvinyl-labelled phosphatidylcholine, ApPC; methyl 4-pyrenylbutyrate, MPB) or in the polar region (rhodamine 19 oleyl ester, OR19) of the bilayer were used as probes. The binding of iodide to the bilayers of different compositions was studied. Based on the anion-induced quenching of the fluorescence, the isotherm of adsorption of the quencher (iodide) to the membrane was plotted. For anions, which are non-quenchers or weak quenchers (thiocyanate, perchlorate or trichloroacetate), the binding parameters were obtained from the data of the competitive displacement of iodide by these anions. The association constants of the anion binding to the bilayer (Ka) were determined for the stoichiometry of 1 ion/1 lipid and also for the case of independent anion binding. At the physiological concentration of the salt, which does not bind noticeably to the membrane (150 mM NaCl), anion binding could be satisfactorily described by the Langmuir isotherm. The approach applied here offers new possibilities for the studies of ion-membrane interactions using fluorescent probes.     

Impact of membrane-anchored fluorescent probes on the mechanical properties of lipid bilayers

Fluorescent probes are used in membrane biophysics studies to provide information about physical properties such as lipid packing, polarity and lipid diffusion or to visualize membrane domains. However, our understanding of the effects the dyes themselves may induce on the membrane structure and properties are sparse. As mechanical properties like bending elasticity were already shown to be highly sensitive to the addition of “impurities” into the membranes, we have investigated the impact of six different commonly used fluorescent membrane probes (LAURDAN, TR-DPPE, Rh-DPPE, DiIC18, Bodipy-PC and NBD-PC) on the bending elasticity of dye containing POPC GUVs as compared to single component POPC GUVs. Small changes in the membrane bending elasticity compared to single POPC bilayers are observed when 2 mol% of Rh-DPPE, Bodipy-PC or NBD-PC are added in POPC membranes. These binary membranes are showing non reproducible mechanical properties attributed to a photo-induced peroxidation processes that may be controlled by a reduction of the fluorescent dye concentration. For TR-DPPE, a measurable decrease of the bending elasticity is detected with reproducible bending elasticity measurements. This is a direct indication that this dye, when exposed to illumination by a microscope lamp and contrary to Rh-DPPE, does not induce chemical degradation. At last, LAURDAN and DiIC18 probes mixed with POPC do not significantly affect the bending elasticity of pure POPC bilayers, even at 2 mol%, suggesting these latter probes do not induce major perturbations on the structure of POPC bilayers.     

Effect of ethanol-induced lipid interdigitation on the membrane solubility of Prodan, Acdan, and Laurdan.

The effect of ethanol-induced lipid interdigitation on the partition coefficient (Kp) of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and its two derivatives, 6-acetyl-2-(dimethylamino)naphthalene (Acdan) and 6-lauroyl-2-(dimethylamino)naphthalene (Laurdan), in L-alpha-dipalmitoylphosphatidylcholine (DPPC) vesicles has been examined by a precipitation method over the ethanol concentration range of 0-1.8 M. At 20 degrees C and in the absence of ethanol, the Kp values for Acdan, Prodan, and Laurdan are 2.0 x 10(3), 2.8 x 10(4), and 4.7 x 10(6), respectively. This result suggests that the Kp of Prodan and its derivatives is not simply a linear function of the polymethylene units. As DPPC undergoes the ethanol-induced phase transition from the noninterdigitated to the fully interdigitated gel state, Kp for Prodan and Acdan decreases by a factor of 5 and 2, respectively, whereas Kp for Laurdan exhibits no detectable changes with ethanol. The differences in Kp are in parallel with the differences in the fluorescence emission spectra of these probes over the ethanol concentration range examined. Previous fluorescence and infrared data indicated that membrane perturbation caused by the probes increases in the order: Laurdan > Prodan > Acdan. Thus, the degree of membrane perturbation also seems to be in parallel with Kp. Among these three probes, Prodan fluorescence reflects most correctly the ethanol-induced lipid interdigitation. In conclusion, the partitioning of small solutes in lipid membranes is significantly reduced by ethanol-induced lipid interdigitation, probably as a result of an increased membrane surface density due to the increased intramolecular lipid acyl chain ordering and a tighter overall intermolecular packing.     

