Solutes in small amounts provide for lipid-bilayer softness: cholesterol,short-chain lipids,and bola lipids

The effect of the incorporation of small amounts (∼1 mole%) of amphiphilic solutes, such as cholesterol, a short-chain lipid (DC₁₀PC), and a bola lipid, into multilamellar DMPC bilayers is studied by small-angle neutron scattering and differential-scanning calorimetry. The anomalous swelling behavior observed in the transition region of pure DMPC bilayers is interpreted as an indication of bilayer softening and thermally reduced bending rigidity. Small amounts of the solutes are found to maintain or even enhance the bilayer softness. In the case of cholesterol, a systematic study shows that the well-known rigidification effect is observed only for cholesterol concentrations above 3–4 mole%. The results are discussed in relation to the physical properties of internal cell membranes. Received: 28 May 1996 / Accepted: 27 July 1996     

Study of the interaction of an α-helical transmembrane peptide with phosphatidylcholine bilayer membranes by means of densimetry and ultrasound velocimetry

We applied precise densimetry and ultrasound velocimetry methods to study the interaction of a synthetic α-helical transmembrane peptide, acetyl-K₂-L₂₄-K₂-amide (L₂₄), with model bilayer lipid membranes. The large unilamellar vesicles (LUVs) utilized were composed of a homologous series of n-saturated diacylphosphatidylcholines (PCs). PCs whose hydrocarbon chains contained from 13 to 16 carbon atoms, thus producing phospholipid bilayers of different thicknesses and gel to liquid-crystalline phase transition temperatures. This allowed us to analyze how the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer influences the thermodynamical and mechanical properties of the membranes. We showed that the incorporation of L₂₄ decreases the temperature and cooperativity of the main phase transition of all LUVs studied. The presence of L₂₄ in the bilayer also caused an increase of the specific volume and of the volume compressibility in the gel state bilayers. In the liquid crystalline state, the peptide decreases the specific volume at relatively higher peptide concentration (mole ratio L₂₄:PC = 1:50). The overall volume compressibility of the peptide-containing lipid bilayers in the liquid-crystalline state was in general higher in comparison with pure membranes. There was, however, a tendency for the volume compressibility of these lipid bilayers to decrease with higher peptide content in comparison with bilayers of lower peptide concentration. For one lipid composition, we also compared the thermodynamical and mechanical properties of LUVs and large multilamellar vesicles (MLVs) with and without L₂₄. As expected, a higher cooperativity of the changes of the thermodynamical and mechanical parameters took place for MLVs in comparison with LUVs. These results are in agreement with previously reported DSC and ²H NMR spectroscopy study of the interaction of the L₂₄ and structurally related peptides with phosphatidylcholine bilayers. An apparent discrepancy between ²H NMR spectroscopy and compressibility data in the liquid crystalline state may be connected with the complex and anisotropic nature of macroscopic mechanical properties of the membranes. The observed changes in membrane mechanical properties induced by the presence of L₂₄ suggest that around each peptide a distorted region exists that involves at least 2 layers of lipid molecules.     

Interaction of a bacterial monorhamnolipid secreted by Pseudomonas aeruginosa MA01 with phosphatidylcholine model membranes

This work presents a biophysical study on the interactions of a monorhamnolipid (monoRL) produced by Pseudomonas aeruginosa MA01 with model phosphatidylcholine membranes. The molecular characterization of the biological activities, including the modulation of phospholipid membranes structure, of this monoRL biosurfactant is of importance for the validation of this particular Pseudomonas aeruginosa strain as a useful biosurfactant producer. The marked amphiphilic structure of monoRL is expected to result in strong interactions with the phospholipid constituents of membrane bilayers. Incorporation of monoRL into DMPC completely abolished the pretransition, and the main gel to liquid-crystalline phase transition was progressively broadened and shifted to lower temperatures, as observed by differential scanning calorimetry. Partial phase diagrams for DPPC and DSPC indicated near-ideal behavior. However, the DMPC diagram indicated fluid phase immiscibility. X-ray diffraction showed and apparent increase in d-value for DPPC containing monoRL, which might be the result of an effective increase in the bilayer thickness, or in the thickness of the hydration layer between bilayers. FTIR indicated that interaction of monoRL with the phospholipid acyl chains did not result in a large additional disordering of the acyl chain region of the fluid bilayer. Analysis of the CO stretching band of DPPC indicated an important effect of monoRL on the interfacial region of phosphatidylcholine bilayers, which might contribute to explain some of the biological activities of this glycolipid.     

