Location of the TEMPO moiety of TEMPO-PC in phosphatidylcholine bilayers is membrane phase dependent

The (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) moiety tethered to the headgroup of phosphatidylcholine (PC) lipid is employed in spin labeling electron paramagnetic resonance spectroscopy to probe the water dynamics near lipid bilayer interfaces. Due to its amphiphilic character, however, TEMPO spin label could partition between aqueous and lipid phases, and may even be stabilized in the lipid phase. Accurate assessment of the TEMPO-PC configuration in bilayer membranes is essential for correctly interpreting the data from measurements. Here, we carry out all-atom molecular dynamics (MD) simulations of TEMPO-PC probe in single-component lipid bilayers at varying temperatures, using two standard MD force fields. We find that, for a dipalmitoylphosphatidylcholine (DPPC) membrane whose gel-to-fluid lipid phase transition occurs at 314 K, while the TEMPO spin label is stabilized above the bilayer interface in the gel phase, there is a preferential location of TEMPO below the membrane interface in the fluid phase. For bilayers made of unsaturated lipids, 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which adopt the fluid phase at ambient temperature, TEMPO is unequivocally stabilized inside the bilayers. Our finding of membrane phase-dependent positioning of the TEMPO moiety highlights the importance of assessing the packing order and fluidity of lipids under a given measurement condition.     

Cholesterol hydroxyl group is found to reside in the center of a polyunsaturated lipid membrane

Cholesterol and saturated lipid species preferentially partition into liquid ordered microdomains, such as lipid rafts, away from unsaturated lipid species for which the sterol has less affinity in the surrounding liquid-disordered membrane. To observe how cholesterol interacts with unsaturated phospholipids, we have determined, from one-dimensional neutron scattering length density profiles, the depth of cholesterol in phosphatidylcholine (PC) bilayers with varying amounts of acyl chain unsaturation. Through the use of [2,2,3,4,4,6-(2)H(6)]-labeled cholesterol, we show that in 1-palmitoyl-2-oleoylphosphatidylcholine (16:0-18:1 PC), 1,2-dioleoylphosphatidylcholine (18:1-18:1 PC), and 1-stearoyl-2-arachidonylphosphatidylcholine (18:0-20:4 PC) bilayers the center of mass of the deuterated sites is approximately 16 A from the bilayer center. This location places the hydroxyl group of the sterol moiety at the hydrophobic/hydrophilic bilayer interface, which is the generally accepted position. In dramatic contrast, for 20:4-20:4 PC membranes the hydroxyl group is found, unequivocally, sequestered in the bilayer center. We attribute the change in location to the high disorder of polyunsaturated fatty acids (PUFA) that is incompatible with close proximity to the steroid moiety in its usual "upright" orientation.     

Thermotropic and barotropic phase transitions in bilayer membranes of ether-linked phospholipids with varying alkyl chain lengths

The bilayer phase transitions of a series of ether-linked phospholipids, 1,2-dialkylphosphatidylcholines containing linear saturated alkyl chain (C(n)=12, 14, 16 and 18), were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The thermodynamic quantities of the pre- and main-transitions for the ether-linked PC bilayer membranes were calculated and compared with those of a series of ester-linked PCs, 1,2-diacylphosphatidylcholines. The thermodynamic quantities of the main transition for the ether-linked PC bilayers showed distinct dependence on alkyl-chain length and were slightly different from those of the ester-linked PC bilayers. From the comparison of thermodynamic quantities for the main transition between both PC bilayers, we revealed that the attractive interaction in the gel phase for the ether-linked PC bilayers is weaker than that for the ester-linked PC bilayers. Regarding the pretransition, although changes in enthalpy and entropy for both PC bilayers were comparable to each other, the volume changes of the ether-linked PC bilayers roughly doubled those of the ester-linked PC bilayers. The larger volume change results from the smallest partial molar volume of the ether-linked PC molecule in the interdigitated gel phase. Further, we constructed the temperature-pressure phase diagrams for the ether-linked PC bilayers by using the phase-transition data. The region of the interdigitated gel phase in the phase diagrams was extended by applying pressure and by increasing the alkyl-chain length of the molecule. Comparing the phase diagrams with those for the ester-linked PC bilayers, it was proved that the phase behavior of the ester-linked PC bilayers under high temperature and pressure is almost equivalent to that of the ether-linked PC bilayers in the vicinity of ambient pressure.     