A model for phase transitions in lipid bilayers and biological membranes

We wish to present an order-disorder model for the observed phase transitions in lipid bilayers and biological membranes. We show that the model may, under certain circumstances, exhibit two phase transitions, one corresponding to positional disordering of entire lipid molecules, and the other corresponding to orientational disordering in the hydrocarbon chains. We then give results of our numerical analysis of the model and compare them with experimental data. Shortcomings of the model and future directions for analyses of this type are also discussed.     

Hexagonal phase forming propensity detected in phospholipid bilayers with fluorescent probes.

The fluorescence emission spectrum of N epsilon-dansyl-L-Lys undergoes a marked blue shift when incorporated from aqueous solution into phospholipid bilayers. This shift is greater for membranes composed of dipalmitoleoylphosphatidylcholine than for membranes of dipalmitoleoylphosphatidylethanolamine. With the latter but not the former lipid, the fluorescence emission from N epsilon-dansyl-L-Lys is markedly temperature-dependent. The marked temperature dependence of N epsilon-dansyl-L-Lys fluorescence in bilayers of dipalmitoleoylphosphatidylethanolamine is greatest as the sample is heated close to the bilayer to hexagonal phase transition temperature. The fluorescence emission properties of another probe of membrane surface hydrophobicity, Laurdan, also exhibit marked changes at temperatures just below the bilayer to hexagonal phase transition temperature. At these temperatures, the generalized polarization begins to increase rather than decrease with temperature, and the emission intensity decreases markedly. Such effects are not observed over the same temperature range with phosphatidylcholine. Thus, both dansyl-L-lysine and Laurdan provide probes to measure changes in the physical properties of membrane bilayers which occur when these bilayers are heated close to the temperature required for transition to the hexagonal phase.     

Influence of cholesterol on phospholipid bilayers phase domains as detected by Laurdan fluorescence.

Coexisting gel and liquid-crystalline phospholipid phase domains can be observed in synthetic phospholipid vesicles during the transition from one phase to the other and, in vesicles of mixed phospholipids, at intermediate temperatures between the transitions of the different phospholipids. The presence of cholesterol perturbs the dynamic properties of both phases to such an extent as to prevent the detection of coexisting phases. 6-Lauroyl-2-dimethylaminopahthalene (Laurdan) fluorescence offers the unique advantage of well resolvable spectral parameters in the two phospholipid phases that can be used for the detection and quantitation of coexisting gel and liquid-crystalline domains. From Laurdan fluorescence excitation and emission spectra, the generalized polarization spectra and values were calculated. By the generalized polarization phospholipid phase domain coexistence can be detected, and each phase can be quantitated. In the same phospholipid vesicles where without cholesterol domain coexistence can be detected, above 15 mol% and, remarkably, at physiological cholesterol concentrations, > or = 30 mol%, no separate Laurdan fluorescence signals characteristic of distinct domains can be observed. Consequences of our results on the possible size and dynamics of phospholipid phase domains and their biological relevance are discussed.     

Investigation of phase transitions of saturated phosphocholine lipid bilayers via molecular dynamics simulations

Lipid bilayers play an important role in biological systems as they protect cells against unwanted chemicals and provide a barrier for material inside a cell from leaking out. In this paper, nearly 30 μs of molecular dynamics (MD) simulations were performed to investigate phase transitions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-phosphocholine (DPPC) lipid bilayers from the liquid crystalline (L_α) to the ripple (P_β) and to the gel phase (L_β). Our MD simulations accurately predict the main transition temperature for the single-component bilayers. A key focus of this work is to quantify the structure of the P_β phase for DMPC and compare with measures from x-ray experiments. The P_β major arm has similar structure to that of the L_β, while the thinner minor arm has interdigitated chains and the transition region between these two regions has large chain splay and disorder. At lower temperatures, our MD simulations predict the formation of the L_β phase with tilted fatty acid chains. The P_β and L_β phases are studied for mixtures of DMPC and DPPC and compare favorably with experiment. Overall, our MD simulations provide evidence for the relevancy of the CHARMM36 lipid force field for structures and add to our understanding of the less-defined P_β phase.     

