Effects of hydrostatic pressure on the location of PRODAN in lipid bilayers and cellular membranes

The effects of hydrostatic pressure on the location of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN), an environmentally sensitive fluorescent probe, in phosphatidylcholine lipid bilayers and in goldfish brain synaptic membranes have been studied by fluorescence spectroscopy over the pressure range of 0.001-2 kbar. The emission spectrum of PRODAN in all the membrane systems examined exhibits two local maxima: one centers at around 435 nm and the other at about 510 nm. The intensity ratio of these two peaks, F435/F510, increases as pressure increases; in the particular case of dimyristoyl-L-alpha-phosphatidylcholine multilamellar vesicles [DMPC(MLV)], a dramatic change in F435/F510 appears at the lipid phase transition pressure. As pressure varies, an isoemissive point is seen in both egg yolk phosphatidylcholines and goldfish brain synaptic membranes; however, no isoemissive point is observed in DMPC(MLV). The presence of an isoemissive point is attributed to a pressure-induced relocation of PRODAN from the "polar" disposition (the 510-nm peak) to the "less polar" disposition (the 435-nm peak). The absence of an isoemissive point in the case of DMPC(MLV) is probably due to the lack of void space in the lipid matrix, as a result of tight lipid packing. Apparently, the probe relocation takes place in unsaturated systems, and PRODAN favors a more hydrophobic environment under pressure. However, on the basis of the emission spectra, PRODAN seems to remain more or less at the interfacial region over the pressure range examined. In goldfish brain synaptic membranes, the PRODAN polarization increases with pressure, giving dT/dP values of 15-20 degrees C kbar-1 for both dispositions.(ABSTRACT TRUNCATED AT 250 WORDS)     

A model for phase transitions in lipid bilayers and biological membranes

We wish to present an order-disorder model for the observed phase transitions in lipid bilayers and biological membranes. We show that the model may, under certain circumstances, exhibit two phase transitions, one corresponding to positional disordering of entire lipid molecules, and the other corresponding to orientational disordering in the hydrocarbon chains. We then give results of our numerical analysis of the model and compare them with experimental data. Shortcomings of the model and future directions for analyses of this type are also discussed.     

Some similarities between processes at biological membranes and lipid bilayers

Several processes at biological membranes can be simulated by experiments with artificial lipid bilayer membranes. Three selected examples are discussed: The uncoupler induced proton permeability of lipid bilayers, the initiation of action potential like voltage responses in lipid membranes, and the reconstitution of active cation pumps across planar lipid bilayers or lipid vesicles.Paper presented at the Biomembrane Symposium of the Deutsche Gesellschaft für Biophysik, Freiburg, April 1975.Supported by Deutsche Forschungsgemeinschaft, SFB 38 Membranforschung.     

The effect of hydrostatic pressure on S-layer-supported lipid membranes

We report on the behavior of unsupported and surface layer (S-layer)-supported lipid membranes at the application of a uniform hydrostatic pressure. At a hydrostatic pressure gradient higher than 6 N/m(2), unsupported lipid membranes, independent from which side pressurized and S-layer-supported lipid membranes pressurized from the lipid-faced side revealed a pronounced increase in capacitance. A maximal hydrostatic pressure gradient of 11.0 N/m(2) resulted in an almost doubling of the capacitance of the (composite) membranes. S-layer-supported lipid membranes showed a hysteresis in the capacitance versus pressure plot, indicating that this composite structure required a certain time to reorient when the pressure gradient acting from the lipid-faced side was balanced. By contrast, the S-layer-supported lipid membrane pressurized from the protein-faced side revealed only a minute increase in capacitance (C/C(0,max)=1.17+/-0.05), reflecting only minor pressure-induced area expansion. In addition, no hysteresis could be observed, indicating that no rearrangement of the composite membrane occurred. The maximal induced tension was with 4.3+/-0.2 mN/m, significantly higher than that of unsupported (2.5+/-0.3 mN/m) and S-layer-supported lipid membranes pressurized from the lipid-faced side (2.6+/-0.1 mN/m).     

A study of water autodiffusion in model biological membranes, oriented lipid bilayers

The autodiffusion of water in a multibilayer structure formed by dipalmitoyl phosphatidylcholine and oriented on glass plates was studied by the method of NMR with magnetic field pulse gradient. It was shown that water molecules occur in several states differing in the degree of interaction with lipid molecules. A spectrum of the coefficients of water autodiffusion in a direction transversal to bilayers was found. The use of samples with different distances between the plates and an analysis of the dependence of the mode of diffuse decay of spin echo on diffusion time and the orientation of the sample, as well as measurements at temperatures above and below the gel-liquid crystal phase transition in cholesterol-containing samples enabled one to discriminate the diffuse decay component responsible for the transbilayer movement of water. The coefficient of bilayer permeability was estimated using the Tanner model. It was shown that the formation of mechanical defects ("cracks") in plane oriented bilayers is the most probable reason for the presence of the water component with the relatively high coefficient of diffusion.     