Thermotropic and barotropic phase transitions in bilayer membranes of ether-linked phospholipids with varying alkyl chain lengths

The bilayer phase transitions of a series of ether-linked phospholipids, 1,2-dialkylphosphatidylcholines containing linear saturated alkyl chain (C_n = 12, 14, 16 and 18), were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The thermodynamic quantities of the pre- and main-transitions for the ether-linked PC bilayer membranes were calculated and compared with those of a series of ester-linked PCs, 1,2-diacylphosphatidylcholines. The thermodynamic quantities of the main transition for the ether-linked PC bilayers showed distinct dependence on alkyl-chain length and were slightly different from those of the ester-linked PC bilayers. From the comparison of thermodynamic quantities for the main transition between both PC bilayers, we revealed that the attractive interaction in the gel phase for the ether-linked PC bilayers is weaker than that for the ester-linked PC bilayers. Regarding the pretransition, although changes in enthalpy and entropy for both PC bilayers were comparable to each other, the volume changes of the ether-linked PC bilayers roughly doubled those of the ester-linked PC bilayers. The larger volume change results from the smallest partial molar volume of the ether-linked PC molecule in the interdigitated gel phase. Further, we constructed the temperature-pressure phase diagrams for the ether-linked PC bilayers by using the phase-transition data. The region of the interdigitated gel phase in the phase diagrams was extended by applying pressure and by increasing the alkyl-chain length of the molecule. Comparing the phase diagrams with those for the ester-linked PC bilayers, it was proved that the phase behavior of the ester-linked PC bilayers under high temperature and pressure is almost equivalent to that of the ether-linked PC bilayers in the vicinity of ambient pressure.     

Structure of polymerizable lipid bilayers IV. Mixtures of long chain diacetylenic and short chain saturated phosphatidylcholines and analogous asymmetric isomers.

Polymerization of 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) is enhanced by addition of short-chain saturated phosphatidylcholines such as 1,2-dinonanoyl-sn-glycero-3- phosphocholine (DNPC). Because of well established constraints on the topochemical polymerisation process, we undertook structure-based experiments to determine the nature of this effect. Two hypotheses were tested: (a) that the DNPC crystalized the proximal (m) and disordered the distal (n) methylene segments of DC8,9PC, thus providing flexibility to accommodate the conformational change upon polymer formation, or (b) that the DNPC forced lateral displacement of DC8,9PC, which would then allow interdigitation of these segments with those of the opposing monolayer and potentially more crystalline alignment of the diacetylene. Low angle X-ray diffraction studies do not support the interdigiated chain model. However, these measurements also indicate that the two lipid species may be phase separated under many conditions. An analogous structure, 1-(tricosa-10,12-diynoyl)2-nonanoyl-sn-glycero-3- phosphocholine (C8,9NPC) did not polymerize, and low angle X-ray diffraction studies indicate that bilayers of this lipid were interdigitated such that the terminal methyl group of the tricosadiynoyl chain on each lipid in the bilayer was adjacent to the diacetylenic moiety of a lipid on the opposing monolayer. Implications of these findings pertinent to identifying significant factors in polymerization of diacetylenic phospholipid bilayers are discussed.     

Morphological behavior of acidic and neutral liposomes induced by basic amphiphilic alpha-helical peptides with systematically varied hydrophobic-hydrophilic balance