PEG blocking of single pores arising on phase transitions in unmodified lipid bilayers

Changes in ionic permeability of bilayer lipid membranes (BLM) from dipalmitoyl phosphatidylcholine at temperature of phase transition in 1 M LiCl solution in the presence of polyethyleneglycols (PEG) of various molecular masses are studied. The transition of ionic membrane channels from conducting to blocked nonconducting state using polymers makes it possible to calibrate lipid pores. It is shown that low-molecular weight glycerol and PEG with molecular weights of 300 and 600 decrease the amplitude of current fluctuations through the membrane, the decrease being proportional to the size of the polymer molecule incorporated. The addition of PEG with molecular masses of 1450, 2000, and 3350 decrease the current fluctuations to the basal noise level. The result is considered as a complete blockade of ion channel conductivity. In the presence of rather large polymers, such as PEG with molecular masses of 6000 and 20000, which are hardly incorporated in the pore, single current fluctuations occur again; however, their amplitudes are somewhat smaller than in the absence of PEG. It is assumed that a complete blockade of the conductivity of lipid ionic channels by PEG with molecular masses of 1450, 2000, and 3350 is due to dehydration of the pore gap and the conversion of the hydrophilic pore to a hydrophobic one.     

Critical effects of polar fluorescent probes on the interaction of DHA with POPC supported lipid bilayers

The understanding of lipid bilayer structure and function has been advanced by the application of molecular fluorophores. However, the effects of these probe molecules on the physicochemical properties of membranes being studied are poorly understood. A quartz crystal microbalance with dissipation monitoring instrument was used in this work to investigate the impact of two commonly used fluorescent probes, 1‑palmitoyl‑2‑{12‑[(7‑nitro‑2‑1,3‑benzoxadiazol‑4‑yl)amino]dodecanoyl}‑sn‑glycero‑3‑phosphocholine (NBD-PC) and 1,2‑dipalmitoyl‑sn‑glycero‑3‑phosphoethanolamine‑n‑(lissamine rhodamine‑B‑sulfonyl) (Lis-Rhod PE), on the formation and physicochemical properties of a 1‑palmitoyl‑2‑oleoyl‑sn‑glycero‑3‑phosphocholine supported lipid bilayer (POPC-SLB). The interaction of the POPC-SLB and fluorophore-modified POPC-SLB with docosahexaenoic acid, DHA, was evaluated. The incorporation of DHA into the POPC-SLB was observed to significantly decrease in the presence of the Lis-Rhod PE probe compared with the POPC-SLB. In addition, it was observed that the small concentration of DHA incorporated into the POPC:NBD-PC SLB can produce rearrangement processes followed by the lost not only of DHA but also of POPC or NBD-PC molecules or both during the washing step. This work has significant implications for the interpretation of data employing fluorescent reporter molecules within SLBs.     

Orientation and rotational freedom of fluorescent probes in lecithin bilayers

Summary The fluorescence polarization properties of lecithin bilayers stained with 2,6-MANS and 1,8-ANS under applied potential steps have been studied. The fluorescence signal components of both dyes were found to have different sign and relative amplitude, suggesting that 1,8-ANS and 2,6-MANS behave differently when bound to black lipid membranes. In order to determine the location and the extent of rotational brownian motions of the bound chromophores, the experimental data were analyzed by using a simplified physico-mathematical model. According to it 2,6-MANS appears to have a ratio /gt higher than 1,8-ANS ( being the rotational relaxation of in plane rotations and the lifetime of the excited singlet state of the bound molecules), suggesting that the former chromophore is more tightly held inside the bilayers. Furthermore, 2,6-MANS is found to possess the absorption and emission oscillators more closely oriented to the normal of membrane surface, while 1,8-ANS has both oscillators almost near the plane of the bilayers. The results furnish also a fair estimate of the random molecular motion own by the phospholipid molecules at room temperature. The comparison of the present data with those obtained from squid axon membranes confirms the validity of the proposed physical model, yielding a rough estimate of the axon membrane-area covered by integral protein macromolecules. These preliminary results derived from lecithin model membranes suggest that fluorescence polarization techniques can provide valuable informations if applied to study the macromolecular organization of in vitro reconstituted membranes.Abbreviations 2,6-MANS 2-n-methylanilinonaphthalene-6-sulfonate - 1,8-ANS 1-anilinonaphthalene-8-sulfonate     

Liquid ordered and gel phases of lipid bilayers: fluorescent probes reveal close fluidity but different hydration