Effects of (+)-totarol, a diterpenoid antibacterial agent, on phospholipid model membranes

(+)-Totarol, a highly hydrophobic diterpenoid isolated from Podocarpus spp., is inhibitory towards the growth of diverse bacterial species. (+)-Totarol decreased the onset temperature of the gel to liquid-crystalline phase transition of DMPC and DMPG membranes and was immiscible with these lipids in the fluid phase at concentrations greater than 5 mol%. Different (+)-totarol/phospholipid mixtures having different stoichiometries appear to coexist with the pure phospholipid in the fluid phase. At concentrations greater than 15 mol% (+)-totarol completely suppressed the gel to liquid-crystalline phase transition in both DMPC and DMPG vesicles. Incorporation of increasing amounts of (+)-totarol into DEPE vesicles induced the appearance of the H(II) hexagonal phase at low temperatures in accordance with NMR data. At (+)-totarol concentrations between 5 and 35 mol% complex thermograms were observed, with new immiscible phases appearing at temperatures below the main transition of DEPE. Steady-state fluorescence anisotropy measurements showed that (+)-totarol decreased and increased the structural order of the phospholipid bilayer below and above the main gel to liquid-crystalline phase transition of DMPC respectively. The changes that (+)-totarol promotes in the physical properties of model membranes, compromising the functional integrity of the cell membrane, could explain its antibacterial effects.     

Interaction of pulmonary surfactant protein SP-A with DPPC/egg-PG bilayers

In the mixture of lipids and proteins which comprise pulmonary surfactant, the dominant protein by mass is surfactant protein A (SP-A), a hydrophilic glycoprotein. SP-A forms octadecamers that interact with phospholipid bilayer surfaces in the presence of calcium. Deuterium NMR was used to characterize the perturbation by SP-A, in the presence of 5 mM Ca²⁺, of dipalmitoyl phosphatidylcholine (DPPC) properties in DPPC/egg-PG (7:3) bilayers. Effects of SP-A were uniformly distributed over the observed DPPC population. SP-A reduced DPPC chain orientational order significantly in the gel phase but only slightly in the liquid-crystalline phase. Quadrupole echo decay times for DPPC chain deuterons were sensitive to SP-A in the liquid-crystalline mixture but not in the gel phase. SP-A reduced quadrupole splittings of DPPC choline β-deuterons but had little effect on choline α-deuteron splittings. The observed effects of SP-A on DPPC/egg-PG bilayer properties differ from those of the hydrophobic surfactant proteins SP-B and SP-C. This is consistent with the expectation that SP-A interacts primarily at bilayer surfaces.     

The effect of cholesterol in small amounts on lipid-bilayer softness in the region of the main phase transition

The temperature dependence of the small-angle neutron scattering from aqueous multilammellar DMPC lipid bilayers, containing small amounts of cholesterol, is analyzed near the main phase transition by means of a simple geometric model which yields the lamellar repeat distance, the hydrophobic thickness of the bilayer, the interlamellar aqueous spacing, as well as fluctuation parameters. The observation of anomalous swelling behavior in the transition region is interpreted as an indication of bilayer softening and thermally reduced bending rigidity. Our results indicate that the effect of small amounts of cholesterol, ≲3 mole%, is a softening of the bilayers in the transition region, whereas cholesterol contents above this range lead to the well-known effect of rigidification. The possible biological relevance of this result is discussed. Received: 24 October 1996 / Accepted: 9 December 1996     

Effect of acyl chain structure and bilayer phase state on binding and penetration of a supported lipid bilayer by HPA3

The effect of acyl chain structure and bilayer phase state on binding and penetration by the peptide HPA3 was studied using dual polarisation interferometry. This peptide is an analogue of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1) which has been shown to have antimicrobial and cell-penetrating properties. The binding of HPA3 to zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitolyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and negatively charged membranes composed of DMPC and 1,2-dimyristoyl-sn-glycero-3-(phosphor-rac-(1-glycerol)) (DMPG) or POPC and 1-palmitolyl-2-oleyl-sn-glycero-3-(phosphor-rac-(1-glycerol)) (POPG) was determined using dual polarisation interferometry (DPI). Mass and birefringence were measured in real time, enabling the creation of birefringence–mass plots for detailed analysis of the changes in lipid bilayer order during the peptide-binding process. HPA3 bound to all four lipids and the binding progressed as a single phase for the saturated gel phase bilayers DMPC and DMPC–DMPG. However, the binding process involved two or more phases, with penetration of the unsaturated fluid phase POPC and POPC–POPG bilayers. Structural changes in the saturated bilayer were partially reversible whereas binding to the unsaturated bilayer resulted in irreversible changes in membrane structure. These results demonstrate that more disordered unsaturated bilayers are more susceptible to further disorganisation and have a lower capacity to recover from peptide-induced structural changes than saturated ordered bilayers. In addition, this study further establishes DPI as powerful tool for analysis of multiphase peptide-insertion processes associated with complex structural changes in the liquid-crystalline membrane.     

