Cholesterol-Induced Nanoscale Variations in the Thickness of Phospholipid Membranes.

Graphene-induced energy transfer (GIET) is a recently developed fluorescence-spectroscopic technique that achieves subnanometric optical localization of fluorophores along the optical axis of a microscope. GIET is based on the near-field energy transfer from an optically excited fluorescent molecule to a single sheet of graphene. It has been successfully used for estimating interleaflet distances of single lipid bilayers and for investigating the membrane organization of living mitochondria. In this study, we use GIET to measure the cholesterol-induced subtle changes of membrane thickness at the nanoscale. We quantify membrane thickness variations in supported lipid bilayers (SLBs) as a function of lipid composition and increasing cholesterol content. Our findings demonstrate that GIET is an extremely sensitive tool for investigating nanometric structural changes in biomembranes.

Investigating the competitive effects of cholesterol and melatonin in model lipid membranes.

We have studied the impact of cholesterol and/or melatonin on the static and dynamical properties of bilayers made of DPPC or DOPC utilizing neutron scattering techniques, Raman spectroscopy and molecular dynamics simulations. While differing in the amplitude of the effect due to cholesterol or melatonin when comparing their interactions with the two lipids, their addition ensued recognizable changes to both types of bilayers. As expected, based on the two-component systems of lipid/cholesterol or lipid/melatonin studied previously, we show the impact of cholesterol and melatonin being opposite and competitive in the case of three-component systems of lipid/cholesterol/melatonin. The effect of cholesterol appears to prevail over that of melatonin in the case of structural properties of DPPC-based bilayers, which can be explained by its interactions targeting primarily the saturated lipid chains. The dynamics of hydrocarbon chains represented by the ratio of trans/gauche conformers reveals the competitive effect of cholesterol and melatonin being somewhat more balanced. The additive yet opposing effects of cholesterol and melatonin have been observed also in the case of structural properties of DOPC-based bilayers. We report that cholesterol induced an increase in bilayer thickness, while melatonin induced a decrease in bilayer thickness in the three-component systems of DOPC/cholesterol/melatonin. Commensurately, by evaluating the projected area of DOPC, we demonstrate a lipid area decrease with an increasing concentration of cholesterol, and a lipid area increase with an increasing concentration of melatonin. The demonstrated condensing effect of cholesterol and the fluidizing effect of melatonin appear in an additive manner upon their mutual presence.

Condensing Effect of Cholesterol on hBest1/POPC and hBest1/SM Langmuir Monolayers.

Human bestrophin-1 protein (hBest1) is a transmembrane channel associated with the calcium-dependent transport of chloride ions in the retinal pigment epithelium as well as with the transport of glutamate and GABA in nerve cells. Interactions between hBest1, sphingomyelins, phosphatidylcholines and cholesterol are crucial for hBest1 association with cell membrane domains and its biological functions. As cholesterol plays a key role in the formation of lipid rafts, motional ordering of lipids and modeling/remodeling of the lateral membrane structure, we examined the effect of different cholesterol concentrations on the surface tension of hBest1/POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and hBest1/SM Langmuir monolayers in the presence/absence of Ca2+ ions using surface pressure measurements and Brewster angle microscopy studies. Here, we report that cholesterol: (1) has negligible condensing effect on pure hBest1 monolayers detected mainly in the presence of Ca2+ ions, and; (2) induces a condensing effect on composite hBest1/POPC and hBest1/SM monolayers. These results offer evidence for the significance of intermolecular protein-lipid interactions for the conformational dynamics of hBest1 and its biological functions as multimeric ion channel.

Modulating the crystallization of phytosterols with monoglycerides in the binary mixture systems: mixing behavior and eutectic formation.