Hydration and fluidity of lipid bilayers in different phase states were studied using fluorescent probes selectively located at the interface. The probe of hydration was a recently developed 3-hydroxyflavone derivative, which is highly sensitive to the environment, whereas the probe of fluidity was the diphenylhexatriene derivative, 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene. By variation of the cholesterol content and temperature in large unilamellar vesicles composed of sphingomyelin or dipalmitoylphosphatidlycholine, we generated different phases: gel, liquid ordered (raft), liquid crystalline, and liquid disordered (considered as liquid crystalline phase with cholesterol). For these four phases, the hydration increases in the following order: liquid ordered < gel approximately liquid disordered < liquid crystalline. The membrane fluidity shows a somewhat different trend, namely liquid ordered approximately gel < liquid disordered < liquid crystalline. Thus, gel and liquid ordered phases exhibit similar fluidity, whereas the last phase is significantly less hydrated. We expect that cholesterol due to its specific H-bonding interactions with lipids and its ability to fill the voids in lipid bilayers expels efficiently water molecules from the highly ordered gel phase to form the liquid ordered phase. In this study, the liquid ordered (raft) and gel phases are for the first time clearly distinguished by their strong difference in hydration.     

Perturbations to lipid bilayers by spectroscopic probes as determined by dielectric measurements

Dielectric measurements on lecithin/cholesterol bimolecular lipid membranes have indicated that the series of extrinsic fluorescent probe molecules, the $\text{n-(9-}\text{anthroyloxy}\text{)}$ 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 1:10 000. The technique therefore detects perturbation of structure at probe levels which are lower than those commonly used in fluorescence-labelling experiments.     

Solvent relaxation in lipid bilayers with dansyl probes

The solvent relaxation properties of the dansyl group attached to two lipids (dansylphosphatidylethanolamine and dansylphosphatidylserine), a fatty acid (dansylundecanoic acid), and two drugs (dansylbenzocaine and dansylpropranolol) were compared in a variety of different lipid systems. Several methods for characterising solvent relaxation were compared in detail for dansylpropranolol in bilayer vesicles of egg phosphatidylcholine. It was shown that the relaxation process is non-monoexponential; nevertheless, for comparative purposes, a model was adopted in which the lifetime associated with the negative exponent in a two exponential decay analysis, obtained at a particular energy on the red edge of emission, was taken as an approximation to a 'solvent relaxation' rate. A negative exponent, indicative of solvent relaxation processes, occurring in the nanosecond time-scale, was found only for dansylpropranolol, dansylPE and dansylundecanoic acid. On addition of the spin probe, 5-doxylstearate, the negative exponent was unaffected in liquid-crystalline phase lipids but was no longer found in gel-phase lipid in the case of dansylpropranolol, while for dansylPE the relaxation time was reduced. On the basis of these types of measurement it was possible to distinguish between different lipid environments using the same probe or between different dansyl environments of the different probes in the same lipid in cases where this would have been difficult or impossible solely on the basis of steady-state or fluorescence lifetime measurements.     

Cold-induced lipid phase transitions

The structural organization of biological membranes is largely determined by the weak interactions existing between their components and between these components and their aqueous environment. These interactions are particularly sensitive to changes in temperature and hydration. The factors influencing membrane lipid phase behaviour are briefly reviewed and used to develop a phase-separation model describing the response of biological membranes to stress. The factors affecting the interaction of cryoprotectants with membrane lipids are explored and their role in the stabilization of membrane organization at low temperatures discussed. It is suggested that the basis of their protective action lies in an ability to preserve the balance of interactions between membrane components at low temperatures at a level similar to that existing under physiological conditions.     

The organisation of cholesterol and ergosterol in lipid bilayers based on studies using non-perturbing fluorescent sterol probes.

The fluorescence properties of dehydroergosterol and cholesta-5,7,9-trien-3 beta-ol have been studied in organic solution, in aqueous dispersions and incorporated into aqueous lipid dispersions. The absorption spectra of aqueous dispersions of the probes are very different to those in organic solution, and aqueous dispersions are non-fluorescent. This can be attributed to micelle formation with dimerisation and/or aggregation in the micelles. Concentration quenching also occurs when sterols are incorporated into lipid bilayers, but relatively high fluorescence is observed even at a 1 : 1 steroid:lipid molar ratio. Further, the fluorescence is still polarized at these high molar ratios. We attribute this to the formation of ordered arrays of sterol molecules in the lipid bilayers. In these arrays the sterol molecules are organised in an end-to-end fashion, and face-to-face overlap of the sterols is prevented by the lipid molecules. Possible structures for 1 : 1 mixtures are presented.