Tetraether Lipid Membranes Research Articles

Vesicular and Planar Membranes of Archaea Lipids: Unusual Physical Properties and Biomedical Applications.

Liposomes and planar membranes made of archaea or archaea-like lipids exhibit many unusual physical properties compared to model membranes composed of conventional diester lipids. Here, we review several recent findings in this research area, which include (1) thermosensitive archaeosomes with the capability to drastically change the membrane surface charge, (2) MthK channel’s capability to insert into tightly packed tetraether black lipid membranes and exhibit channel activity with surprisingly high calcium sensitivity, and (3) the intercalation of apolar squalane into the midplane space of diether bilayers to impede proton permeation. We also review the usage of tetraether archaeosomes as nanocarriers of therapeutics and vaccine adjuvants, as well as the biomedical applications of planar archaea lipid membranes. The discussion on archaeosomal therapeutics is focused on partially purified tetraether lipid fractions such as the polar lipid fraction E (PLFE) and glyceryl caldityl tetraether (GCTE), which are the main components of PLFE with the sugar and phosphate removed.

Improved Isolation of SlaA and SlaB S-layer proteins in Sulfolobus acidocaldarius.

The Sulfolobus acidocaldarius S-layer is composed of two main proteins: SlaA, which forms the ordered structure of the S-layer matrix, and SlaB, which supports and anchors the S-layer into the tetraether lipid membrane. While SlaA has previously been purified by exploiting its thermotolerance and high resistance to detergents, SlaB has resisted isolation, particularly from the cell membrane. Removal of proteins other than those of the S-layer is especially difficult if large batch-scale culture volumes are unavailable. Here, we describe a benchtop-scale protocol for the purification of SlaA from S. acidocaldarius, enabling isolation of SlaB using size exclusion chromatography (gel filtration). Using this protocol, we were able to identify for the first time tetraether lipids strongly attached to SlaB via heat- and detergent-resistant interactions.

Sulfolobus acidocaldarius Microvesicles Exhibit Unusually Tight Packing Properties as Revealed by Optical Spectroscopy.

In this study, we used optical spectroscopy to characterize the physical properties of microvesicles released from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sa-MVs). The most abundant proteins in Sa-MVs are the S-layer proteins, which self-assemble on the vesicle surface forming an array of crystalline structures. Lipids in Sa-MVs are exclusively bipolar tetraethers. We found that when excited at 275 nm, intrinsic protein fluorescence of Sa-MVs at 23 °C has an emission maximum at 303 nm (or 296 nm measured at 75 °C), which is unusually low for protein samples containing multiple tryptophans and tyrosines. In the presence of 10–11 mM of the surfactant n-tetradecyl-β-d-maltoside (TDM), Sa-MVs were disintegrated, the emission maximum of intrinsic protein fluorescence was shifted to 312 nm, and the excitation maximum was changed from 288 nm to 280.5 nm, in conjunction with a significant decrease (>2 times) in excitation band sharpness. These data suggest that most of the fluorescent amino acid residues in native Sa-MVs are in a tightly packed protein matrix and that the S-layer proteins may form J-aggregates. The membranes in Sa-MVs, as well as those of unilamellar vesicles (LUVs) made of the polar lipid fraction E (PLFE) tetraether lipids isolated from S. acidocaldarius (LUVPLFE), LUVs reconstituted from the tetraether lipids extracted from Sa-MVs (LUVMV) and LUVs made of the diester lipids, were investigated using the probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The generalized polarization (GP) values of Laurdan in tightly packed Sa-MVs, LUVMV, and LUVPLFE were found to be much lower than those obtained from less tightly packed DPPC gel state, which echoes the previous finding that the GP values from tetraether lipid membranes cannot be directly compared with the GP values from diester lipid membranes, due to differences in probe disposition. Laurdan’s GP and red-edge excitation shift (REES) values in Sa-MVs and LUVMV decrease with increasing temperature monotonically with no sign for lipid phase transition. Laurdan’s REES values are high (9.3–18.9 nm) in the tetraether lipid membrane systems (i.e., Sa-MVs, LUVMV and LUVPLFE) and low (0.4–5.0 nm) in diester liposomes. The high REES and low GP values suggest that Laurdan in tetraether lipid membranes, especially in the membrane of Sa-MVs, is in a very motionally restricted environment, bound water molecules and the polar moieties in the tetraether lipid headgroups strongly interact with Laurdan’s excited state dipole moment, and “solvent” reorientation around Laurdan’s chromophore in tetraether lipid membranes occurs very slowly compared to Laurdan’s lifetime.

