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BBA - Biomembranes (v.1758, #2)

Editorial Board (pp. ii).

Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH by Grzegorz Terlecki; Elżbieta Czapiñska; Katarzyna Rogozik; Marek Lisowski; Jan Gutowicz (pp. 133-144).

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values <6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.

Keywords: Lipid–protein interaction; Lactate dehydrogenase; Acidic phospholipids; Phosphatidylserine; Cardiolipin

Rapid phase change of lipid microdomains in giant vesicles induced by conversion of sphingomyelin to ceramide by Yukinori Taniguchi; Tetsuhiko Ohba; Hidetake Miyata; Kazuo Ohki (pp. 145-153).

To understand the role of sphingomyelinase (SMase) in the function of biological membranes, we have investigated the effect of conversion of sphingomyelin (SM) to ceramide (Cer) on the assembly of domains in giant unilamellar vesicles (GUVs). The GUVs were prepared from mixture of 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC), N-palmitoly-d- erythro-sphingosine (C₁₆Cer), N-palmitoyl-d- erythro-sphingosylphosphorylcholine (C₁₆SM) and cholesterol. The amounts of DOPC, sum of C₁₆Cer and C₁₆SM, and cholesterol were kept constant (the ratio of these four lipids is shown as 1: X:1– X:1 (molar ratio), i.e., X is C₁₆Cer/(C₁₆Cer+C₁₆SM)). Shape and distribution of domains formed in the GUVs were monitored by a fluorescent lipid, Texas Red 1,2-dihexadecanoyl- sn-glycero-3-phosphoethanolamine (0.1 mol%). In GUVs containing low C₁₆Cer ( X=0 and 0.25), round-shaped domains labeled by the fluorescent lipid were present, suggesting coexistence of liquid-ordered and disordered domains. In GUVs containing intermediate Cer concentration ( X=0.5), the fluorescent domain covered most of GUV surface, which was surrounded by gel-like domains. Differential scanning calorimetry of multilamellar vesicles prepared in the presence of higher Cer concentration ( X≥0.5) suggested existence of a Cer-enriched gel phase. Video microscopy showed that the enzymatic conversion of SM to Cer caused rapid change in the domain structure: several minutes after the SMase addition, the fluorescent region spread over the GUV surface, within which regions with darker contrast existed. Image-based measurement of generalized polarization (GP) of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan), which is related to the acyl chain ordering of the lipids, was performed. Before the SMase treatment domains with high (0.65) and low (below 0.4) GP values coexisted, presumably reflecting the liquid-ordered and disordered domains; after the SMase treatment regions with intermediate GP values (0.5) and smaller regions with higher GP values (0.65) were present. Generation of Cer thus caused a phase transition from liquid-ordered and disordered phases to a gel and liquid phase.

Keywords: Sphingomyelinase; Lipid raft; Lipid microdomain; Giant liposome; Generalized polarization imaging

Cell selectivity correlates with membrane-specific interactions: A case study on the antimicrobial peptide G15 derived from granulysin by Ayyalusamy Ramamoorthy; Sathiah Thennarasu; Anmin Tan; Dong-Kuk Lee; Carol Clayberger; Alan M. Krensky (pp. 154-163).

A 15-residue peptide dimer G15 derived from the cell lytic protein granulysin has been shown to exert potent activity against microbes, including E. coli, but not against human Jurkat cells [Z. Wang, E. Choice, A. Kaspar, D. Hanson, S. Okada, S.C. Lyu, A.M. Krensky, C. Clayberger, Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J. Immunol. 165 (2000) 1486–1490]. We investigated the target membrane selectivity of G15 using fluorescence, circular dichroism and³¹P NMR methods. The ANS uptake assay shows that the extent of E. coli outer membrane disruption depends on G15 concentration.³¹P NMR spectra obtained from E. coli total lipid bilayers incorporated with G15 show disruption of lipid bilayers. Fluorescence binding studies on the interaction of G15 with synthetic liposomes formed of E. coli lipids suggest a tight binding of the peptide at the membrane interface. The peptide also binds to negatively charged POPC/POPG (3:1) lipid vesicles but fails to insert deep into the membrane interior. These results are supported by the peptide-induced changes in the measured isotropic chemical shift and T1 values of POPG in 3:1 POPC:POPG multilamellar vesicles while neither a non-lamellar phase nor a fragmentation of bilayers was observed from NMR studies. The circular dichroism studies reveal that the peptide exists as a random coil in solution but folds into a less ordered conformation upon binding to POPC/POPG (3:1) vesicles. However, G15 does not bind to lipid vesicles made of POPC/POPG/Chl (9:1:1) mixture, mimicking tumor cell membrane. These results explain the susceptibility of E. coli and the resistance of human Jurkat cells to G15, and may have implications in designing membrane-selective therapeutic agents.