Bilayer polarity and its thermal dependency in the ?o and ?d phases of binary phosphatidylcholine/cholesterol mixtures

Diverse variations in membrane properties are observed in binary phosphatidylcholine/cholesterol mixtures. These mixtures are nonideal, displaying single or phase coexistence, depending on chemical composition and other thermodynamic parameters. When compared with pure phospholipid bilayers, there are changes in water permeability, bilayer thickness and thermomechanical properties, molecular packing and conformational freedom of phospholipid acyl chains, in internal dipolar potential and in lipid lateral diffusion. Based on the phase diagrams for DMPC/cholesterol and DPPC/cholesterol, we compare the equivalent polarity of pure bilayers with specific compositions of these mixtures, by using the Py empirical scale of polarity. Besides the contrast between pure and mixed lipid bilayers, we find that liquid-ordered (?_o) and liquid-disordered (?_d) phases display significantly different polarities. Moreover, in the ?_o phase, the polarities of bilayers and their thermal dependences vary with the chemical composition, showing noteworthy differences for cholesterol proportions at 35, 40, and 45 mol%. At 20 °C, for DMPC/cholesterol at 35 and 45 mol%, the equivalent dielectric constants are 21.8 and 23.8, respectively. Additionally, we illustrate potential implications of polarity in various membrane-based processes and reactions, proposing that for cholesterol containing bilayers, it may also go along with the occurrence of lateral heterogeneity in biological membranes.     

Structure, topology, and tilt of cell-signaling peptides containing nuclear localization sequences in membrane bilayers determined by solid-state NMR and molecular dynamics simulation studies

Cell-signaling peptides have been extensively used to transport functional molecules across the plasma membrane into living cells. These peptides consist of a hydrophobic sequence and a cationic nuclear localization sequence (NLS). It has been assumed that the hydrophobic region penetrates the hydrophobic lipid bilayer and delivers the NLS inside the cell. To better understand the transport mechanism of these peptides, in this study, we investigated the structure, orientation, tilt of the peptide relative to the bilayer normal, and the membrane interaction of two cell-signaling peptides, SA and SKP. Results from CD and solid-state NMR experiments combined with molecular dynamics simulations suggest that the hydrophobic region is helical and has a transmembrane orientation with the helical axis tilted away from the bilayer normal. The influence of the hydrophobic mismatch, between the hydrophobic length of the peptide and the hydrophobic thickness of the bilayer, on the tilt angle of the peptides was investigated using thicker POPC and thinner DMPC bilayers. NMR experiments showed that the hydrophobic domain of each peptide has a tilt angle of 15 +/- 3 degrees in POPC, whereas in DMPC, 25 +/- 3 degree and 30 +/- 3 degree tilts were observed for SA and SKP peptides, respectively. These results are in good agreement with molecular dynamics simulations, which predict a tilt angle of 13.3 degrees (SA in POPC), 16.4 degrees (SKP in POPC), 22.3 degrees (SA in DMPC), and 31.7 degrees (SKP in DMPC). These results and simulations on the hydrophobic fragment of SA or SKP suggest that the tilt of helices increases with a decrease in bilayer thickness without changing the phase, order, and structure of the lipid bilayers.     