Biological Membranes in Extreme Conditions: Anionic Tetraether Lipid Membranes and Their Interactions with Sodium and Potassium.

Archaea such as Sulfolobus acidocaldarius tolerate extreme temperatures and high acidity and can grow in the presence of toxic metals and low concentrations of Na+ or K+. It is believed that their unique tetraether membranes protect them from harsh environments and allow their survival under such conditions. We used molecular dynamics simulations to study membranes comprising glycerol dialkylnonitol tetraether lipids, which are the main component of S. acidocaldarius membranes, in solutions containing different concentrations of NaCl and KCl or with Na+ or K+ counterions (trace cations, 0 M). Anionic binding sites on the membranes were almost 50% occupied in the presence of counterions. The free energy of cation-phosphate complexation and the residence times of ions near the membranes were found to be both ion- and concentration-dependent. Sodium ions had more favorable interactions with the membranes and a longer residence time, whereas higher cation concentrations led to shorter ion residence times. When only counterions were present in the solutions, large residence times suggested that the membrane may function as a cation-attracting reservoir. The results suggested that the ions can be easily transferred to the cytoplasm as needed, explaining the growth curves of S. acidocaldarius under different salinities and pH.

Biological Membranes in Extreme Conditions: Simulations of Anionic Archaeal Tetraether Lipid Membranes.

In contrast to the majority of organisms that have cells bound by di-ester phospholipids, archaeal membranes consist of di- and tetraether phospholipids. Originating from organisms that withstand harsh conditions (e.g., low pH and a wide range of temperatures) such membranes have physical properties that make them attractive materials for biological research and biotechnological applications. We developed force-field parameters based on the widely used Generalized Amber Force Field (GAFF) to enable the study of anionic tetraether membranes of the model archaean Sulfolobus acidocaldarius by computer simulations. The simulations reveal that the physical properties of these unique membranes depend on the number of cyclopentane rings included in each lipid unit, and on the size of cations that are used to ensure charge neutrality. This suggests that the biophysical properties of Sulfolobus acidocaldarius cells depend not only on the compositions of their membranes but also on the media in which they grow.

Effect of Headgroups on Small-Ion Permeability across Archaea-Inspired Tetraether Lipid Membranes.

This paper examines the effects of four different polar headgroups on small-ion membrane permeability from liposomes comprised of Archaea-inspired glycerolmonoalkyl glycerol tetraether (GMGT) lipids. We found that the membrane-leakage rate across GMGT lipid membranes varied by a factor of ≤1.6 as a function of headgroup structure. However, the leakage rates of small ions across membranes comprised of commercial bilayer-forming 1-palmitoyl-2-oleoyl-sn-glycerol (PO) lipids varied by as much as 32-fold within the same series of headgroups. These results demonstrate that membrane leakage from GMGT lipids is less influenced by headgroup structure, making it possible to tailor the structure of the polar headgroups on GMGT lipids while retaining predictable leakage properties of membranes comprised of these tethered lipids.

Biomimetic surface modification with bolaamphiphilic archaeal tetraether lipids via liposome spreading.

Through investigations of the self-assembly behavior of three different tetraether lipids, the authors successfully established a solid supported, biomimetic tetraether lipid membrane via liposome spreading. These bolaamphiphilic lipids are the main compound in membranes of archaea, extremophile microorganisms, which underwent an enormous adaptation to extreme conditions in their natural environment with regard to temperature, pH, and high salt concentrations. Starting from a mathematical point of view, the authors calculated hydrophilic-lipophilic balance values for each lipid and recognized a wide difference in self-assembly potentials relying on size and hydrophilic properties of the lipid head groups. These results were in good accordance with data generated by lipid experiments at the air-water interface applying a Langmuir-Blodgett film balance so that the self-assembly potential of two different tetraether lipids was found to be sufficient to form stable liposomes in aqueous media. Liposomes composed of the main phospholipid of the archaea strain Sulfolobus acidocaldarius fused covalently on silanized glass substrates and formed a monomolecular lipid layer with upright standing molecules at film consistent thicknesses of approximately 5 nm determined by ellipsometry and atomic force microscopy. This work can be considered as a basic strategy to find optimized lipid properties in terms of liposome formation and spreading in water, and it is the first report about archaeal liposome fusing on surfaces to establish a solid supported lipid monolayer.