Effects of hydrostatic pressure on the location of PRODAN in lipid bilayers: a FT-IR study

The effects of hydrostatic pressure on the location of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN), an environmentally sensitive fluorescent probe, in phosphatidylcholine lipid bilayers have been studied by Fourier-transform infrared spectroscopy (FT-IR) over the pressure range of 0.001-25 kbar. The results derived from the PRODAN C = O stretching band, the correlation field splitting of the methylene scissoring mode, and the methylene symmetric stretching mode as well as the absorption of the naphthalene ring show that in the sample of 4% (w/w) PRODAN in dimyristoyl-L-alpha-phosphatidylcholine (DMPC) at pH 6.8, most of the PRODAN molecules are embedded in the bilayers. In contrast, at pH 3.0, PRODAN was found to reside either on the membrane surface or dispersed in water. Compared to DMPC, egg yolk phosphatidylcholine (egg PC), which contains a substantial amount of unsaturated fatty acyl chains, is more susceptible to PRODAN permeation. The present study shows that the pressure dependence of the location of PRODAN in lipid membranes is different from that of tetracaine, a local anesthetic, in lipid bilayers. The model regarding the PRODAN location in lipid bilayers derived from the present infrared data has been compared with that obtained with previous fluorescence studies.     

Electron spin resonance study on the permeability of superoxide radicals in lipid bilayers and biological membranes.

The permeability of lipid bilayers and biological membranes to superoxide free radicals was examined by using superoxide dismutase (SOD)-loaded lipid vesicles and SOD-loaded erythrocyte ghosts. After exposing SOD lipid vesicles and SOD ghosts to enzymatically produced superoxide radicals and using spin-trapping and electron spin resonance (ESR) techniques, we found that SOD entrapped within erythrocyte ghosts effectively scavenges external O2.- while SOD inside the lipid bilayers has no effect. These results confirm that O2.- is able to cross through a biological plasma membrane but not across a pure lipid bilayer. The data provide instruction as to how and where anti-oxidant therapy is to be approached relative to the site of oxygen free radical production.     

Evaluation of the thickness of lipid bilayers of biological membranes by measurement of specific capacity

The estimate of the thickness of the hydrophobic part of biological membranes by means of its formula for plane capacitor leads to a necessity of very high values of protein dielectric permeability. The current estimate can be performed if the membrane possesses a wavy cross-section. Due to the existence of integral proteins which occupy about 1/3 of the bilayer surface and have dielectric permeability epsilon = 6 divided by 8 and also due to some peculiarities of intermembrane electric field the thickness of hydrophobic bilayer region can be estimated from membrane specific capacity as 3 divided by 3.5 nm, this value coincides with X-ray data.     

Structural studies of polymer-cushioned lipid bilayers.

The structure of softly supported polymer-cushioned lipid bilayers, prepared in two different ways at the quartz-solution interface, were determined using neutron reflectometry. The polymer cushion consisted of a thin layer of branched, cationic polyethyleneimine (PEI), and the bilayers were formed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles. When vesicles were first allowed to adsorb to a bare quartz substrate, an almost perfect bilayer formed. When the polymer was then added to the aqueous solution, it appeared to diffuse beneath this bilayer, effectively lifting it from the substrate. In contrast, if the polymer layer is adsorbed first to the bare quartz substrate followed by addition of vesicles to the solution, there is very little interaction of the vesicles with the polymer layer, and the result is a complex structure most likely consisting of patchy multilayers or adsorbed vesicles.     