Lipid-peptide interaction has been investigated using cationic amphiphilic alpha-helical peptides and systematically varying their hydrophobic-hydrophilic balance (HHB). The influence of the peptides on neutral and acidic liposomes was examined by 1) Trp fluorescence quenched by brominated phospholipid, 2) membrane-clearing ability, 3) size determination of liposomes by dynamic light scattering, 4) morphological observation by electron microscopy, and 5) ability to form planar lipid bilayers from channels. The peptides examined consist of hydrophobic Leu and hydrophilic Lys residues with ratios 13:5, 11:7, 9:9, 7:11, and 5:13 (abbreviated as Hels 13-5, 11-7, 9-9, 7-11, and 5-13, respectively; Kiyota, T., S. Lee, and G. Sugihara. 1996. Biochemistry. 35:13196-13204). The most hydrophobic peptide (Hel 13-5) induced a twisted ribbon-like fibril structure for egg PC liposomes. In a 3/1 (egg PC/egg PG) lipid mixture, Hel 13-5 addition caused fusion of the liposomes. Hel 13-5 formed ion channels in neutral lipid bilayer (egg PE/egg PC = 7/3) at low peptide concentrations, but not in an acidic bilayer (egg PE/brain PS = 7/3). The peptides with hydrophobicity less than Hel 13-5 (Hels 11-7 and Hel 9-9) were able to partially immerse their hydrophobic part of the amphiphilic helix in lipid bilayers and fragment liposome to small bicelles or micelles, and then the bicelles aggregated to form a larger assembly. Peptides Hel 11-7 and Hel 9-9 each formed strong ion channels. Peptides (Hel 7-11 and Hel 5-13) with a more hydrophilic HHB interacted with an acidic lipid bilayer by charge interaction, in which the former immerses the hydrophobic part in lipid bilayer, and the latter did not immerse, and formed large assemblies by aggregation of original liposomes. The present study clearly showed that hydrophobic-hydrophilic balance of a peptide is a crucial factor in understanding lipid-peptide interactions.     

Insertion of bacteriorhodopsin into polymerized diacetylenic phosphatidylcholine bilayers

We have developed a method to incorporate the membrane protein bacteriorhodopsin into polymerized bilayers composed of a diacetylenic phosphatidylcholine, 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) and a non-polymerizable phospholipid, dinonanoylphosphatidylcholine (DNPC). The extent of DC8,9PC polymerization in the bilayer was significantly improved when 2:1 mole ratio DNPC-DC8,9PC was used. Octyl glucopyranoside-solubilized bacteriorhodopsin was inserted into the polymerized DNPC-DC8,9PC bilayers by overnight incubation at 4 degrees C followed by dialysis to remove the detergent. The protein was inserted into the membranes after photo-polymerization to avoid inactivation of the protein due to the UV irradiation. The insertion of bacteriorhodopsin into the polymerized DNPC-DC8,9PC membranes was confirmed by density gradient centrifugation, UV/visible spectroscopy, and freeze fracture electron microscopy. The polymerized DNPC-DC8,9PC membranes containing bacteriorhodopsin were about 10% protein by weight. These results suggest that mixed lipid systems such as the DNPC-DC8,9PC can be used to improve both the extent of polymerization and the efficiency of membrane protein incorporation in the polymerized bilayer.     

Structure of polymerizable lipid bilayers. I--1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine, a tubule-forming phosphatidylcholine

This report presents the first X-ray diffraction data on diacetylenic phospholipids. The tubule-forming polymerizable lipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC), was studied by low angle X-ray diffraction from partially dehydrated oriented multibilayers in both polymerized and unpolymerized form. Bilayers of this material were found to be highly ordered, yielding as many as 16 orders of lamellar diffraction, in both the polymerized and unpolymerized states. The unit cell dimension was very small for a lipid of this size. In addition to the features usually observed in the electron density profile structure of phospholipid bilayers, the electron-dense diacetylenic portions of the fatty acyl chain produced electron density maxima at two well-defined levels on each side of the bilayer approximately 15 A and 9 A from the bilayer midplane. A model molecular conformation deduced from the one-dimensional electron density map features all-trans acyl chains tilted at approximately 28 degrees from the bilayer normal that are interdigitated with chains of the opposing monolayer by approximately two carbons at the bilayer center. The linear diacetylene moieties on beta- and gamma-chains appear at different positions along the bilayer normal axis and are roughly parallel to the bilayer surface. This model is discussed in terms of a polymerization mechanism.     

