Inverted Hexagonal Phase Research Articles

Cr(vi) permanently binds to the lipid bilayer in an inverted hexagonal phase throughout the reduction process.

Cr(vi) is a harmful, carcinogenic agent with a high permeability rate throughout the lipid membranes. In an intracellular environment and during interactions with cellular membranes, it undergoes an instant reduction to lower oxidation states throughout radical states, recognized as the most dangerous factor for cells. The cellular membrane is the most visible cellular organelle in the interior and exterior of a cell. In this study, liposomes and non-lamellar inverted hexagonal phase lipid structures based on phosphoethanolamine (PE) were used as model cellular bilayers because of their simple composition, preparation procedure, and the many other properties of natural systems. The lipid membranes were subjected to 0.075 mM Cr(vi) for 15 min, after which the Cr content was removed via dialysis. This way, the remaining Cr content could be studied qualitatively and quantitatively. Using the combined XRF/XAS/EPR approach, we revealed that some Cr content (Cr(iii) and Cr(vi)) was still present in the samples even after long-term dialysis at a temperature significantly above the phase transition for the chosen liposome. The amount of bound Cr increased with increasing PE and -C[double bond, length as m-dash]C- bond content in lipid mixtures. Internal membrane order decreased in less fluid membranes, while in more liquified ones, internal order was only slightly changed after subjecting them to the Cr(vi) agent. The results suggest that the inverted hexagonal phase of lipid structures is much more sensitive to oxidation than the lamellar lipid phase, which can play an important role in the strong cytotoxicity of Cr(vi).

Induction of non-lamellar phases in archaeal lipids at high temperature and high hydrostatic pressure by apolar polyisoprenoids

It is now well established that cell membranes are much more than a barrier that separate the cytoplasm from the outside world. Regarding membrane's lipids and their self-assembling, the system is highly complex, for example, the cell membrane needs to adopt different curvatures to be functional. This is possible thanks to the presence of non-lamellar-forming lipids, which tend to curve the membrane. Here, we present the effect of squalane, an apolar isoprenoid molecule, on an archaea-like lipid membrane. The presence of this molecule provokes negative membrane curvature and forces lipids to self-assemble under inverted cubic and inverted hexagonal phases. Such non-lamellar phases are highly stable under a broad range of external extreme conditions, e.g. temperatures and high hydrostatic pressures, confirming that such apolar lipids could be included in the architecture of membranes arising from cells living under extreme environments.

PH-Sensitive N,N-Dimethylalkane-1-amine N-Oxides in DNA Delivery: From Structure to Transfection Efficiency.

pH-sensitive liposomes composed of homologues of series of N,N-dimethylalkane-1-amine N-oxides (CnNO, n = 8-18, where n is the number of carbon atoms in the alkyl substituent) and neutral phospholipid dioleoylphosphatidylethanolamine (DOPE) were prepared at two molar ratios (CnNO/DOPE = 0.4:1 and 1:1) and tested for their in vitro transfection activity. Several techniques (SAXS/WAXS, UV-vis, zeta potential measurements, confocal microscopy) were applied to characterize the system in an effort to unravel the relationship among the transfection efficiency, structure, and composition of the lipoplexes. The transfection efficiency of CnNO/DOPE for plasmid DNA in U2OS cells follows a quasi-parabolic dependence on CnNO's alkyl substituent length with a maximum at n = 16. The transfection efficiency of CnNO/DOPE (n = 12-18) lipoplexes was found to be higher than that of commercially available Lipofectamine 2000. C16NO/DOPE also positively transfected HEK 293T and HeLa cells. Small-angle X-ray scattering (SAXS) shows large structural diversity depending on the complex's composition and pH. Transfection efficiencies mediated by two structures, either a condensed lamellar (Lαc) or epitaxially connected Lαc and a condensed inverted hexagonal (HIIc) phase (Lαc & HIIc), were found to be very similar. The change in pH from acidic to neutral induces phase transition Lαc & HIIc → QII + Lα, with cubic phase QII of the Pn3m space group. QII detected in lipoplexes of most efficient composition CnNO/DOPE (n = 16 and 18) facilitates DNA release and promotes its internalization in the cell.