Ethanol causes decreased partitioning into biological membranes without changes in lipid order

One of the adaptive responses of cell membranes to chronic ethanol consumption is the acquisition of a resistance to fluidization or disordering of the lipids by ethanol in vitro and a reduced partitioning of ethanol into the membrane (membrane tolerance). The degree to which the effects on partitioning and lipid disordering share common features has not previously been explored and in addition the relevance of the value of lipid order in the absence of added ethanol (baseline lipid order) to membrane tolerance has not been established. The location in the bilayer and the nature of the modification underlying these effects is also unknown. The effect of chronic ethanol treatment was examined using 5-doxyl decane as a model hydrophobic compound. Its partitioning into the membranes was determined by utilizing its ability to quench fluorophores (1,6-diphenyl-2,3,5-hexatriene and 3- and 12-anthroyl stearates) by collisional quenching. The partition coefficient of 5-doxyl decane into the bilayer central region was reduced as a result of the chronic ethanol treatment. The effect could also be demonstrated in vesicles of phospholipids and was lost 4 days after withdrawal of the ethanol from the diet. These results closely parallel those relating to resistance to lipid disordering and suggest that both techniques detect a common modification. Lipid order was assessed using fluorescence anisotropy measurements of a range of fluorophores, including those used to determine the partitioning properties of the membrane. No effect of chronic ethanol treatment on lipid order was found, either in the intact membranes or in vesicles of extracted phospholipids. This suggests that changes in baseline order are not critical features of membrane tolerance in liver microsomes. In addition it appears that the altered partitioning of the 5-doxyl decane into the central region of the membrane is not related to lipid order changes in this region. The reduced partitioning of 5-doxyl decane may be a reflection of a redistribution in the lipid bilayer, perhaps due to modifications in other locations in the membrane, such as the lipid head group region.     

The physicochemical properties of cardiolipin bilayers and cardiolipin-containing lipid membranes

In this review article, we summarize the current state of biophysical knowledge concerning the phase behavior and organization of cardiolipin (CL) and CL-containing phospholipid bilayer model membranes. We first briefly consider the occurrence and distribution of CL in biological membranes and its probable biological functions therein. We next consider the unique chemical structure of the CL molecule and how this structure may determine its distinctive physical properties. We then consider in some detail the thermotropic phase behavior and organization of CL and CL-containing lipid model membranes as revealed by a variety of biophysical techniques. We also attempt to relate the chemical properties of CL to its function in the biological membranes in which it occurs. Finally, we point out the requirement for additional biophysical studies of both lipid model and biological membranes in order to increase our currently limited understanding of the relationship between CL structure and physical properties and CL function in biological membranes.     

Translocation of histone proteins across lipid bilayers and Mycoplasma membranes

We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).     

Maleimide-functionalized lipids that anchor polypeptides to lipid bilayers and membranes

Two maleimide-containing diacylglycerol derivatives were synthesized to permit the anchoring of short peptides and longer polypeptides to phospholipid bilayers and membranes. The maleimide was introduced at the site normally occupied by a phospholipid headgroup. The first lipid, the dipalmitoyl ester of 1-maleimido-2,3-propanediol, was developed as a membrane anchor for extracellular domains of transmembrane proteins. The second anchoring lipid, in which the 3-position contained a 6-aminohexanoate, was designed for convenient modification with amine-reactive reporter groups. Specifically, the NBD fluorophore, 7-nitrobenzo-2-oxa-1, 3-diazole-aminohexanoic-N-hydroxysuccinimide ester, was attached to give an fluorescent anchoring reagent. Next, these reagents were applied to the anchoring of a C-terminally cysteamine-modified 8 kDa polypeptide that comprises the extracellular N-terminal domain of the human thrombin receptor, a transmembrane protease-activated receptor (PAR-1). Gel filtration and fluorescence analysis showed that the fluorescent lipopolypeptide spontaneously inserted into preformed phospholipid vesicles, but it did not insert into whole cell membranes. In contrast, the dipalmitoyl derivative could only be reconstituted into artificial membranes by mixing the lipopolypeptide and phospholipid before vesicle formation. These results suggest that biophysical interactions governing the lipopolypeptide insertion into artificial and cellular membranes may differ. The thiol-reactive lipidating reagents should be valuable materials for studying the structure and function of peptides and polypeptides at phospholipid bilayer surfaces.     

Structural changes of the prion protein in lipid membranes leading to aggregation and fibrillization

Prion diseases are associated with a major refolding event of the normal cellular prion protein, PrP(C), where the predominantly alpha-helical and random coil structure of PrP(C) is converted into a beta-sheet-rich aggregated form, PrP(Sc). Under normal physiological conditions PrP(C) is attached to the outer leaflet of the plasma membrane via a GPI anchor, and it is plausible that an interaction between PrP and lipid membranes could be involved in the conversion of PrP(C) into PrP(Sc). Recombinant PrP can be refolded into an alpha-helical structure, designated alpha-PrP isoform, or into beta-sheet-rich states, designated beta-PrP isoform. The current study investigates the binding of beta-PrP to model lipid membranes and compares the structural changes in alpha- and beta-PrP induced upon membrane binding. beta-PrP binds to negatively charged POPG membranes and to raft membranes composed of DPPC, cholesterol, and sphingomyelin. Binding of beta-PrP to raft membranes results in substantial unfolding of beta-PrP. This membrane-associated largely unfolded state of PrP is slowly converted into fibrils. In contrast, beta-PrP and alpha-PrP gain structure with POPG membranes, which instead leads to amorphous aggregates. Furthermore, binding of beta-PrP to POPG has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents, whereas raft vesicles are not destabilized upon binding of beta-PrP.     