Keywords: Abbreviations; ANS; anilinonaphthalene-8-sulfonic acid; CD; circular dichroism; Chl; cholesterol; H; _II; inverted hexagonal phase; MIC; minimum inhibitory concentration; MAS; magic angle spinning; NMR; nuclear magnetic resonance; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylcholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylglycerol; SUVs; small unilamellar vesiclesGranulysin; Antimicrobial peptide; Membrane-disruption; Lipid-peptide; NMR; Fluorescence

Yeast protein kinase Ptk2 localizes at the plasma membrane and phosphorylates in vitro the C-terminal peptide of the H⁺-ATPase by Pilar Eraso; María J. Mazón; Francisco Portillo (pp. 164-170).

Glucose triggers posttranslational modifications that increase the activity of the Saccharomyces cerevisiae plasma membrane H⁺-ATPase (Pma1). Glucose activation of yeast H⁺-ATPase results from the change in two kinetic parameters: an increase in the affinity of the enzyme for ATP, depending on Ser⁸⁹⁹, and an increase in the V_max involving Thr⁹¹². Our previous studies suggested that Ptk2 mediates the Ser⁸⁹⁹-dependent part of the activation. In this study we find that Ptk2 localized to the plasma membrane in a Triton X-100 insoluble fraction. In vitro phosphorylation assays using a recombinant GST-fusion protein comprising 30 C-terminal amino acids of Pma1 suggest that Ser⁸⁹⁹ is phosphorylated by Ptk2. Furthermore, we show that the Ptk2 carboxyl terminus is essential for glucose-dependent Pma1 activation and for the phosphorylation of Ser⁸⁹⁹.

Keywords: H; ⁺; -ATPase; Plasma membrane; Ptk2; Phosphorylation; Glucose activation

Effects of phospholipid hydrolysis on the aggregate structure in DPPC/DSPE-PEG₂₀₀₀ liposome preparations after gel to liquid crystalline phase transition by Ludger M. Ickenstein; Maria C. Sandström; Lawrence D. Mayer; Katarina Edwards (pp. 171-180).

Upon storage of phospholipid liposome samples, lysolipids, fatty acids, and glycerol-3-phosphatidylcholine are generated as a result of acid- or base-catalyzed hydrolysis. Accumulation of hydrolysis products in the liposome membrane can induce fusion, leakage, and structural transformations of the liposomes, which may be detrimental or beneficial to their performance depending on their applications as, e.g., drug delivery devices. We investigated in the present study the influence of phospholipid hydrolysis on the aggregate morphology of DPPC/DSPE-PEG₂₀₀₀ liposomes after transition of the phospholipid membrane from the gel phase to liquid crystalline phase using high performance liquid chromatography (HPLC) in combination with static light scattering, dynamic light scattering, and cryo-transmission electron microscopy (cryo-TEM). The rates of DPPC hydrolysis in DPPC/DSPE-PEG₂₀₀₀ liposomes were investigated at a pH of 2, 4, or 6.5 and temperatures of 22 °C or 4 °C. Results indicate that following phase transition, severe structural reorganizations occurred in liposome samples that were partially hydrolyzed in the gel phase. The most prominent effect was an increasing tendency of liposomes to disintegrate into membrane discs in accordance with an increasing degree of phospholipid hydrolysis. Complete disintegration occurred when DPPC concentrations had decreased by, in some cases, as little as 3.6%. After extensive phospholipid hydrolysis, liposomes and discs fused to form large bilayer sheets as well as other more complex bilayer structures apparently due to a decreased ratio of lysolipid to palmitic acid levels in the liposome membrane.

Keywords: Phospholipid hydrolysis; Disc formation; PEG-lipid; Lysolipid; Fatty acid

Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed¹³C NMR by Kazutoshi Yamamoto; Satoru Tuzi; Hazime Saitô; Izuru Kawamura; Akira Naito (pp. 181-189).

¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled D85N mutant of bacteriorhodopsin (bR) reconstituted in egg PC or DMPC bilayers were recorded to gain insight into their secondary structures and dynamics. They were substantially suppressed as compared with those of 2D crystals, especially at the loops and several transmembrane α_II-helices. Surprisingly, the¹³C NMR spectra of [3-¹³C]Ala-D85N turned out to be very similar to those of [3-¹³C]Ala-bR in lipid bilayers, in spite of the presence of globular conformational and dynamics changes in the former as found from 2D crystalline preparations. No further spectral change was also noted between the ground (pH 7) and M-like state (pH 10) as far as D85N in lipid bilayers was examined, in spite of their distinct changes in the 2D crystalline state. This is mainly caused by that the resulting¹³C NMR peaks which are sensitive to conformation and dynamics changes in the loops and several transmembrane α_II-helices of the M-like state are suppressed already by fluctuation motions in the order of 10⁴–10⁵ Hz interfered with frequencies of magic angle spinning or proton decoupling. However,¹³C NMR signal from the cytoplasmic α-helix protruding from the membrane surface is not strongly influenced by 2D crystal or monomer. Deceptively simplified carbonyl¹³C NMR signals of the loop and transmembrane α-helices followed by Pro residues in [1-¹³C]Val-labeled bR and D85N in 2D crystal are split into two peaks for reconstituted preparations in the absence of 2D crystalline lattice. Fortunately,¹³C NMR spectral feature of reconstituted [1-¹³C]Val and [3-¹³C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.

Keywords: Site-directed; ¹³; C NMR; Conformation and dynamics change; 2D crystalline lattice; Bacteriorhodopsin; D85N

Detergent solubilization of phosphatidylcholine bilayers in the fluid state: Influence of the acyl chain structure by Hasna Ahyayauch; Banafshe Larijani; Alicia Alonso; Félix M. Goñi (pp. 190-196).

Seventeen different, chemically defined phosphatidylcholines, dispersed in aqueous medium in the form of large unilamellar vesicles, have been tested for solubilization by the non-ionic detergent Triton X-100. The temperatures (either 20 °C or 45 °C) were such that the bilayers were always in the liquid-disordered state. For each case, the solubilization parameters, D_on (total detergent: lipid mole ratio producing the onset of solubilization) and D₅₀ (total detergent: lipid mole ratio producing 50% solubilization), were determined under equilibrium conditions. Both parameters varied generally in parallel. When double bonds were introduced to the acyl chains, other factors remaining constant, solubilization became more difficult, i.e., more detergent was required. Cis-unsaturated phospholipids required more detergent than the corresponding trans-isomers. Increasing chain length in saturated phospholipids between C12 and C16 decreased moderately the detergent/lipid ratios causing solubilization. Acyl and alkyl phospholipids were equally susceptible to Triton X-100 solubilization. Lipid chain order, as measured by DPH fluorescence polarization, seemed to facilitate solubilization, perhaps because more ordered bilayers have a smaller capacity to accommodate detergent monomers without breaking down into lipid–detergent mixed micelles.

Keywords: D; _on; total detergent: lipid mole ratio producing the onset of solubilization; D; ₅₀; total detergent: lipid mole ratio causing 50% solubilization; DPH; diphenylhexatriene; LUV; large unilamellar vesicles; DAPC; diarachidonoyl phosphatidylcholine; DEPC; dielaidoylphosphatidylcholine; DLaPC; dilauroyl phosphatidylcholine; DLPC; dilinoleoyl phosphatidylcholine; DLnPC; dilinolenoyl phosphatidylcholine; DMPC; dimyristoyl phosphatidylcholine; DMoPC; dimyristoleoyl phosphatidylcholine; DOPC; dioleoyl phosphatidylcholine; DPPC; dipalmitoyl phosphatidylcholine; DPePC; dipentadecanoyl phosphatidylcholine; DPoPC; dipalmitoleoyl phosphatidylcholine; DPhPC; diphytanoyl phosphatidylcholine; DPsPC; dipetroselenoyl phosphatidylcholine; DTPC; ditridecanoyl phosphatidylcholine; HAPC; 1-hexadecyl-2-arachidonoyl phosphatidylcholine; PAPC; 1-palmitoyl-2-arachidonoyl phosphatidylcholine; PC; phosphatidylcholine; POPC; 1-palmitoyl-2-oleoyl phosphatidylcholineMembrane solubilization; Detergent; Surfactant; Lipid phase; Lipid order

Permeation and inhibition of polycystin-l channel by monovalent organic cations by Xiao-Qing Dai; Edward Karpinski; Xing-Zhen Chen (pp. 197-205).