Phospholipid vesicle fusion on micropatterned polymeric bilayer substrates

As an approach to create versatile model systems of the biological membrane we have recently developed a novel micropatterning strategy of substrate-supported planar lipid bilayers (SPBs) based on photolithographic polymerization of a diacetylene phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine. The micropatterned SPBs are composed of a polymeric bilayer matrix and embedded fluid lipid bilayers. In this study, we investigated the incorporation of fluid bilayers into micropatterned polymeric bilayer matrices through the adsorption and reorganization of phospholipid vesicles (vesicle fusion). Total internal reflection fluorescence microscopy observation showed that vesicle fusion started at the boundary of polymeric bilayers and propagated into the central part of lipid-free regions. On the other hand, quartz crystal microbalance with dissipation monitoring revealed that the transformation from adsorbed vesicles into SPBs was significantly accelerated for substrates with micropatterned polymeric bilayers. These results indicate that the edges of polymeric bilayers catalyze the formation of SPBs by destabilizing adsorbed vesicles and also support the premise that polymeric bilayers and embedded fluid bilayers are forming a continuous hybrid bilayer membrane, sealing energetically unfavorable bilayer edges.     

Structural effect of cationic amphiphiles in diacetylenic photopolymerizable membranes

Liposomes have been an excellent option as drug delivery systems, since they are able of incorporating lipophobic and/or lipophilic drugs, reduce drug side effects, increase drug targeting, and control delivery. Also, in the last years, their use reached the field of gene therapy, as non-viral vectors for DNA delivery. As a strategy to increase system stability, the use of polymerizable phospholipids has been proposed in liposomal formulations. In this work, through differential scanning calorimetry (DSC) and electron spin resonance (ESR) of spin labels incorporated into the bilayers, we structurally characterize liposomes formed by a mixture of the polymerizable lipid diacetylenic phosphatidylcholine 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC(8,9)PC) and the zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), in a 1:1 molar ratio. It is shown here that the polymerization efficiency of the mixture (c.a. 60%) is much higher than that of pure DC(8,9)PC bilayers (c.a. 20%). Cationic amphiphiles (CA) were added, in a final molar ratio of 1:1:0.2 (DC(8,9)PC:DMPC:CA), to make the liposomes possible carriers for genetic material, due to their electrostatic interaction with negatively charged DNA. Three amphiphiles were tested, 1,2-dioleoyl-3-trimetylammonium-propane (DOTAP), stearylamine (SA) and trimetyl (2-miristoyloxietyl) ammonium chloride (MCL), and the systems were studied before and after UV irradiation. Interestingly, the presence of the cationic amphiphiles increased liposomes polymerization, MCL displaying the strongest effect. Considering the different structural effects the three cationic amphiphiles cause in DC(8,9)PC bilayers, there seem to be a correlation between the degree of DC(8,9)PC polymerization and the packing of the membrane at the temperature it is irradiated (gel phase). Moreover, at higher temperatures, in the bilayer fluid phase, more polymerized membranes are significantly more rigid. Considering that the structure and stability of liposomes at different temperatures can be crucial for DNA binding and delivery, we expect the study presented here contributes to the production of new carrier systems with potential applications in gene therapy.     

Coarse-grained simulation studies of peptide-induced pore formation

We investigate the interactions between lipid bilayers and amphiphilic peptides using a solvent-free coarse-grained simulation technique. In our model, each lipid is represented by one hydrophilic and three hydrophobic beads. The amphiphilic peptide is modeled as a hydrophobic-hydrophilic cylinder with hydrophilic caps. We find that with increasing peptide-lipid attraction the preferred state of the peptide changes from desorbed, to adsorbed, to inserted. A single peptide with weak attraction binds on the bilayer surface, while one with strong attraction spontaneously inserts into the bilayer. We show how several peptides, which individually bind only to the bilayer surface, cooperatively insert. Furthermore, hydrophilic strips along the peptide cylinder induce the formation of multipeptide pores, whose size and morphology depend on the peptides’ overall hydrophilicity, the distribution of hydrophilic residues, and the peptide-peptide interactions. Strongly hydrophilic peptides insert less readily, but prove to be more destructive to bilayer integrity.     