Physical Properties of Archaeal Tetraether Lipid Membranes As Revealed by Differential Scanning and Pressure Perturbation Calorimetry, Molecular Acoustics, and Neutron Reflectometry: Effects of Pressure and Cell Growth Temperature

The polar lipid fraction E (PLFE) is a major tetraether lipid component in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Using differential scanning and pressure perturbation calorimetry as well as ultrasound velocity and density measurements, we have determined the compressibilities and volume fluctuations of PLFE liposomes derived from different cell growth temperatures (T(g) = 68, 76, and 81 °C). The compressibility and volume fluctuation values of PLFE liposomes, which are substantially less than those detected from diester lipid membranes (e.g., DPPC), exhibit small but significant differences with T(g). Among the three T(g)s employed, 76 °C leads to the least compressible and most tightly packed PLFE membranes. This temperature is within the range for optimal cell growth (75-80 °C). It is known that a decrease in T(g) decreases the number of cyclopentane rings in archael tetraether lipids. Thus, our data enable us to present the new view that membrane packing in PLFE liposomes varies with the number of cyclopentane rings in a nonlinear manner, reaching maximal tightness when the tetraether lipids are derived from cells grown at optimal T(g)s. In addition, we have studied the effects of pressure on total layer thickness, d, and neutron scattering length density, ρ(n), of a silicon-D(2)O interface that is covered with a PLFE membrane using neutron reflectometry (NR). At 55 °C, d and ρ(n) are found to be rather insensitive to pressure up to 1800 bar, suggesting minor changes of the thickness of the membrane's hydrophobic core and headgroup orientation upon compression only.

On physical properties of tetraether lipid membranes: effects of cyclopentane rings.

This paper reviews the recent findings related to the physical properties of tetraether lipid membranes, with special attention to the effects of the number, position, and configuration of cyclopentane rings on membrane properties. We discuss the findings obtained from liposomes and monolayers, composed of naturally occurring archaeal tetraether lipids and synthetic tetraethers as well as the results from computer simulations. It appears that the number, position, and stereochemistry of cyclopentane rings in the dibiphytanyl chains of tetraether lipids have significant influence on packing tightness, lipid conformation, membrane thickness and organization, and headgroup hydration/orientation.

A molecular dynamics study of an archaeal tetraether lipid membrane: Comparison with a dipalmitoylphosphatidylcholine lipid bilayer

Molecular dynamics simulations of an archaeal membrane made up of bipolar tetraether lipids and a dipalmitoylphosphatidylcholine (DPPC) lipid membrane were performed and compared for the first time. The simulated archaeal membrane consists of a pure monolayer of asymmetrical lipids, analogous to the main polar lipid [MPL; Swain, M., Brisson, J.-R., Sprott, G.D., Cooper, F.P., and Patel, G.B., (1997) Identification of beta-L-Gulose as the Sugar Moiety of the Main Polar Lipid of Thermoplasma acidophilum, Biochim. Biophys. Acta 1345, 56-64] found in T. acidophilum, an extremophile archaeal organism. This simulated membrane lipid contains two cyclopentane rings located on one of the two aliphatic chains of the lipid. The archaeal membrane is simulated at 62degreesC, slightly above the optimal growth temperature of T. acidophilum. We compared the organization of this tetraether lipid monolayer with a DPPC bilayer simulated at 50degreesC, both of them being modeled in a partially hydrated state. Our results assess the singularity of the tetraether lipid organization, in particular the influence of the spanning structure on the molecular ordering within the archaeal membrane.

Structure and Conformation of Bipolar Tetraether Lipid Membranes Derived from Thermoacidophilic Archaeon Sulfolobus acidocaldarius as Revealed by Small-Angle X-ray Scattering and High-Pressure FT-IR Spectroscopy