Phospholipase A2 induced effects on the structural organization and physical properties of pea chloroplast membranes

Phospholipase A₂ (PLA₂)-induced effects on the membrane organization, fluidity properties and surface charge density of pea chloroplasts were investigated. It was observed that lipolytic treatment with PLA₂ altered the chloroplast structure having as a result a swelling of thylakoids and a total destruction of normal granal structure. In spite of this, the thylakoid membranes remained in close contact. At the same time, a slight decrease of surface charge density was registered, thus explaining the adhesion of swelled membranes. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was measured during PLA₂ treatment. A pronounced decrease of DPH fluorescence polarization was found, indicating that phospholipase treatment resulted in considerable disordering and/or fluidization of the thylakoid membranes. The increased fluidity could be attributed to the destabilizing effect of the products of enzymatic hydrolysis of the phospholipids (free fatty acids, lysophospholipids) on the bilayer structure of thylakoids membranes.Abbreviations 9-AA 9-aminoacridine - BSA bovine serium albumin - DCMU 3-/3,4-dichlorophenyl-1,1-dimethyl/urea - DPH 1,6-diphenyl-1,3,5-hexatriene - EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - LHC light harvesting chlorophyll a/b-protein complex of PS II - MES 2/N-morpholine/ethanesulfonic acid - PLA₂ phospholipase A₂ - PS I, PS II photosystem I and photosystem II, respectively - S lipid structural order parameter - THF tetrahydrofuran - TRICINE N-/tris/hydroxymethyl/methyl/glicine     

Effect of the aminoacid composition of model α-helical peptides on the physical properties of lipid bilayers and peptide conformation: a molecular dynamics simulation

The interaction of a model Lys flanked α-helical peptides K₂-X₂₄-K₂, (X = A,I,L,L+A,V) with lipid bilayers composed of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) both, in a gel and in a liquid-crystalline state, has been studied by molecular dynamics simulations. It has been shown that these peptides cause disordering of the lipid bilayer in the gel state but only small changes have been monitored in a liquid-crystalline state. The peptides affect ordering of the surrounding lipids depending on the helix stability which is determined by amino acid side chains – their volume, shape, etc. We have shown that the helix does not keep the linear shape in all simulations but often bends or breaks. During some simulations with a very small difference between hydrophobic length of peptide and membrane thickness the peptide exhibits negligible tilt. At the same time changes in peptide conformations during simulations resulted in appearance of superhelix.     

Calorimetric and spectroscopic studies of the phase behavior and organization of lipid bilayer model membranes composed of binary mixtures of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol

The thermotropic phase behavior of hydrated bilayers derived from binary mixtures of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) was investigated by differential scanning calorimetry, Fourier-transform infrared spectroscopy and ³¹P-nuclear magnetic resonance spectroscopy. Binary mixtures of DMPC and DMPG that have not been annealed at low temperatures exhibit broad, weakly energetic pretransitions (∼11-15 °C) and highly cooperative, strongly energetic gel/liquid-crystalline phase transitions (∼23-25 °C). After low temperature incubation, these mixtures also exhibit a thermotropic transition form a lamellar-crystalline to a lamellar gel phase at temperatures below the onset of the gel/liquid-crystalline phase transition. The midpoint temperatures of the pretransitions and gel/liquid-crystalline phase transitions of these lipid mixtures are both maximal in mixtures containing ∼30 mol% DMPG but the widths and enthalpies of the same thermotropic events exhibit no discernable composition dependence. In contrast, thermotropic transitions involving the L_c phase exhibit a very strong composition dependence, and the midpoint temperatures and transition enthalpies are both maximal with mixtures containing equimolar amounts of the two lipids. Our spectroscopic studies indicate that the L_c phases formed are structurally similar as regards their modes of hydrocarbon chain packing, interfacial hydration and hydrogen-bonding interactions, as well as the range and amplitudes of the reorientational motions of their phosphate headgroups. Our results indicate that although DMPC and DMPG are highly miscible, their mixtures do not exhibit ideal mixing. We attribute the non-ideality in their mixing behavior to the formation of preferential PC/PG contacts in the L_c phase due to the combined effects of steric crowding of the DMPC headgroups and charge repulsion between the negatively charged DMPG molecules.     

Solid-State H NMR Shows Equivalence of Dehydration and Osmotic Pressures in Lipid Membrane Deformation

Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state ²H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our ²H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S_CD) of DMPC approach very large values of ≈0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state ²H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10−100 atm or lower. This research demonstrates the applicability of solid-state ²H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.     