Effect of Imidazolium-Based Ionic Liquids on the Structure and Phase Behavior of Palmitoyl-oleoyl-phosphatidylethanolamine.

Among various applications, ionic liquids (ILs) have been used as antimicrobial agents in laboratories, possibly through induction of the leakage of bacteria. A molecular-level understanding of the mechanism that describes how ILs enhance the permeation of membranes is still lacking. In this study, the effects of imidazolium-based ILs with different alky chain lengths on the structure and phase behavior of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), which is a representative bacteria-membrane-rich lipid, have been investigated. By employing differential scanning calorimetry and synchrotron small- and wide-angle X-ray scattering techniques, we found that ILs with longer alkyl chains influenced the phase behavior more effectively, and lower IL concentrations are needed to induce phase separation for both lamellar liquid crystalline phase and nonlamellar inverted hexagonal phase of POPE. Interestingly, the IL with an alkyl chain of 12 carbon atoms ([C12mim]Cl) shows a difference. It exhibits a stronger disturbing effect on the POPE bilayer structure than [C16mim]Cl, indicating that the ability of ILs to influence the membrane structures is dependent not only on the alkyl chain length of ILs, but also on the degree of matching of the alkyl chain lengths of ILs and lipids. The new lamellar and nonlamellar structures induced by ILs both have smaller repeat distances than that of pure POPE, implying thinner membrane structures. Data of the fluorescence-based vesicle dye leakage assay are consistent with these results, particularly the defects caused by IL-induced phase separation can enhance the membrane permeability markedly. The present work may shed light on our understanding of the antimicrobial mechanism of ILs.

Understanding the Nanoscale Structure of Inverted Hexagonal Phase Lyotropic Liquid Crystal Polymer Membranes.

Periodic, nanostructured porous polymer membranes made from the cross-linked inverted hexagonal phase of self-assembled lyotropic liquid crystals (LLCs) are a promising class of materials for selective separations. In this work, we investigate an experimentally characterized LLC polymer membrane using atomistic molecular modeling. In particular, we compare simulated X-ray diffraction (XRD) patterns with experimental XRD data to quantify and understand the differences between simulation and experiment. We find that the nanopores are likely composed of five columns of stacked LLC monomers which surround each hydrophilic core. Evidence suggests that these columns likely move independently of each other over longer time scales than accessible via atomistic simulation. We also find that wide-angle X-ray scattering structural features previously attributed to monomer tail tilt are likely instead due to ordered tail packing. Although this system has been reported as dry, we show that small amounts of water are necessary to reproduce all features from the experimental XRD pattern because of asymmetries introduced by hydrogen bonds between the monomer head groups and water molecules. Finally, we explore the composition and structure of the nanopores and reveal that there exists a composition gradient rather than an abrupt partition between the hydrophilic and hydrophobic regions. A caveat is that the time scales of the dynamics are extremely long for this system, resulting in simulated structures that appear too ordered, thus requiring careful examination of the metastable states observed in order to draw any conclusions. The clear picture of the nanoscopic structure of these membranes provided in this study will enable a better understanding of the mechanisms of small-molecule transport within these nanopores.

Pathological transitions in myelin membranes driven by environmental and multiple sclerosis conditions

Multiple sclerosis (MS) is an autoimmune disease, leading to the destruction of the myelin sheaths, the protective layers surrounding the axons. The etiology of the disease is unknown, although there are several postulated environmental factors that may contribute to it. Recently, myelin damage was correlated to structural phase transition from a healthy stack of lamellas to a diseased inverted hexagonal phase as a result of the altered lipid stoichiometry and low myelin basic protein (MBP) content. In this work, we show that environmental conditions, such as buffer salinity and temperature, induce the same pathological phase transition as in the case of the lipid composition in the absence of MBP. These phase transitions have different transition points, which depend on the lipid's compositions, and are ion specific. In extreme environmental conditions, we find an additional dense lamellar phase and that the native lipid composition results in similar pathology as the diseased composition. These findings demonstrate that several local environmental changes can trigger pathological structural changes. We postulate that these structural modifications result in myelin membrane vulnerability to the immune system attacks and thus can help explain MS etiology.