The phase behavior, conformation, and structure of bipolar tetraether liposomes composed of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius have been studied by small-angle X-ray scattering (SAXS) and high-pressure Fourier transform infrared spectroscopy (FT-IR) at pD 2.15. The SAXS data on PLFE multilamellar vesicles in 85 wt % D 2 O showed two orders of lamellar Bragg reflections over the temperature range 5-75 °C. The lamellar repeat distances d indicate three lamellar regions: 5-50 °C (d, ∼49-50 A), 50-60 °C (∼50-54 A), and 60-74 °C (∼54-56 A). The 4-A increase in d-spacing from 50 to 60 °C is probably due to an increase in hydration at the PLFE polar headgroups and/or a stretch of the polar headgroups toward the bulk aqueous phase. At 74-75 °C, a lamellar-to-cubic phase transition occurs. The reciprocal spacings of the cubic phases correspond to the coexistence of the inverse bicontinuous cubic phases Q I I D and Q I I P with lattice constants of 105 and 82 A, respectively. At atmospheric pressure, the vibrational frequency of the CH 2 symmetric stretching mode showed transitions at ∼46-48, 61, and 74 °C, in agreement with the SAXS data. The overall 2 cm - 1 increase in wavenumber between 61 and 74 °C indicates a transition from a rigid, gellike lipid conformation to a chain conformation with considerable disorder, as expected from cubic phases which appear above ∼74 °C. The pressure dependence (up to 15 kbar) of the symmetric CH 2 stretching vibrational wavenumber at various temperatures has also been examined. A variety of new gellike phases are observed at elevated pressures, showing again a rich polymorphism in PLFE liposomes. The most prominent pressure-induced transition involves a ∼2 cm - 1 increase in wavenumber, which appears at 8.0 kbar at 60 °C, 8.4 kbar at 43 °C, and 10.8 kbar at 20 °C, giving an unusual negative dT/dp value. The possible source of this anomaly has been discussed in terms of the temperature attenuation of the hydrogen bond network in the polar headgroup region.

New Method for Generating Tetraether Lipid Membranes on Porous Supports

The present paper describes a new and reproducible method to generate stable lipid membranes on porous supports. The support was produced by the deposition of a smooth layer of crystalline bacterial cell surface (S-layer) protein lattices on a microfiltration membrane (SUM). The commercially available main phospholipid of Thermoplasma acidophilum (MPL), a membrane-spanning tetraether lipid, but also mixtures of MPL with diphytanoylphosphatidylcholine (DPhPC) at molar ratios of MPL/DPhPC = 1:1 and 5:1 and pure DPhPC were spread at the air/water interface. The monomolecular films were transferred by one (MPL and mixtures) or two (DPhPC) steps on the SUM. In addition, folded membranes were generated with DPhPC and the lipid mixtures. The specific capacitance of the tight SUM-supported membranes increased continuously with increasing MPL to DPhPC ratio from 0.62 μF/cm2 (pure DPhPC) over 0.66 μF/cm2 (equimolar DPhPC/MPL) to 0.76 μF/cm2 for pure MPL membranes. SUM-supported MPL membranes showed a lifetime of 8.3 ± 2.9 h. An additional monomolecular S-layer protein lattice recrystallized on the lipid-faced side increased the lifetime significantly to 21.2 ± 3.1 h. To prove the functionality of the membranes, gramicidin was reconstituted. All folded and SUM-supported membranes allowed reconstitution of gramicidin, and high-resolution measurements on single pores could be performed.

Two-Dimensional Self-Organization of the Light-Harvesting Polypeptides/BChlaComplex into a Thermostable Liposomal Membrane

The detergent-isolated light-harvesting polypeptide (LH)/bacteriochlorophyll a (BChl a) complex from the photosynthetic bacterium Rhodospirillum rubrum was organized in thermostable liposomal membranes comprising membrane-spanning tetraether lipids from Sulfolobus acidocaldarius to develop a two-dimensional self-organization of the LH/BChl a complex in the lipid membrane. UV−vis absorption spectral data showed that the LH/BChl a complex was stable in the tetraether lipid membrane. As indicated by the Qy band of BChl a in the complex, the thermostability of the complex in liposomal membranes made from different lipids increased in the following order: dioleoylphospholipids < distearoylphospholipids ≪ tetraether lipids. The increased thermostability of the complex in the tetraether lipid membrane was probably due to hydrophobicity and microviscosity of the isoprenoid chains of the tetraether lipid. Linear dichroism spectra indicated that the BChl a in the complex incorporated into the tetraether lipid membrane was perpendicularly oriented and was homogeneous to the transmembrane axis. This implies that the orientation of the Qy band of BChl a in the complex was parallel to the membrane plane, consistent with the orientation in the photosynthetic membrane.