Phytosterols (PSs) are insoluble in water and poorly soluble in oil, which hampers their potential as cholesterol level regulator in human. To mitigate this problem, monoglycerides (MGs) were used to modulates the crystallization behavior of PSs. Therefore, the understanding on mixing behavior provides the insight into different aspects of crystallization and the resultant effects. The effects on thermal, morphology, diffraction, and spectroscopy behavior were investigated for binary mixtures of 11 different ratios (100:0 to 0:100 MGs:PSs). The phase behavior of binary mixtures of commercial MGs and PSs exhibited complexity with the formation of eutectic mixtures at 90:10 and 80:20 (MGs:PSs) combinations. These combinations revealed a single melting profile and reduced melting enthalpy, though after a month of storage at 5 °C. Conversely, two separate melting regions were observed in others. Furthermore, powder X-ray diffraction (PXRD) analysis of selected combinations revealed a change in crystalline forms with changes in the peaks located between 18-19° (2θ) and 25-26° (2θ). Accordingly, Raman spectroscopy results revealed changes in intensities and peak shape. Therefore, the change in crystalline forms or behavior correlated well to the change in thermal properties. Overall, the characterizations revealed the formation of eutectic mixtures between MGs and PSs at 90:10 and 80:20 (MGs:PSs) in which MGs modified the crystallization of PSs and changed the crystal forms thus, thermal behaviors. This study provides new insight into the mixing behavior of MGs and PSs which supports other research. Therefore, the results of this study are beneficial for the improvement of formulation of phytosterols in food and pharmaceutical products. Nonetheless, this study reveals a simple technique to alter crystal forms of phytosterols through simple complexation with monoglycerides.

Effects of Sterol-Like Additives on Phase Transition Behavior of Ion-Pair Amphiphile Bilayers.

The incorporation of additive in lipid bilayers is one of the ordinary approaches for modulating their properties. Additive effect on phase transition of ion-pair amphiphile (IPA) bilayers, however, is not known. In this work, four double-chained IPAs with different hydrocarbon chain lengths and symmetry were designed and synthesized from single-chained cationic and anionic surfactants by the precipitation method. By using differential scanning calorimetry (DSC), the thermotropic transition behavior from gel phase (Lß) through rippled phase (Pß') if any to liquid-crystalline phase (Lα) was studied for bilayers of these lipid-like IPAs in excess water. The effects of three sterol-like additives (cholesterol, α-tocopherol, and α-tocopheryl acetate) in IPA bilayers on thermal phase behavior were then systematically investigated. The experimental results revealed that with increasing concentration of additive, the phase transition temperatures were unaffected on the one hand and the enthalpies of phase transition were decreased on the other hand. When the addition of additive exceeded a specific amount, the phase transition disappeared. More hasty disappearance of phase transition was found for IPAs with lower total number of carbon atoms in the hydrocarbon chains. For IPAs with the same total number of carbon atoms in the hydrocarbon chains, the disappearance of phase transition is more hasty for the asymmetric one than for the symmetric one. Similar effects on thermal phase behavior of four IPA bilayers were exhibited by the three additives with similar chemical structures. Possible mechanism of additive effects on phase transition of IPA bilayers was then proposed in line with that of lipid bilayers.

Alteration of lipid membrane structure and dynamics by diacylglycerols with unsaturated chains.

Diacylglycerols (DAGs) with unsaturated acyl chains play many important roles in biomembranes, such as a second messenger and activator for protein kinase C. In this study, three DAGs of distinctly different chain unsaturations (i.e. di16:0DAG (DPG), 16:0-18:1DAG (POG), and di18:1DAG (DOG)) are studied using atomistic MD simulation to compare their roles in the structure and dynamics of 16:0-18:1phosphatidylcholine (POPC) membranes. All three DAGs are able to produce the so-called 'condensing effect' in POPC membranes: decreasing area-per-lipid, and increasing acyl chain order and bilayer thickness. Our visual and quantitative analyses clearly show that DAG with unsaturated chains induce larger spacing between POPC headgroups, compared with DAG with saturated chains; this particular effect has long been hypothesized to be crucial for activating enzymes and receptors in cell membranes. DAGs with unsaturated chains are also located closer to the bilayer/aqueous interface than DPG and are more effective in slowing down lateral diffusion of molecules. We show that DAG molecules seek the "umbrella coverage" from neighboring phospholipid headgroups - similar to cholesterol. Unlike cholesterol, DAGs also hide their chains from water by laterally inserting their chains into the surrounding. Thus, acyl chains of DAG are more spread and disordered than those of PC due to the insertion. By calculating the potential of mean force (PMF) for POPC in POPC/DAG bilayers, we found that all three DAGs can significantly increase the free energy barrier for POPC to flip-flop, but only DAGs with unsaturated chains can additionally increase the free energy of POPC desorption.