Liquid-crystalline Phosphatidylcholine Bilayers Research Articles

Unperturbed hydrocarbon chains and liquid phase bilayer lipid chains: a computer simulation study

In this work, the properties of saturated and unsaturated fatty acid acyl chains 16:0, 18:0, 18:1(n-9)cis, 18:2(n-6)cis, 18:3(n-3)cis, 18:4(n-3)cis, 18:5(n-3)cis, 20:4(n-6)cis, 20:5(n-3)cis and 22:6(n-3)cis in a bilayer liquid crystalline state and similar hydrocarbon chains (with CH_3 terminal groups instead of C=O groups) in the unperturbed state characterised by a lack of long-range interaction were investigated. The unperturbed hydrocarbon chains were modelled by Monte Carlo simulations at temperature T = 303 K; sixteen fully hydrated homogeneous liquid crystalline phosphatidylcholine bilayers containing these chains were studied by molecular dynamics simulations at the same temperature. To eliminate effects of the simulation parameters, the molecular dynamics and Monte Carlo simulations were carried out using the same structural data and force field coefficients. From these computer simulations, the average distances between terminal carbon atoms of the chains (end-to-end distances) were calculated and compared. The trends in the end-to-end distances obtained for the unperturbed chains were found to be qualitatively similar to those obtained for the same lipid chains in the bilayers. So, for understanding of a number of processes in biological membranes (e.g., changes in fatty acid composition caused by environmental changes such as temperature and pressure), it is possible to use, at least as a first approximation, the relationships between the structure and properties for unperturbed or isolated hydrocarbon chains.

Influence of Cholesterol Microstructures on Fluctuation Spikes in Nystatin Channel Currents in Phospholipid/Cholesterol Bilayers

Nystatin (NYS) is an antifungal agent that preferentially forms ion channels in membranes containing ergosterol (ERG) or cholesterol (CHOL). In phospholipid bilayers ERG or CHOL segregate into ordered (Lo) and disordered (Ld) domains. We prepared POPC bilayers containing CHOL mol fraction 0.18 ≤ χERG ≤ 0.50 separating two chambers containing KCl solutions. The concentration gradient between chambers was 435/150 mM KCl. We allowed the CHOL/POPC bilayer to settle 15 min then added 19 or 38 microM NYS to the reference (Cis) chamber, imposed 50 mV across the bilayer, and stirred the Cis chamber at 4 Hz. The NYS molecules adhered to the bilayer forming channels on the boundaries of the Lo domains. [1] The resultant bilayer current exhibited prominent spikes of very short duration. A plot of the frequency of these spikes against χERG revealed prominent spikes at χERG = 0.19, 0.25 and 0.40. These correspond well to the dips in dehydroergosterol fluorescence observed by Chong [2], which he understood in terms of a superlattice. We conclude that fluctuations in NYS channels on the perimeter of the Lo CHOL domains depend strongly on Lo domain structure.1. Helrich, C. S., J. A. Schmucker, and D. J. Woodbury 2006. Evidence that Nystatin Channels Form at the Boundaries, Not the Interiors of Lipid Domains. Biophys. J. 91: 1116-1127.2. Chong, P.L-G. 1994. Evidence for regular distribution of sterols in liquid crystalline phosphatidylcholine bilayers, Proc. Nati. Acad. Sci. USA 91:10069-10073.

Fluorescence evidence for cholesterol regular distribution in phosphatidylcholine and in sphingomyelin lipid bilayers

Our previous studies indicated that sterols (including cholesterol and dehydroergosterol) can be regularly distributed into hexagonal superlattices in the plane of liquid-crystalline phosphatidylcholine bilayers. It was suggested that regular and irregular regions coexist in the membrane. In the present study, we report supporting evidence for our sterol regular distribution model. We have examined the fractional concentration dependencies of dehydroergosterol (a naturally occurring cholesterol analogue) fluorescence intensity and lifetime in various phosphatidylcholine and sphingomyelin bilayers. Fluorescence intensity and lifetime dips have been observed at specific sterol mole fractions. At those mole fractions, the acrylamide quenching rate constant of dehydroergosterol fluorescence reaches a local maximum. Those mole fractions match the critical sterol mole fractions at which sterol molecules are expected to be regularly distributed into hexagonal superlattices. The results support the idea that the sterols in the regular region are embedded in the bilayer less deep than those in the irregular regions. We have also examined the fractional cholesterol concentration dependencies of diphenylhexatriene (DPH) fluorescence intensity, lifetime, and polarization in DMPC vesicles. DPH fluorescence intensity and polarization also exhibit distinct dips and peaks, respectively, at critical sterol mole fractions for hexagonal superlattices. However, DPH lifetime changes little with sterol mole fraction. As a comparison, the fluorescence properties of DHE and DPH behave differently in response to the formation of sterol regular distribution. Furthermore, finding evidence for sterol regular distribution in both phosphatidylcholine and sphingomyelin membranes raises the possibility that sterol regular distribution may occur within phospholipid/cholesterol enriched domains of real biological membranes.

Evidence for regular distribution of sterols in liquid crystalline phosphatidylcholine bilayers.

To investigate the lateral organization of sterols in membranes, the fluorescence intensity of dehydroergosterol at different mole fractions in liquid crystalline dimyristoyl phosphatidylcholine bilayers was examined. A number of intensity drops were observed at specific mole fractions, as predicted from a hexagonal super-lattice model. The fluorescence dips provide compelling evidence that a naturally occurring sterol is regularly distributed at fixed compositional fractions, consistent with the presence of hexagonal super-lattices in the fluid membranes. Regularly distributed regions, however, coexist with irregularly distributed regions. The extent of regular distribution varies periodically with sterol mole fraction and, consequently, similar variations take place in the membrane volume and lipid packing. This level of modulation in local membrane structure by minute changes in sterol concentration should have profound implications for the functional role of cholesterol content in cell membranes.