Molecular dynamics study of changes in physico-chemical properties of DMPC lipid bilayers by addition of nonionic surfactant C12E10

Changes in physico-chemical properties of dimyristoyl phosphatidylcholine (DMPC) lipid bilayers caused by the addition of 9.4 mol% nonionic surfactant decaoxyethylene monododecyl ethers (C₁₂E₁₀) have been investigated by molecular dynamics calculations. In spite of addition of single chain C₁₂E₁₀, the lipid bilayers showed an increase of membrane area. Isothermal area compressibility, which is a measure of membrane softness in lateral direction, also increased by 50% for DMPC/C₁₂E₁₀ mixed bilayers. Furthermore, the order parameter of C–H vector for DMPC acyl tails decreased. We found that these changes are caused by the hydrophilic head groups of C₁₂E₁₀ which are located near the glycerol backbone of the DMPC molecules and have bulky random coil conformation without any preferential ordered structures.     

Combined influence of cholesterol and synthetic amphiphillic peptides upon bilayer thickness in model membranes.

Deuterium (2H) NMR was used to study bilayer hydrophobic thickness and mechanical properties when cholesterol and/or synthetic amphiphillic polypeptides were added to deuterated POPC lipid bilayer membranes in the liquid-crystalline (fluid) phase. Smoothed acyl chain orientational order profiles were used to calculate bilayer hydrophobic thickness. Addition of 30 mol% cholesterol to POPC at 25 degrees C increased the bilayer thickness from 2.58 to 2.99 nm. The peptides were chosen to span the bilayers with more or less mismatch between the hydrophobic peptide length and membrane hydrophobic thickness. The average thickness of the pure lipid bilayers was significantly perturbed upon addition of peptide only in cases of large mismatch, being increased (decreased) when the peptide hydrophobic length was greater (less) than that of the pure bilayer, consistent with the "mattress" model of protein lipid interactions (Mouritsen, O.G., and M. Bloom. 1984. Biophys. J. 46:141-153). The experimental results were also used to examine the combined influence of the polypeptides and cholesterol on the orientational order profile and thickness expansivity of the membranes. A detailed model for the spatial distribution of POPC and cholesterol molecules in the bilayers was proposed to reconcile the general features of these measurements with micromechanical measurements of area expansivity in closely related systems. Experiments to test the model were proposed.     

Antioxidant activity of probucol and its effects on phase transitions in phosphatidylcholine liposomes

The effect of probucol on the phase behavior of dimyristoylphosphatidylcholine (DMPC) was examined by fluorescence polarization and differential scanning calorimetry (DSC). Probucol broadens and shifts the temperature of the main phase transition of DMPC liposomes as measured by fluorescence polarization with diphenylhexatriene and trimethyl-ammonium-diphenylhexatrine at concentrations as low as 5 mole%. As measured by DSC, probucol reduces the transition temperature of the gel----liquid-crystalline phase transition of DMPC by approx. 2 C degrees at all concentrations above about 5 mole% probucol and eliminates the pretransition at less than 1 mole%. In addition, the phase transition of DMPC is broadened and the enthalpy of the transition reduced by approx. 50%. Even at high concentrations of probucol, the gel----liquid-crystalline phase transition of DMPC is not eliminated. Similar effects are observed with dipalmitoylphosphatidylcholine liposomes. Based on these DSC measurements, measurements of the melting of probucol in dry mixtures with DMPC and observations of probucol mixtures with DMPC under polarizing optics, the maximum solubility of probucol in DMPC is approx. 10 mole%. This concentration exceeds that required (approx. 0.5 mole%) to prevent peroxidation of 10 mole% arachidonic acid in DMPC liposomes for 30 min in the presence of 0.05 mM Fe(NH4)(SO4)2 at 4 degrees C. Thus, probucol has a limited solubility in saturated phosphatidylcholine bilayers, but is an effective antioxidant at concentrations lower than its maximum solubility.     

Fluid Mechanical Matching of H-ATP Synthase Subunit c-Ring with Lipid Membranes Revealed by H Solid-State NMR

The F₁F_o-ATP synthase utilizes the transmembrane H⁺ gradient for the synthesis of ATP. F_o subunit c-ring plays a key role in transporting H⁺ through F_o in the membrane. We investigated the interactions of Escherichia coli subunit c with dimyristoylphosphatidylcholine (DMPC-d₅₄) at lipid/protein ratios of 50:1 and 20:1 by means of ²H-solid-state NMR. In the liquid-crystalline state of DMPC, the ²H-NMR moment values and the order parameter (S_CD) profile were little affected by the presence of subunit c, suggesting that the bilayer thickness in the liquid-crystalline state is matched to the transmembrane hydrophobic surface of subunit c. On the other hand, hydrophobic mismatch of subunit c with the lipid bilayer was observed in the gel state of DMPC. Moreover, the viscoelasticity represented by a square-law function of the ²H-NMR relaxation was also little influenced by subunit c in the fluid phase, in contrast with flexible nonionic detergents or rigid additives. Thus, the hydrophobic matching of the lipid bilayer to subunit c involves at least two factors, the hydrophobic length and the fluid mechanical property. These findings may be important for the torque generation in the rotary catalytic mechanism of the F₁F_o-ATPse molecular motor.     

