How to link pyrene to its host lipid to minimize the extent of membrane perturbations and to optimize pyrene dimer formation.

We study how lipid probes based on pyrene-labeling could be designed to minimize perturbations in lipid bilayers, and how the same design principles could be exploited to develop probes which gauge lipid dynamics primarily within a single lipid monolayer or between them. To this end, we use atomistic molecular dynamics simulations to consider membranes where pyrene moieties are attached to lipid acyl chains in varying positions. We find that in a DOPC bilayer the conformational ordering of lipids around di-pyrenyl-PC probes is altered to a largely similar extent regardless of where the pyrene moiety is attached to the hydrocarbon chain. This is in contrast to saturated membranes, where pyrene-induced perturbations have been observed to be more prominent. Meanwhile, the formation of pyrene dimers depends on the linkage point between pyrene and its host lipid. Membrane-spanning dimers between lipids in different membrane leaflets are observed only if the pyrene moiety is attached to the latter half of the acyl chain. A seemingly minor change to link pyrene to an acyl chain that is two carbons shorter leads to a situation where membrane-spanning dimers are no longer observed. Further, simulations suggest that formation of dimers is a slow process, where the rate is limited by both lateral diffusion and the dimerization process once the two probes are neighbors to one another. Typical lifetimes of pyrene dimers turn out be of the order of nanoseconds. The results are expected to pave the way for designing ways to consider experimentally topics such as intraleaflet lateral diffusion, motion of lipids within and between membrane domains, and membrane domain registration across bilayers.

Effects of DPH on DPPC-cholesterol membranes with varying concentrations of cholesterol: from local perturbations to limitations in fluorescence anisotropy experiments.

We have used atomistic molecular dynamics simulations to consider 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescent probes in a fluid dipalmitoylphosphatidylcholine bilayer with 5 and 20 mol % cholesterol (CHOL). We show that while DPH affects a number of membrane properties, the perturbations induced by DPH depend on the concentration of cholesterol in a membrane. For example, we find DPH to influence the mass density distribution of lipids across the membrane and to promote the ordering of acyl chains around the probe. Yet, these perturbations get relatively weaker for increasing cholesterol concentration. Meanwhile, we also find that the commonly used analysis in terms of the Brownian rotational diffusion (BRD) model with Legendre polynomials to interpret fluorescence anisotropy (FA) experiments is sensitive to the amount of cholesterol. For small concentrations of cholesterol, the analysis of FA turns out to yield a relatively good approximation of the correct orientational distribution of DPH. However, for a CHOL concentration of 20 mol %, we find that the FA analysis fails to yield the true orientational distribution of DPH, the disagreement being substantial. The results suggest that in highly ordered membrane domains, the view given by FA analysis using the BRD model is likely reliable in a qualitative sense, but the quantitative description deviates substantially from the correct one. The present results imply that FA studies for the orientational distribution of DPH should be interpreted with great care.

Probing biomembranes with positrons.

Free volume pockets inside a cell membrane play a prominent role in a variety of dynamic processes such as the permeability of small molecules across membranes and the diffusion of, e.g., lipids, drugs, and electron carriers in the plane of the membrane. Nonetheless, by now the chances for characterizing free volume voids in a nonperturbative manner through experiments have been very limited. Here we use lipid membranes as an example to show how positron annihilation spectroscopy (PALS) together with atomistic simulations can be employed to gauge changes in free volume pockets in biological macromolecular complexes. The measurements show that PALS is a viable technique to probe free volume in biomolecular systems. As examples, we consider the gel-to-fluid transition and the role of increasing cholesterol concentration in a lipid membrane. Further applications proposed in this work for PALS are likely to provide a great deal of insight into the understanding of the role of free volume in the dynamics of biomolecular complexes.

Modeling of the triglyceride-rich core in lipoprotein particles.

Triglycerides are a major component of many important biological entities such as lipoproteins and lipid droplets. This work focuses on two common triglycerides, tripalmitin and triolein, which have been simulated through atomistic molecular dynamics at temperatures of 310 and 350 K for 300-700 ns. In these systems, both structural and dynamical properties have been characterized, paying particular attention to understanding the packing of triglyceride molecules and their molecular conformations. Additionally, we study the liquid-to-crystalline phase transition of tripalmitin through a temperature quench from the high-temperature isotropic liquid phase to 310 K, corresponding to a polymorphic, crystalline-like phase. The transition is characterized in detail through density, average molecular shape, and, in particular, the relevant order parameter describing the transition.

BODIPY-cholesterol: a new tool to visualize sterol trafficking in living cells and organisms.