1-Palmitoyl-2-pyrenedecanoyl glycerophospholipids as membrane probes: evidence for regular distribution in liquid-crystalline phosphatidylcholine bilayers.

We have synthesized 1-palmitoyl-2-pyrenedecanoyl-sn-glycero derivatives of 3-phosphatidylcholine, 3-phosphatidylethanolamine, 3-phosphatidylserine, 3-phosphatidylglycerol, 3-phosphatidylinositol, and 3-phosphatidic acid and investigated their behavior in monolayers and in neat and mixed bilayers. Fluorescence spectroscopy of neat pyrene phospholipid dispersions revealed a well-defined thermotropic transition at 13.5-19 degrees C depending on the polar head group. An endotherm coinciding with this transition was observed with differential scanning calorimetry, indicating it to be due to the melting of the lipid acyl chains. For pyrenephosphatidylethanolamine, the endotherm was observed at a much higher temperature (70 degrees C). Compression isotherms obtained at an argon/water interface revealed that the pyrene moiety somewhat increases the mean molecular area of a phospholipid molecule but does not prevent the expression of head-group-dependent packing behavior. Partition of the pyrene lipids between coexisting fluid and solid phases was investigated with fluorometry and calorimetry. Both techniques indicate that these lipids prefer the fluid phase and that this preference is independent of the head group. The rates and apparent activation energies of lateral diffusion in fluid bilayers were found to be similar for most pyrene lipids, suggesting that the lateral movement of phospholipids is not critically dependent on interactions at the head-group level. Lateral distribution of the pyrene lipids in gel and fluid phosphatidylcholine bilayers was studied with the excimer technique and calorimetry. In gel-state dipalmitoylphosphatidylcholine bilayers, the pyrene lipids form clusters. These clusters, however, do not consist of pure pyrene lipid but of aggregates (compounds) of the labeled and unlabeled lipid.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of lysophosphatidylcholines on phosphatidylcholine and phosphatidylcholine/cholesterol liposome systems as revealed by 31P-NMR, electron microscopy and permeability studies

(1) The effect of incorporation of different lysophosphatidylcholine species on the structure, barrier properties and dynamics of bilayers made of various phosphatidylcholines both the presence and absence of cholesterol have been investigated by 31P-NMR, freeze-fracture electron microscopy and K +-permeability measurements. (2) In a dispersion of lysophosphatidylcholine : cholesterol (1 : 1) the lipids are organized in extended bilayers. Upon cooling a micellar solution of 1-palmitoyllysophosphatidylcholine below the chain-melting temperature a transtion to a lamellar, most likely interdigitating organization is observed. 31P-NMR shows in both situations a marked decrease in effective chemical shift anisotropy. (3) 1-Palmitoyllysophosphatidylcholine can be incorporated up to 30 mol% into liquid crystalline bilayers of dipalmitoylphosphatidylcholine and up to 35 mol% into dioleoylphosphatidylcholine bilayers. Above this concentration micellization of the bilayers occurs. In the gel state, bilayer structure is maintained up to 60 mol% of the lysocompound. (4) 1-Oleoyllysophosphatidylcholine can be incorporated to higher concentrations into liquid crystalline phosphatidylcholine bilayers than the palmitoyl analogue, which can be explained by the more cylindrical shape of the 1-oleoyllysophosphatidylcholine. (5) In marked contrast, incorporation of only 1 mol% of 1-oleoyllysophosphatidylcholine into gel state dipalmitoylphosphatidylcholine already destabilizes bilayer structure and makes the membranes completely permeable for K +. These results are discussed with respect to the mixing properties of the various lysophosphatidylcholines. (6) In general these effects are accompanied by a loss of the K +-permeability barrier, which however occurs at lower lysophosphatidylcholine concentrations than needed for the start of micellization. (7) Cholesterol incorporation counteracts the bilayer destabilizing role of lysophosphatidylcholines. (8) 31P-NMR demonstrates with increasing lysophosphatidylcholine concentrations in the bilayers of phosphatidylcholines a decrease in the effective chemical shift anisotropy. As the rigid lattice spectra of lysophosphatidylcholine and phosphatidylcholine are identical, this reflects a change in the conformational and/or motional properties of the phospholipid head groups. This phenomenon might play a role in the observed permeability changes.

Use of fluorescent probes in the study of phospholipid--sterol bilayers.

1. The transfer of excitation energy between the fluorescent probes 1,6-diphenylhexa-1,3,5-triene and 12-(9-anthroyl)stearic acid and the cholesterol analogue cholesta-4,6-dien-3-one in phosphatidylcholine liposomes has been investigated. 2. The results indicate that probes and steroid are randomly distributed in the bilayer at steroid concentrations up to 35 mol%. 3. The degree of polarization of diphenylhexatriene fluorescence increases with increasing cholesterol content. Other sterols, differing in structure in the region of the polar group or in the side chain at position-17, produce similar but not identical effects. 4. the results are consistent with the proposal that diphenylhexatriene gives a general picture of the state of the bilayer and that there is no segregation of sterols in liquid-crystalline phosphatidylcholine bilayers.

Clustering of fatty acids in phospholipid bilayers

From electrophoresis experiments it is concluded that acidic phospholipids incorporated in liquid crystalline phosphatidylcholine bilayers at neutral pH are randomly distributed. The same is true for spin-labelled fatty acids. In contrast, long chain fatty acids are not fully ionized at neutral pH and appear to be clustered, i.e. they segregate out into patches. Only at pH>11 is the fatty acid-COOH group fully ionized and charge repulsion leads to a random distribution of the fatty acid within the plane of the bilayer.