Polycystin-L (PCL), homologous to polycystin-2 (71% similarity in protein sequence), is the third member of the polycystin family of proteins. Polycystin-1 and -2 are mutated in autosomal dominant polycystic kidney disease, but the physiological role of PCL has not been determined. PCL acts as a Ca-regulated non-selective cation channel permeable to mono- and divalent cations. To further understand the biophysical and pharmacological properties of PCL, we examined a series of organic cations for permeation and inhibition, using single-channel patch clamp and whole-cell two-microelectrode voltage clamp techniques in conjunction with Xenopus oocyte expression. We found that PCL is permeable to organic amines, methlyamine (MA, 3.8 Å), dimethylamine (DMA, 4.6 Å) and triethylamine (TriEA, 6 Å), and to tetra-alkylammonium cation (TAA) tetra-methylammonium (TMA, 5.5–6.4 Å). TAA compounds tetra-ethylammonium (TEA, 6.1–8.2 Å) and tetra-propylammonium (TPA, 9.8 Å) were impermeable through PCL and exhibited weak inhibition on PCL (IC₅₀ values>13 mM). Larger TAA cations tetra-butylammonium (TBA, 11.6 Å) and tetra-pentylammonium (TPeA, 13.2 Å) were impermeable through PCL as well and showed strong inhibition (IC₅₀ values of 2.7 mM and 1.3 μM, respectively). Inhibition by TBA was on decreasing the single-channel current amplitude and exhibited no effect on open probability (NP_o) or mean open time (MOT), suggesting that it blocks the PCL permeation pathway. In contract, TEA, TPA and TPeA reduced NP_o and MOT values but had no effect on the amplitude, suggesting their binding to a different site in PCL, which affects the channel gating. Taken together, our studies revealed that PCL is permeable to organic amines and TAA cation TMA, and that inhibition of PCL by large TAA cations exhibits two different mechanisms, presumably through binding either to the pore pathway to reduce permeant flux or to another site to regulate the channel gating. These data allow to estimate a channel pore size of ∼7 Å for PCL.

Keywords: Patch clamp; Voltage clamp; Xenopus; oocyte; Polycystic kidney disease; Pore size; Organic amine; Tetra-alkylammonium

A divalent-ion binding site on the 16-kDa proton channel from Nephrops norvegicus—revealed by EPR spectroscopy by Tibor Páli; Malcolm E. Finbow; Derek Marsh (pp. 206-212).

As purified from the hepatopancreas of Nephrops norvegicus, the 16-kDa proton channel proteolipid is found to contain an endogenous divalent ion binding site that is occupied by Cu²⁺. The EPR spectrum has g-values and hyperfine splittings that are characteristic of type 2 Cu²⁺. The copper may be removed by extensive washing with EDTA. Titration with Ni²⁺ then induces spin–spin interactions with nitroxyl spin labels that are attached either to the unique Cys⁵⁴, or to fatty acids intercalated in the membrane. Paramagnetic relaxation enhancement by the fast-relaxing Ni²⁺ is used to characterise the binding and to estimate distances from the dipolar interactions. The Ni²⁺-binding site on the protein is situated around 14–18 Å from the spin label on Cys⁵⁴, and is at a similar distance from a lipid chain spin-labelled on the 5 C-atom, but is more remote from the C-9 and C-14 positions of the lipid chains.

Keywords: Abbreviations; V-ATPase; vacuolar H; ⁺; -ATPase; 5-MSL; 3-maleimido-2,2,5,5-tetramethylpyrrolidine-; N; -oxyl; n; -SASL; n; -(4,4-dimethyloxazolidine-; N; -oxyl)stearic acid; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA; ethylenediamine tetraacetic acid; EPR; electron paramagnetic resonance; ST-EPR; saturation transfer EPR; V; ₁; first-harmonic absorption EPR spectrum detected in phase with respect to the static magnetic field modulation; V; ₂; ^′; second-harmonic absorption EPR spectrum detected 90°-out-of-phase with respect to the static magnetic field modulation16-kDa proteolipid; Vacuolar ATPase; Proteolipid subunit c; Spin label; Cu; ²⁺; Saturation transfer EPR

Thermodynamic and structural study of the main phospholipid components comprising the mitochondrial inner membrane by Òscar Domènech; Fausto Sanz; M. Teresa Montero; Jordi Hernández-Borrell (pp. 213-221).

