Diphenylhexatriene membrane probes DPH and TMA-DPH: A comparative molecular dynamics simulation study

Fluorescence spectroscopy andmicroscopy have been utilized as tools in membrane biophysics for decades now. Because phospholipids are non-fluorescent, the use of extrinsic membrane probes in this context is commonplace. Among the latter, 1,6-diphenylhexatriene (DPH) and its trimethylammonium derivative (TMA-DPH) have been extensively used. It is widely believed that, owing to its additional charged group, TMA-DPH is anchored at the lipid/water interface and reports on a bilayer region that is distinct from that of the hydrophobic DPH. In this study, we employ atomistic MD simulations to characterize the behavior of DPH and TMA-DPH in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and POPC/cholesterol (4:1) bilayers. We show that although the dynamics of TMA-DPH in thesemembranes is noticeably more hindered than that of DPH, the location of the average fluorophore of TMA-DPH is only ~3–4 Å more shallow than that of DPH. The hindrance observed in the translational and rotational motions of TMA-DPH compared to DPH is mainly not due to significant differences in depth, but to the favorable electrostatic interactions of the former with electronegative lipid atoms instead. By revealing detailed insights on the behavior of these two probes, our results are useful both in the interpretation of past work and in the planning of future experiments using themasmembrane reporters.

Diphenylhexatriene membrane probes DPH and TMA-DPH: A comparative molecular dynamics simulation study

Fluorescence spectroscopy andmicroscopy have been utilized as tools in membrane biophysics for decades now. Because phospholipids are non-fluorescent, the use of extrinsic membrane probes in this context is commonplace. Among the latter, 1,6-diphenylhexatriene (DPH) and its trimethylammonium derivative (TMA-DPH) have been extensively used. It is widely believed that, owing to its additional charged group, TMA-DPH is anchored at the lipid/water interface and reports on a bilayer region that is distinct from that of the hydrophobic DPH. In this study, we employ atomistic MD simulations to characterize the behavior of DPH and TMA-DPH in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and POPC/cholesterol (4:1) bilayers. We show that although the dynamics of TMA-DPH in thesemembranes is noticeably more hindered than that of DPH, the location of the average fluorophore of TMA-DPH is only ~3–4 Å more shallow than that of DPH. The hindrance observed in the translational and rotational motions of TMA-DPH compared to DPH is mainly not due to significant differences in depth, but to the favorable electrostatic interactions of the former with electronegative lipid atoms instead. By revealing detailed insights on the behavior of these two probes, our results are useful both in the interpretation of past work and in the planning of future experiments using themasmembrane reporters.

Fluorescence of nitrobenzoxadiazole (NBD)-labeled lipids in model membranes is connected not to lipid mobility but to probe location

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 (red-edge excitation shift or 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 (Dm) of NBD upon excitation. Previous calculations of the value of Dm 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 Dm and verified that it is rather small (B2 D). Fluorescence measurements confirmed that the value of REES is B16 nm 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 and a reinterpretation of its REES in particular.

Liquid Mixtures Involving Hydrogenated and Fluorinated Alcohols: Thermodynamics, Spectroscopy, and Simulation

This article reports a combined thermodynamic, spectroscopic, and computational study on the interactions and structure of binary mixtures of hydrogenated and fluorinated substances that simultaneously interact through strong hydrogen bonding. Four binary mixtures of hydrogenated and fluorinated alcohols have been studied, namely, (ethanol + 2,2,2-trifluoroethanol (TFE)), (ethanol + 2,2,3,3,4,4,4-heptafluoro-1-butanol), (1-butanol (BuOH) + TFE), and (BuOH + 2,2,3,3,4,4,4-heptafluoro-1-butanol). Excess molar volumes and vibrational spectra of all four binary mixtures have been measured as a function of composition at 298 K, and molecular dynamics simulations have been performed. The systems display a complex behavior when compared with mixtures of hydrogenated alcohols and mixtures of alkanes and perfluoroalkanes. The combined analysis of the results from different approaches indicates that this results from a balance between preferential hydrogen bonding between the hydrogenated and fluorinated alcohols and the unfavorable dispersion forces between the hydrogenated and fluorinated chains. As the chain length increases, the contribution of dispersion increases and overcomes the contribution of H-bonds. In terms of the liquid structure, the simulations suggest the possibility of segregation between the hydrogenated and fluorinated segments, a hypothesis corroborated by the spectroscopic results. Furthermore, a quantitative analysis of the infrared spectra reveals that the presence of fluorinated groups induces conformational changes in the hydrogenated chains from the usually preferred all-trans to more globular arrangements involving gauche conformations. Conformational rearrangements at the CCOH dihedral angle upon mixing are also disclosed by the spectra.

Vapor pressure and liquid density of fluorinated alcohols:Experimental, simulation and GC-SAFT-VR predictions

The vapor pressure of four liquid 1H,1H-perfluoroalcohols (CF3(CF2)n(CH2)OH, n ¼ 1, 2, 3, 4), often called odd-fluorotelomer alcohols, was measured as a function of temperature between 278 K and 328 K. Liquid densities were also measured for a temperature range between 278 K and 353 K. Molar enthalpies of vaporization were calculated from the experimental data. The results are compared with data from the literature for other perfluoroalcohols as well as with the equivalent hydrogenated alcohols. The results were modeled and interpreted using molecular dynamics simulations and the GC-SAFT-VR equation of state.