Relationship between the structure of amphiphilic copolymers and their ability to disturb lipid bilayers

Nonionic amphiphiles and particularly block copolymers of ethylene oxide and propylene oxide (Pluronics) cause pronounced chemosensitization of tumor cells that exhibit multiple resistance to antineoplastic drugs. This effect is due to inhibition of P-glycoprotein (P-gp) responsible for drug efflux. It was suggested that the inhibition of P-gp might be due to changes in its lipid surrounding. Indeed, high dependence of P-gp activity on the membrane microviscosity was demonstrated [Regev et al. (1999) Eur. J. Biochem. 259, 18-24], suggesting that the ability of Pluronics to affect the P-gp activity is mediated by their effect on the membrane structure. We have found recently that adsorption of Pluronics on lipid bilayers induced considerable disturbance of the lipid packing [Krylova et al. (2003) Chemistry 9, 3930-3936]. In the present paper, we studied 19 amphiphilic copolymers, including newly synthesized hyperbranched polyglycerols, Pluronic and Brij surfactants, for their ability to accelerate flip-flop and permeation of antitumor drug doxorubicin (DOX) in liposomes. It was found that not only bulk hydrophobicity but also the chemical microstructure of the copolymer determines its membrane disturbing ability. Copolymers containing polypropylene oxide caused higher acceleration of flip-flop and DOX permeation than polysurfactants containing aliphatic chains. The effects of copolymers containing hyperbranched polyglycerol "corona" were more pronounced, as compared to the copolymers with linear poly(ethylene oxide) chains, indicating that a bulky hydrophilic block induces additional disturbances in the lipid bilayer. A good correlation between the copolymer flippase activity and a linear combination of copolymer bulk hydrophobicity and the van der Waals volume of its hydrophobic block was found. The relationship between the structure of a copolymer and its ability to disturb lipid membranes presented in this paper may be useful for the design of novel amphiphilic copolymers capable of affecting the activity of membrane transporters in living cells.     

Mixing behavior of symmetric chain length and mixed chain length phosphatidylcholines in two-component multilamellar bilayers: evidence for gel and liquid-crystalline phase immiscibility

The mixing behavior of symmetric chain length and mixed chain length phosphatidylcholines in two-component multilamellar bilayers has been investigated by high-sensitivity differential scanning calorimetry. Phase diagrams have been constructed for two-component bilayers composed of C(18)C(18)PC and either C(18)C(16)PC, C(18)C(14)PC, C(18)C(12)PC, or C(18)C(10)PC. It is found that C(18)C(18)PC-C(18)C(16)PC and C(18)C(18)PC-C(18)C(14)PC mixed bilayers exhibit complete miscibility of the components in both the gel and liquid-crystalline phases. Whereas this mixing is observed to be nearly ideal for the C(18)C(18)PC-C(18)C(16)PC binary system, the intermixing of the lipids is highly nonideal in the gel phase of the C(18)C(18)PC-C(18)C(14)PC binary mixture. The C(18)C(18)PC-C(18)C(12)PC and C(18)C(18)PC-C(18)C(10)PC mixed bilayers are characterized by partial immiscibility of the phosphatidylcholine components in the bilayer gel phase. Over a large compositional range, these bilayers appear to consist of phase-separated regions of interdigitated and noninterdigitated gel phases. In addition, the C(18)C(18)PC-C(18)C(10)PC two-component bilayer displays a limited region of liquid-liquid immiscibility in the liquid-crystalline bilayer phase. The phase separation of the mixed chain length phosphatidylcholines revealed in these mixed bilayers may represent a three-dimensional phase separation of the lipid components where the phosphatidylcholines are both laterally separated within the plane of the bilayer and conformationally coupled across the bilayer. Such phase-separated domains could have profound effects on membrane structure and function if they were to occur in biological membranes.     

Interaction of biologically active molecules with phospholipid membranes: I. Fluorescence depolarization studies on the effect of polymeric biocide bearing biguanide groups in the main chain

Interaction of poly(hexamethylene biguanide hydrochloride) (PHMB), which is a polymeric biocide bearing biguanide groups in its main chain, with phospholipid bilayers was studied by the fluorescence depolarization method. A strong interaction of PHMB with negatively charged bilayers composed of phosphatidylglycerol(PG) alone or of PG and phosphatidylcholine (PC) was observed, whereas neutral PC bilayers were not affected. On adding PHMB, the fluorescence polarization of diphenylhexatriene embedded in the negatively charged bilayers was reduced to a great extent, especially in the gel phase. This was interpreted in terms of PHMB-induced expansion and fluidization of the bilayer, which enables the probe molecule to undergo less-hindered torsional motion. Similarity between PHMB and polymyxin B in the structure, the mode of action against bacteria and the interaction with lipid membranes is discussed.     