Cardiolipin (CL) is a phospholipid found in the energy-transducing membranes of bacteria and mitochondria and it is thought to be involved in relevant biological processes as apoptosis. In this work, the mixing properties of CL and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocoline (POPC) and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE) at the air–water interface, have been examined using the thermodynamic framework analysis of compression isotherms. Accordingly, the values of the Gibbs energy of mixing, the more stable monolayers assayed were: POPC:CL (0.6:0.4, mol:mol) and POPE:CL (0.8:0.2, mol:mol). The results reflect that attractive forces are the greatest contributors to the total interaction in these compositions. Supported planar bilayers (SPBs) with such compositions were examined using atomic force microscopy (AFM) at different temperatures. With the POPC:CL mixture, rounded and featureless SPBs were obtained at 4 °C and 24 °C. In contrast, the extension of the POPE:CL mixture revealed the existence of different lipid domains at 24 °C and 37 °C. Three lipid domains coexisted which can be distinguished by measuring the step height difference between the uncovered mica and the bilayer. While the low and intermediate domains were temperature dependent, the high domain was composition dependent. When cytochrome c (cyt c) was injected into the fluid cell, the protein showed a preferential adsorption onto the high domain of the POPC:CL. These results suggest that the high domain is mainly formed by CL.

Keywords: Monolayers; Supported planar bilayers; Atomic force microscopy

Preparation of submicron unilamellar liposomes by freeze-drying double emulsions by Ting Wang; Yingjie Deng; Yehui Geng; Zibin Gao; Jianping Zou; Zhixuan Wang (pp. 222-231).

A novel method is described for the preparation of sterile submicron unilamellar liposomes. The method is based on the lyophilization of double emulsions containing disaccharides as lyoprotectants in both the inner and outer aqueous phase. Using various phospholipids or mixtures of lipids as emulsifiers, the double emulsions can be prepared by a two-step emulsification, including hydrophilic agents in the inner aqueous phase or lipophilic agents in the oil phase. Then, the double emulsions are lyophilized after sterilization by passing them through a 0.22-μm pore filter. Rehydration of the lyophilized products results in liposomes with a relatively high encapsulation efficiency (for calcein, 87%; 5-fluorouracil, 19%; flurbiprofen, 93%) and a size below 200 nm measured by the dynamic light scattering technique (DLS) and the atomic force microscopy (AFM). The liposomes were found to be unilamellar from freeze-fracture electron micrographs and X-ray diffraction patterns. In addition, the liposomes can be reconstituted just before use by rehydration of the lyophilized products which are relatively stable. Thus, this reproducible and simple technique can be used to prepare sterilized, submicron unilamellar liposomes with a relatively high encapsulation efficiency, and excellent stability during long-term storage.

Keywords: Liposome; Preparation; Freeze-drying; Double emulsion; Encapsulation efficiency; Particle size

In situ characterization of lipid A interaction with antimicrobial peptides using surface X-ray scattering by Frances Neville; Chris S. Hodges; Chao Liu; Oleg Konovalov; David Gidalevitz (pp. 232-240).

Lipid A structure at the air–aqueous interface has been studied using pressure-area isotherm methods coupled with the surface X-ray scattering techniques of X-ray reflectivity (XR) and grazing incidence X-ray diffraction (GIXD). Lipid A monolayers were formed at the air–aqueous interface to represent the lipid moiety of the outer membrane of Gram-negative bacteria. Lipid A structure was characterized at surface pressures between 10 and 35 mN/m. Interactions of α-helical antimicrobial peptides LL-37, SMAP-29 and D2A22 with lipid A monolayers were subsequently studied. Although insertion into the lipid A monolayers was observed with the α-helical peptides, little change was seen from the X-ray data, suggesting that the lipid A hydrocarbon chains are involved in reorientation during insertion and that the hydrocarbon chains have a relatively rigid structure.