Analysis of sterol distribution and transport in living cells has been hampered by the lack of bright, photostable fluorescent sterol derivatives that closely resemble cholesterol. In this study, we employed atomistic simulations and experiments to characterize a cholesterol compound with fluorescent boron dipyrromethene difluoride linked to sterol carbon-24 (BODIPY-cholesterol). This probe packed in the membrane and behaved similarly to cholesterol both in normal and in cholesterol-storage disease cells and with trace amounts allowed the visualization of sterol movement in living systems. Upon injection into the yolk sac, BODIPY-cholesterol did not disturb zebrafish development and was targeted to sterol-enriched brain regions in live fish. We conclude that this new probe closely mimics the membrane partitioning and trafficking of cholesterol and, because of its excellent fluorescent properties, enables the direct monitoring of sterol movement by time-lapse imaging using trace amounts of the probe. This is, to our knowledge, the first cholesterol probe that fulfills these prerequisites.

Free pyrene probes in gel and fluid membranes: perspective through atomistic simulations.

We consider the properties of free pyrene probes inside gel- and fluidlike phospholipid membranes and unravel their influence on membrane properties. For this purpose, we employ atomic-scale molecular dynamics simulations at several temperatures for varying pyrene concentrations. Molecular dynamics simulations show that free pyrene molecules prefer to be located in the hydrophobic acyl chain region close to the glycerol group of lipid molecules. Their orientation is shown to depend on the phase of the membrane. In the fluid phase, pyrenes favor orientations where they are standing upright in parallel to the membrane normal, while, in the gel phase, the orientation is affected by the tilt of lipid acyl chains. Pyrenes are found to locally perturb membrane structure, while the nature of perturbations in the gel and fluid phases is completely different. In the gel phase, pyrenes break the local packing of lipids and decrease the ordering of lipid acyl chains around them, while, in the fluid phase, pyrenes increase the ordering of nearby acyl chains, thus having an opposite effect. Interestingly, this proposes a similarity to effects induced by cholesterol on structural membrane properties above and below the gel-fluid transition temperature. Further studies express a view that the orientational ordering of pyrene is not a particularly good measure of the acyl chain ordering of lipids. While pyrene ordering provides the correct qualitative behavior of acyl chain ordering in the fluid phase, its capability to predict the correct temperature dependence is limited.

Influence of pyrene-labeling on fluid lipid membranes.

We elucidate the influence of pyrene-labeled phospholipids on the structural properties of a fluid dipalmitoylphosphatidylcholine lipid membrane. To this end, we employ extensive atomic-scale molecular dynamics simulations with varying concentrations of pyrene-linked lipids. We find pyrene labeling to perturb the membrane structure significantly in the vicinity of the probe, the correlation length in the bilayer plane being about 1.0-1.5 nm. The local perturbations lead to enhanced ordering and packing of lipid acyl chains located in the vicinity of the probe. Surprisingly, this holds true not only for lipids that reside in the same leaflet as the pyrene-labeled probe but also for lipids in the opposite monolayer. The latter is due to substantial interdigitation of the pyrene moiety into the opposite leaflet, suggesting that occasional excimer formation may take place for probes in different leaflets. As a related issue, we also discuss the location and conformational orientation of the pyrene moieties. In particular, the orientational distribution of pyrene turns out to be more broad and diverse than the distribution of the corresponding acyl tails of nonlabeled lipids.

Influence of DPH on the structure and dynamics of a DPPC bilayer.

We have conducted extensive molecular dynamics (MD) simulations together with differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) experiments to quantify the influence of free 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescent probes on the structure and dynamics of a dipalmitoylphosphatidylcholine bilayer. Atomistic MD simulations show that in the membrane-water interface the influence of DPH is minor, whereas in the acyl-chain region DPH gives rise to major perturbations. In the latter case, DPH is found to influence a wide range of membrane properties, such as the packing and ordering of hydrocarbon tails and the lateral diffusion of lipid molecules. The effects are prominent but of local nature, i.e., the changes observed in the properties of lipid molecules are significant in the vicinity of DPH, but reduce rapidly as the distance from the probe increases. Long-range perturbations due to DPH are hence not expected. Detailed DSC and (2)H NMR measurements support this view. DSC shows only subtle perturbation to the cooperative behavior of the membrane system in the presence of DPH, and (2)H NMR shows that DPH gives rise to a slight increase in the lipid chain order, in agreement with MD simulations. Potential effects of other probes such as pyrene are briefly discussed.

Characterization of molecular structures and properties of polyurethanes using molecular dynamics simulations.

Thermotropic polyurethanes with mesogenic groups in side chains were prepared from two diisocyanates and four diols with stoichiometric ratios of reactive isocyanate (NCO) and hydroxy (OH) groups. Their thermal behavior was determined by differential scanning calorimetry. The effect of structure modifications of the diisocyanates and diols, in particular changes in the mesogen, were investigated. Introduction of mesogenic segments into the polymers suppresses the ordering. Stiff end substituents (phenyl and alkoxy groups) of the mesogens stabilize the mesophases to such an extent that the negative influence of long polymer chains is compensated and the liquid-crystalline properties are recovered. All-atom molecular dynamics simulations in the Cerius2 modeling environment were carried out to characterize the structures of the polymers. Analysis of the dynamic trajectories at 20, 100, 120 and 170 degrees C revealed changes in conformation of macromolecules, which correlate with DSC measurements.