Interaction of biologically active molecules with phospholipid membranes. I. Fluorescence depolarization studies on the effect of polymeric biocide bearing biguanide groups in the main chain

Interaction of poly(hexamethylene biguanide hydrochloride) (PHMB), which is a polymeric biocide bearing biguanide groups in its main chain, with phospholipid bilayers was studied by the fluorescence depolarization method. A strong interaction of PHMB with negatively charged bilayers composed of phosphatidylglycerol(PG) alone or of PG and phosphatidylcholine (PC) was observed, whereas neutral PC bilayers were not affected. On adding PHMB, the fluorescence polarization of diphenylhexatriene embedded in the negatively charged bilayers was reduced to a great extent, especially in the gel phase. This was interpreted in terms of PHMB-induced expansion and fluidization of the bilayer, which enables the probe molecule to undergo less-hindered torsional motion. Similarity between PHMB and polymyxin B in the structure, the mode of action against bacteria and the interaction with lipid membranes is discussed.     

Effect of hydrostatic pressure on the bilayer phase behavior of symmetric and asymmetric phospholipids with the same total chain length

The bilayer phase transitions of palmitoylstearoyl-phosphatidylcholine (PSPC), diheptadecanoyl-PC (C17PC) and stearoylpalmitoyl-PC (SPPC) which have the same total carbon numbers in the two acyl chains were observed by differential scanning calorimetry and high-pressure optical method. As the temperature increased, these bilayers exhibited four phases of the subgel (Lc), lamellar gel (L beta'), ripple gel (P beta') and liquid crystal (L alpha), in turn. The Lc phase was observed only in the first heating scan after cold storage. The temperatures of the phase transitions were almost linearly elevated by applying pressure. The temperature-pressure phase diagrams and the thermodynamic quantities associated with the phase transitions were compared among the lipid bilayers. For all the bilayers studied, the pressure-induced interdigitated gel (L beta I) phase appeared above the critical interdigitation pressure (CIP) between the L beta' and P beta' phases. The CIPs for the PSPC, C17PC and SPPC bilayers were found to be 50.6, 79.1 and 93.0 MPa, respectively. Contribution of two acyl chains to thermodynamic properties for the phase transitions of asymmetric PSPC and SPPC bilayers was not even. The sn-2 acyl chain lengths of asymmetric PCs governed primarily the bilayer properties. The fluorescence spectra of Prodan in lipid bilayers showed the emission maxima characteristic of bilayer phases, which were dependent on the location of Prodan in the bilayers. Second derivative of fluorescent spectrum exhibited the original emission spectrum of Prodan to be composed of the distribution of Prodan into multiple locations in the lipid bilayer. The F'497/F'430 value, a ratio of second derivative of fluorescence intensity at 497 nm to that at 430 nm, is decisive evidence whether bilayer interdigitation will occur. With respect to the L beta'/L beta I phase transition in the SPPC bilayer, the emission maximum of Prodan exhibited the narrow-range red-shift from 441 to 449 nm, indicating that the L beta I phase in the SPPC bilayer has a less polar "pocket" formed by a space between uneven terminal methyl ends of the sn-1 and sn-2 chains, in which the Prodan molecule remains stably.     

Immobilization and activity assay of cytochrome P450 on patterned lipid membranes

We report on a methodology for immobilizing cytochrome P450 on the surface of micropatterned lipid bilayer membranes and measuring the enzymatic activity. The patterned bilayer comprised a matrix of polymeric lipid bilayers and embedded fluid lipid bilayers. The polymeric lipid bilayer domains act as a barrier to confine fluid lipid bilayers in defined areas and as a framework to stabilize embedded membranes. The fluid bilayer domains, on the other hand, can contain lipid compositions that facilitate the fusion between lipid membranes, and are intended to be used as the binding agent of microsomes containing rat CYP1A1. By optimizing the membrane compositions of the fluid bilayers, we could selectively immobilize microsomal membranes on these domains. The enzymatic activity was significantly higher on lipid bilayer substrates compared with direct adsorption on glass. Furthermore, competitive assay experiment between two fluorogenic substrates demonstrated the feasibility of bioassays based on immobilized P450s.     

