Cíntia C. Vequi-Suplicy

Laurdan in Fluid Bilayers: Position and Structural Sensitivity

Laurdan (2-dimethylamino-6-lauroylnaphthalene) is a hydrophobic fluorescent probe widely used in lipid systems. This probe was shown to be highly sensitive to lipid phases, and this sensitivity related to the probe microenvironment polarity and viscosity. In the present study, Laurdan was incorporated in 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), which has a phase transition around 41°C, and DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), which is in the fluid phase at all temperatures studied. The temperature dependence of Laurdan fluorescent emission was analyzed via the decomposition into two gaussian bands, a short- and a long-wavelength band, corresponding to a non-relaxed and a water-relaxed excited state, respectively. As expected, Laurdan fluorescence is highly sensitive to DPPG gel–fluid transition. However, it is shown that Laurdan fluorescence, in DLPC, is also dependent on the temperature, though the bilayer phase does not change. This is in contrast to the rather similar fluorescent emission obtained for the analogous hydrophilic probe, Prodan (2-dimethylamino-6-propionylnaphthalene), when free in aqueous solution, over the same range of temperature. Therefore, Laurdan fluorescence seems to be highly dependent on the lipid bilayer packing, even for fluid membranes. This is supported by Laurdan fluorescence anisotropy and spin labels incorporated at different positions in the fluid lipid bilayer of DLPC. The latter were used both as structural probes for bilayer packing, and as Laurdan fluorescence quenchers. The results confirm the high sensitivity of Laurdan fluorescence emission to membrane packing, and indicate a rather shallow position for Laurdan in the membrane.

Lipid bilayer pre-transition as the beginning of the melting process

We investigate the bilayer pre-transition exhibited by some lipids at temperatures below their main phase transition, and which is generally associated to the formation of periodic ripples in the membrane. Experimentally we focus on the anionic lipid dipalmytoylphosphatidylglycerol (DPPG) at different ionic strengths, and on the neutral lipid dipalmytoylphosphatidylcholine (DPPC). From the analysis of differential scanning calorimetry traces of the two lipids we find that both pre- and main transitions are part of the same melting process. Electron spin resonance of spin labels and excitation generalized polarization of Laurdan reveal the coexistence of gel and fluid domains at temperatures between the pre- and main transitions of both lipids, reinforcing the first finding. Also, the melting process of DPPG at low ionic strength is found to be less cooperative than that of DPPC. From the theoretical side, we introduce a statistical model in which a next-nearest-neighbor competing interaction is added to the usual two-state model. For the first time, modulated phases (ordered and disordered lipids periodically aligned) emerge between the gel and fluid phases as a natural consequence of the competition between lipid-lipid interactions.

Vesicles with charged domains

This work summarizes results obtained on membranes composed of the ternary mixture dioleoylphosphatidylglycerol (DOPG), egg sphingomyelin (eSM) and cholesterol (Chol). The membrane phase state as a function of composition is characterized from data collected with fluorescence microscopy on giant unilamellar vesicles. The results suggest that the presence of the charged DOPG significantly decreases the composition region of coexistence of liquid ordered and liquid disordered phases as compared to that in the ternary mixture of dioleoylphosphatidycholine, sphingomyelin and cholesterol. The addition of calcium chloride to DOPG:eSM:Chol vesicles, and to a lesser extent the addition of sodium chloride, leads to the stabilization of the two-phase coexistence region, which is expressed in an increase in the miscibility temperature. On the other hand, addition of the chelating agent EDTA has the opposite effect, suggesting that impurities of divalent cations in preparations of giant vesicles contribute to the stabilization of charged domains. We also explore the behavior of these membranes in the presence of extruded unilamellar vesicles made of the positively charged lipid dioleoyltrimethylammoniumpropane (DOTAP). The latter can induce domain formation in DOPG:eSM:Chol vesicles with initial composition in the one-phase region.

Molecular Dynamics Investigations of PRODAN in a DLPC Bilayer

Molecular dynamics computer simulations have been performed to identify preferred positions of the fluorescent probe PRODAN in a fully hydrated DLPC bilayer in the fluid phase. In addition to the intramolecular charge-transfer first vertical excited state, we considered different charge distributions for the electronic ground state of the PRODAN molecule by distinct atomic charge models corresponding to the probe molecule in vacuum as well as polarized in a weak and a strong dielectric solvent (cyclohexane and water). Independent on the charge distribution model of PRODAN, we observed a preferential orientation of this molecule in the bilayer with the dimethylamino group pointing toward the membranes center and the carbonyl oxygen toward the membranes interface. However, changing the charge distribution model of PRODAN, independent of its initial position in the equilibrated DLPC membrane, we observed different preferential positions. For the ground state representation without polarization and the in-cyclohexane polarization, the probe maintains its position close to the membranes center. Considering the in-water polarization model, the probe approaches more of the polar headgroup region of the bilayer, with a strong structural correlation with the choline group, exposing its oxygen atom to water molecules. PRODANs representation of the first vertical excited state with the in-water polarization also approaches the polar region of the membrane with the oxygen atom exposed to the bilayers hydration shell. However, this model presents a stronger structural correlation with the phosphate groups than the ground state. Therefore, we conclude that the orientation of the PRODAN molecule inside the DLPC membrane is well-defined, but its position is very sensitive to the effect of the medium polarization included here by different models for the atomic charge distribution of the probe.

Optical characterization of Prodan aggregates in water medium

The fluorescent probe Prodan (2-dimethylamino-6-propionylnaphthalene) has been widely used in biological systems, mainly due to the high sensitivity of its emission spectrum to the medium polarity. Though mostly used as a membrane probe, in lipid dispersions Prodan partitions in water, mainly in the presence of gel-phase bilayers. Here, optical properties of Prodan in aqueous medium are experimentally studied using absorption and emission spectroscopies, and compared with those of the probe in cyclohexane, where it is supposed to be very soluble. In parallel, theoretical calculations of the absorption spectrum of a monomer and aggregated Prodan in water were performed. Moreover, to understand Prodan-water and Prodan-Prodan interactions, solvation free energies of Prodan in water and in liquid Prodan were calculated. A light scattering profile underneath the optical absorption spectrum of Prodan in water clearly indicates the presence of aggregates at very low Prodan concentrations (0.9 μM). Experimental evidence of Prodan aggregation is theoretically supported by solvation free energy calculations, which demonstrate that Prodan molecules interact preferentially with other Prodan molecules than with water molecules. Theoretical calculations for electronic transition energies of monomers and aggregated Prodan in water show that a Prodan optical absorption band at 358 nm is related to the monomeric form of Prodan. This band saturates as Prodan concentration increases, indicating that aggregated Prodan prevails at higher concentrations. The relative increase in Prodan aggregated population is monitored by the increase in an absorption band at higher energies, at around 250 nm, and by the disappearance of a band at around 280 nm. Surprisingly, it was observed that the fluorescent emission spectrum of Prodan is not sensitive to probe aggregation up to around 15 μM. Hence, Prodan aggregation in water medium, even at very low concentrations, must be considered when using this fluorescent probe in biological systems, having in mind that its fluorescence spectrum is rather insensitive to aggregation.

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.