Localization and mobility of coenzyme Q in lipid bilayers and membranes.

We have studied the mobility of coenzyme Q (CoQ) in lipid bilayers and mitochondrial membranes in relation to the control of electron transfer activities. A molecular dynamics computer simulation in the vacuum yielded a folded structure for CoQ10, with a length of only 21 A. Using this information we were able to calculate diffusion coefficients in the range of 10(-6) cm2/s in good agreement with those found experimentally by fluorescence quenching of pyrene derivatives. To investigate if CoQ diffusion may represent the rate-limiting step of electron transfer, we reconstituted complexes I and III and assayed the resulting NADH-cytochrome c reductase activity in presence of different CoQ10 levels and at different distances between complexes; the experimental turnovers were higher than the collision frequencies calculated using diffusion coefficients of 10(-9) cm2/s but compatible with values found by us by fluorescence quenching. Since the experimental turnovers are independent of the distance between complexes, we conclude that CoQ diffusion is not rate-limiting for electron transfer.     

Structural and functional responses of the bullfrog urinary bladder to distension caused by hydrostatic pressure gradients

The responses to mucosal pressure elevation (physiological pressure: PP) were compared to responses to serosal pressure elevation (non-physiological pressure: NPP) in bullfrog urinary bladders (Rana catesbeiana). The bladders were mounted on vertical chambers as flat sheets. Distension was applied with 98.07 Pa. pressure gradients. PP resulted in increases in transepithelial electrical potential difference (TEP) and short-circuit current (SCC). Electrical resistance (R), urea permeability (P(urea)) and net water flux (J( v)) were not effected. NPP resulted in decreases in TEP (38%), SCC (13%), and R (36%). While P(urea) (97%) and J(v) (96%) increased. PP caused little or no change in the electron microscopic structure of frog bladder while NPP caused irreversible dilation of the lateral intercellular spaces. There were no observable changes in tight junctions under PP or NPP. The subepithelial elements of the bladder became detached from the epithelial layer during NPP suggesting a role for them during PP.     

Application of pressure perturbation calorimetry to lipid bilayers

Pressure perturbation calorimetry (PPC) is a new method that measures the heat consumed or released by a sample after a sudden pressure jump. The heat change can be used to derive the thermal volume expansion coefficient, alpha(V), as a function of temperature and, in the case of phase transitions, the volume change, DeltaV, occurring at the phase transition. Here we present the first report on the application of PPC to determine these quantities for lipid bilayers. We measure the volume changes of the pretransition and main transition of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the thermal expansivity of the fluid phase of DMPC and of two unsaturated lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine. The high sensitivity of PPC instrumentation gives accurate data for alpha(V) and DeltaV even upon the application of relatively low pressures of approximately 5 bar.     

Creating biological membranes on the micron scale: forming patterned lipid bilayers using a polymer lift-off technique

We present a new method for creating patches of fluid lipid bilayers with conjugated biotin and other compounds down to 1 microm resolution using a photolithographically patterned polymer lift-off technique. The patterns are realized as the polymer is mechanically peeled away in one contiguous piece in solution. The functionality of these surfaces is verified with binding of antibodies and avidin on these uniform micron-scale platforms. The biomaterial patches, measuring 1 micro m-76 microm on edge, provide a synthetic biological substrate for biochemical analysis that is approximately 100x smaller in width than commercial printing technologies. 100 nm unilamellar lipid vesicles spread to form a supported fluid lipid bilayer on oxidized silicon surface as confirmed by fluorescence photobleaching recovery. Fluorescence photobleaching recovery measurements of DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC(18)(3))) stained bilayer patches yielded an average diffusion coefficient of 7.54 +/- 1.25 microm(2) s(-1), equal to or slightly faster than typically found in DiI stained cells. This diffusion rate is approximately 3x faster than previous values for bilayers on glass. This method provides a new means to form functionalized fluid lipid bilayers as micron-scale platforms to immobilize biomaterials, capture antibodies and biotinylated reagents from solution, and form antigenic stimuli for cell stimulation.