Comparative study on the properties of saturated phosphatidylethanolamine and phosphatidylcholine bilayers: Barrier characteristics and susceptibility to phospholipase A2 degradation

Comparative studies on bilayer systems of saturated phosphatidylcholines and phosphatidylethanolamines revealed a maximum in ionic permeability in phosphatidylcholine bilayers at the temperature of the gel to liquid-crystalline phase transition but such an increase in permeability was not detectable in bilayers of phosphatidylethanolamine. Furthermore, it was found that at the phase transition temperature the phosphatidylcholine bilayers are subject to rapid hydrolysis by pancreatic phospholipase A₂ whereas phosphatidylethanolamine bilayers are not. These differences are discussed in view of detailed information on the molecular organization in the gel and liquid crystalline phases of the two phospholipid classes.     

Transmembrane Peptides Influence the Affinity of Sterols for Phospholipid Bilayers

Cholesterol is distributed unevenly between different cellular membrane compartments, and the cholesterol content increases from the inner bilayers toward the plasma membrane. It has been suggested that this cholesterol gradient is important in the sorting of transmembrane proteins. Cholesterol has also been to shown play an important role in lateral organization of eukaryotic cell membranes. In this study the aim was to determine how transmembrane proteins influence the lateral distribution of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with bilayer membranes of different composition in the presence of designed peptides that mimic the transmembrane helices of proteins. For this purpose we developed an assay in which the partitioning of the fluorescent cholesterol analog CTL between LUVs and mβCD can be measured. Comparison of how cholesterol and CTL partitioning between mβCD and phospholipid bilayers with different composition suggests that CTL sensed changes in bilayer composition similarly as cholesterol. Therefore, the results obtained with CTL can be used to understand cholesterol distribution in lipid bilayers. The effect of WALP23 on CTL partitioning between DMPC bilayers and mβCD was measured. From the results it was clear that WALP23 increased both the order in the bilayers (as seen from CTL and DPH anisotropy) and the affinity of the sterol for the bilayer in a concentration dependent way. Although WALP23 also increased the order in DLPC and POPC bilayers the effects on CTL partitioning was much smaller with these lipids. This indicates that proteins have the largest effect on sterol interactions with phospholipids that have longer and saturated acyl chains. KALP23 did not significantly affect the acyl chain order in the phospholipid bilayers, and inclusion of KALP23 into DMPC bilayers slightly decreased CTL partitioning into the bilayer. This shows that transmembrane proteins can both decrease and increase the affinity of sterols for the lipid bilayers surrounding proteins. This is likely to affect the sterol distribution within the bilayer and thereby the lateral organization in biomembranes.     

Impact of free hydroxylated and methyl-branched fatty acids on the organization of lipid membranes

Differential scanning calorimetry (DSC) has been applied to study the effect of free hydroxylated and methyl-branched fatty acids on the physico-chemical properties of lipid membranes. First, the impact of free hydroxy fatty acids (HFAs) on dimyristoylphosphatidylcholine (DMPC) model membranes was monitored only as a function of chain length and position of the attached hydroxyl group. Second, racemic vs. enantiopure anteiso fatty acids (AFAs) and HFAs were investigated to address the question of which role does a fatty acid's chirality play on its membrane pertubing effect. The DSC thermograms revealed that the main gel to liquid-crystalline phase transition of the DMPC bilayers which results in a disordering effect of the lipid hydrocarbon chains was affected in different ways depending on the nature of the incorporated fatty acid. Long-chain 2- and 3-HFAs stabilized the gel phase by reducing the phase transition temperature (T(m)), whereas short-chain HFAs and long-chain HFAs with the hydroxy group remote from the head group stabilized the more disordered liquid-crystalline state. Additionally, we observed that enantiopure (S)-14-methylhexadecanoic acid ((S)-a17:0) and (R)-2-hydroxy octadecanoic acid and the corresponding racemates had contrary effects upon incorporation into DMPC bilayers. In both cases, the pure enantiomers alleviated the liquid-crystalline state of the biological model membrane.