Mattress model of lipid-protein interactions in membranes.

A thermodynamic model is proposed for describing phase diagrams of mixtures of lipid bilayers and amphiphilic proteins or polypeptides in water solution. The basic geometrical variables of the model are the thickness of the hydrophobic region of the lipid bilayer and the length of the hydrophobic region of the proteins. The model incorporates the elastic properties of the lipid bilayer and the proteins, as well as indirect and direct lipid-protein interactions expressed in terms of the geometrical variables. The concept of mismatch of the hydrophobic regions of the lipids and proteins is an important ingredient of the model. The general phase behavior is calculated using simple real solution theory. The phase behavior turns out to be quite rich and is used to discuss previous experiments on planar aggregations of proteins in phospholipid bilayers and to propose a systematic study of synthetic amphiphilic polypeptides in bilayers of different thicknesses. The model is used to interpret the influence of the lipid-protein interaction on calorimetric measurements and on local orientational order as determined by deuterium nuclear magnetic resonance.     

Regulation of the gating of BKCa channel by lipid bilayer thickness

Transmembrane segments of ion channels tend to match the hydrophobic thickness of lipid bilayers to minimize mismatch energy and to maintain their proper organization and function. To probe how ion channels respond to mismatch with lipid bilayers of different thicknesses, we examined the single channel activities of BK(Ca) (hSlo alpha-subunit) channels in planar bilayers of binary mixtures of DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) with phosphatidylcholines (PCs) of varying chain lengths, including PC 14:1, PC 18:1, PC 22:1, PC 24:1, and with porcine brain sphingomyelin. Bilayer thickness and structure was measured with small angle x-ray diffraction and atomic force microscopy. The open probability (P(o)) of the BK(Ca) channel was finely tuned by bilayer thickness, first decreasing with increases in bilayer thickness from PC 14:1 to PC 22:1 and then increasing from PC 22:1 to PC 24:1 and to porcine brain sphingomyelin. Single channel kinetic analyses revealed that the mean open time of the channel increased monotonically with bilayer thickness and, therefore, could not account for the biphasic changes in P(o). The mean closed time increased with bilayer thickness from PC 14:1 up to PC 22:1 and then decreased with further increases in bilayer thickness to PC 24:1 and sphingomyelin, correlating with changes in P(o). This is consistent with the proposition that bilayer thickness affects channel activity mainly through altering the stability of the closed state. We suggest a simple mechanical model that combines forces of lateral stress within the lipid bilayer with local hydrophobic mismatch between lipids and the protein to account for the biphasic modulation of BK(Ca) gating.     

Effect of Peg Homopolymer and Grafted Amphiphilic Peg-Palmityl on the Thermotropic Phase Behavior of 1,2-Dipalmitoyl-Sn-Glycero-3-Phosphocholine Bilayer

Abstract

Phospholipids covalently attached to polyethylene glycol (PEG-PE) are routinely used for the preparation of long-circulating liposomes. The common preparation procedure for long-circulating liposomes involves use of organic solvent. Although there is a plethora of studies describing the interaction of PEG-PE with bilayers, little is known about the effects of PEG homopolymers and single chain amphiphilic PEG on liposome structure. In the present investigation the interaction of PEG homopolymer and amphiphilic PEG-palmityl conjugate with large multilamellar liposomes composed of 1,2-dipalmitoyl-sn-glycero-phosphocholine was investigated utilizing differential scanning calorimetry. Vesicle and aggregate sizes were determined by dynamic light scattering. DSC thermograms revealed interaction of PEG homopolymer with DPPC when the two are premixed in organic solvent. The data suggest that PEG interacts with the phospholipid acyl chains deep in the bilayer. Several questions are raised regarding the suitability of the current procedure for preparation of long-circulating liposomes which utilizes organic solvent. Incorporation of only 2 mol% 5 kDa PEG-palmityl conjugate completely solubilized DPPC liposomes. Packing geometry of the lipid anchor, irrespective of the polymer molecular weight, is suggested to be the primary factor for successful grafting of hydrophilic polymers on liposomes. Pure PEG-palmityl formed self-assembled organized structures of potential use in the delivery of poorly soluble drugs.