Voltage clamp studies on S-layer-supported tetraether lipid membranes

Isolated subunits from the cell surface proteins (S-layer) of Bacillus coagulans E38–66 have been recrystallized on a glycerol dialkyl nonitol tetraether lipid (GDNT)-monolayer and the electrophysical features of this biomimetic membrane have been investigated in comparison to unsupported GDNT-monolayers. The GDNT-monolayer, spread on a Langmuir-Blodgett trough, was clamped with the tip of a glass patch pipette. In order to investigate the barrier function and potential to incorporate functional molecules, voltage-clamp examinations on plain and S-layer-supported GDNT-monolayers were performed. Our results indicate the formation of a tight GDNT-monolayer sealing the tip of the glass pipette, and a decrease in conductance of the GDNT-monolayer upon recrystallization of the S-layer protein. Thus, the S-layer protein, apparently, did not penetrate or rupture the lipid monolayer. The valinomycin-mediated increase in conductance was less pronounced for the S-layer-supported than for the plain GDNT-monolayer, indicating differences in the accessibility and/or in the fluidity of the lipid membranes. Furthermore, in contrast to plain GDNT-monolayers, S-layer supported GDNT-monolayers with high valinomycin-mediated conductance persisted over long periods of time, indicating enhanced stability. These composite S-layer/lipid films may constitute a new tool for electrophysical and electrophysiological studies on membrane-associated and membrane-integrated biomolecules.

Resolution of the Aerobic Respiratory System of the Thermoacidophilic Archaeon, Sulfolobus sp. Strain 7:: III. THE ARCHAEAL NOVEL RESPIRATORY COMPLEX II (SUCCINATE:CALDARIELLAQUINONE OXIDOREDUCTASE COMPLEX) INHERENTLY LACKS HEME GROUP

An active respiratory complex II (succinate:quinone oxidoreductase) has been purified from tetraether lipid membranes of the thermoacidophilic archaeon, Sulfolobus sp. strain 7. It consists of four different subunits with apparent molecular masses of 66, 37, 33, and 12 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 66-kDa subunit contains a covalently bound flavin, the 37-kDa subunit is a possible iron-sulfur protein carrying three distinct types of EPR-visible FeS cluster, and the 33- and 12-kDa subunits are putative membrane-anchor subunits, respectively. While no heme group is detected in the purified complex II, it catalyzes succinate-dependent reduction of ubiquinone-1 and 2,6-dichlorophenolindophenol in the absence of phenazine methosulfate. The respiratory complex II of Sulfolobus sp. strain 7 appears to be novel in that it functions as a true succinate:caldariellaquinone oxidoreductase, although inherently lacking any heme group. This further indicates that the heme group of several respiratory complexes II may not be involved in the redox intermediates of the electron transfer from succinate to quinone.

Black lipid membranes of tetraether lipids from Thermoplasma acidophilum

Black lipid membranes were formed of tetraether lipids from Thermoplasma acidophilum and compared to the bilayer forming lipids diphytanoylphosphatidylcholine and diphythanylglucosylglycerol. Bilayer-forming lipids varied in thickness of black lipid membranes due to the organic solvent used. Measurements of the specific membrane capacitance ( C m = 0.744 μF/cm 2) showed that the membrane-spanning tetraether lipids from Thermoplasma acidophilum form a monolayer of a constant thickness of 2.5–3.0 nm no matter from which solvent. This finding corresponds to the results of Gliozzi et al. [1] for the lipids of another archaebacterium, Sulfolobus solfataricus. Black lipid membranes were formed at room temperature with a torus from bilayer-forming lipids, however, the torus could also be formed by the tetraetherlipid itself at room temperature and at defined concentration. In these stable black lipid membranes, conductance was measured in the presence of valinomycin, nonactin, and gramicidin. At 10 −7 M concentration, valinomycin mediated higher conductance in membranes from tetraether lipids (200–1200 μS/cm 2) than from bilayer-forming lipids (125–480 μS/cm 2). Nonactin, at 10 −6 M concentration, mediated a 6-fold higher conductance in a tetraether lipid membrane than in a bilayer, whereas conductance, in the presence of 5 · 10 −11 M gramicidin could reach higher values in bilayers than in tetraether lipid monolayers of comparable thickness. Monensin did not increase the conductance of black lipid membranes from tetraether lipids under all conditions applied in our experiments. Poly( l-lysine) destroyed black lipid membranes. Lipopolysaccharides from Thermoplasma acidophilum were not able to form stable black lipid membranes by themselves. The lipopolysaccharide complexes from Thermoplasma acidophilum and from Escherichia coli decreased the valinomycin-mediated conductance of monolayer and bilayer membranes. This influence was stronger than that of the polysaccharide dextran.