Keywords: Lipid A; Langmuir monolayer; Antimicrobial peptide; Lipopolysaccharide; GIXD; Reflectivity

Hydrodynamic properties of porcine bestrophin-1 in Triton X-100 by J. Brett Stanton; Andrew F.X. Goldberg; George Hoppe; Lihua Y. Marmorstein; Alan D. Marmorstein (pp. 241-247).

Bestrophin-1 (Best-1) is an integral membrane protein, defects in which cause Best vitelliform macular dystrophy. Best-1 is proposed to function as a Cl⁻ channel and/or a regulator of Ca⁺⁺ channels. A tetrameric (or pentameric) stoichiometry has been reported for recombinant best-1. Using a combination of gel exclusion chromatography and velocity sedimentation we examined the quaternary structure of native best-1 and found that it migrates as a single species with a Stokes radius of 7.3 nm, sedimentation coefficient ( S_20,w) of 4.9, and partial specific volume ( ν) of 0.80 ml/g. The mass of the protein–detergent complex is calculated to be 206 kDa, with the protein component estimated to be ∼138 kDa. Given a monomeric mass of 68 kDa, we conclude that native best-1 solubilized with Triton X-100 is a homodimer. The differences between this observation and a prior report were examined by comparing recombinant best-1 with tissue derived best-1 using gel exclusion chromatography. Much of the recombinant best-1 eluted in the column void ( V_o) fraction, unlike that extracted from RPE cells. We conclude that the minimal functional unit of best-1 is dimeric. This stoichiometry differs from that previously measured for recombinant best-1, suggesting that further studies are necessary to determine the stoichiometry of functional best-1 in RPE membranes.

Keywords: Ion channel; Calcium; Chloride; Oligomer; Centrifugation; Gel exclusion chromatography

Temperature and pressure effects on structural and conformational properties of POPC/SM/cholesterol model raft mixtures—a FT-IR, SAXS, DSC, PPC and Laurdan fluorescence spectroscopy study by Chiara Nicolini; Julia Kraineva; Monika Khurana; Nagarajan Periasamy; Sérgio S. Funari; Roland Winter (pp. 248-258).

We report on the effects of temperature and pressure on the structure, conformation and phase behavior of aqueous dispersions of the model lipid “raft�? mixture palmitoyloleoylphosphatidylcholine (POPC)/bovine brain sphingomyelin (SM)/cholesterol (Chol) (1:1:1). We investigated interchain interactions, hydrogen bonding, conformational and structural properties as well as phase transformations of this system using Fourier transform-infrared (FT-IR) spectroscopy, small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), and Laurdan fluorescence spectroscopy. The IR spectral parameters in combination with the scattering patterns from the SAXS measurements were used to detect structural and conformational transformations upon changes of pressure up to 7–9 kbar and temperature in the range from 1 to about 80 °C. The generalized polarization function ( GP) values, obtained from the Laurdan fluorescence spectroscopy studies also reveal temperature and pressure dependent phase changes. DSC and PPC were used to detect thermodynamic properties accompanying the temperature-dependent phase changes. In combination with literature fluorescence spectroscopy and microscopy data, a tentative p, T stability diagram of the mixture has been established. The data reveal a broad liquid-order/solid-ordered (l_o+s_o) two-phase coexistence region below 8±2 °C at ambient pressure. With increasing temperature, a l_o+l_d+s_o three-phase region is formed, which extends up to ∼27 °C, where a liquid-ordered/liquid-disordered (l_o+l_d) immiscibility region is formed. Finally, above 48±2 °C, the POPC/SM/Chol (1:1:1) mixture becomes completely fluid-like (liquid-disordered, l_d). With increasing pressure, all phase transition lines shift to higher temperatures. Notably, the l_o+l_d (+s_o) phase coexistence region, mimicking raft-like lateral phase separation in natural membranes, extends over a rather wide temperature range of about 40 °C, and a pressure range, which extends up to about 2 kbar for T=37 °C. Interestingly, in this pressure range, ceasing of membrane protein function in natural membrane environments has been observed for a variety of systems.

Keywords: Lipid bilayer; Model raft mixture; Phase transition; Pressure; SAXS; DSC; PPC; FT-IR

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BBA - Biomembranes (v.1758, #2)