Influence of γ-Lactones on Monolinolein/Water Bulk and Emulsified Mesophases

The emulsification of two flavor compounds of the γ-lactone type with monolinolein liquid crystalline submicron particles is reported. The stabilization is ensured by the copolymer F127. γ-Nonalactone can be loaded in bicontinuous cubic monolinolein particles at a larger level (up to 20 wt %) than γ-decalactone (less than 15 wt %). The phase behavior of the ternary monolinolein/water/γ-nonalactone system was studied. The large γ-nonalactone content solubilized into cubosomes was corroborated by the observation of a wide cubic V2 range in the ternary phase diagram. Surprisingly, no inverted hexagonal phase was found in the system. On the contrary, the incorporation of γ-decalactone in the lipid particles gave rise to a dispersion of inverted hexagonal phase, which corresponds to a classical behavior of an oily additive. We finally determined the internal phase of particles including 10 wt % of γ-nonalactone upon increasing the F127 content. We thus found that γ-nonalactone restricts very significantly the interaction of the emulsifier with the cubosomes' interior.

Characteristic responses of a 1,2-dipalmitoleoyl-sn-glycero-3- phosphoethanolamine molecular layer depending on the number of CH(OH) groups in polyols

To elucidate the effect of polyols on a phospholipid molecular layer, different polyols with the formula H-(CH(OH))m-H (m = 2–6) were added to a phospholipid (1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DPoPE)) solution above and below the phase transition temperature between the lamellar and inverted hexagonal phases. As for the surface pressure (π)−surface area (A) isotherm of the phospholipid monolayer at the air-water interface, π at a constant value of A increased with increasing m, especially for xylitol (m = 5) and sorbitol (m = 6). Small angle X-ray scattering (SAXS) and Fourier transform IR spectrometry (FT-IR) were used to evaluate the interaction between DPoPE and the polyol molecules. The experimental results suggest that the number of CH(OH) groups in the polyol plays an important role in the characteristic interaction between DPoPE and the polyol in both the lamellar and inverted hexagonal phases.

Interaction between Surface Charge-Modified Gold Nanoparticles and Phospholipid Membranes.

This report clarifies the interaction of surface charge-modified gold nanoparticles (AuNPs) with phospholipid membranes, which is helpful to understand the antibacterial mechanism of positive charge-modified AuNPs to Gram-negative bacteria. Although the simulated bacterial cell membranes as a whole are negatively charged, the local electrostatic repulsive interaction between the positive charge-coated AuNPs and the small-sized flexible cationic head group of dioleyl phosphatidylethanolamine molecules induces the phase transformation of the simulated bacterial cell membranes from a lamellar to an inverted hexagonal phase. Transmembrane pores with a diameter of about 3.0 nm in the inverted hexagonal structure would result in the destruction of cell membrane function. Such an interaction of positive charge-modified AuNPs with the membrane mimics provides a promising route to develop new antibacterial agents by modifying positive charges on the surface of nanoparticles.

Erratum to: Implementation of a methodology for determining elastic properties of lipid assemblies from molecular dynamics simulations

The importance of the material properties of membranes for diverse cellular processes is well established. Notably, the elastic properties of the membrane, which depend on its composition, can directly influence membrane reshaping and fusion processes as well as the organisation and function of membrane proteins. Determining these properties is therefore key for a mechanistic understanding of how the cell functions. We have developed a method to determine the bending rigidity and tilt modulus, for lipidic assemblies of arbitrary lipid composition and shape, from molecular dynamics simulations. The method extracts the elastic moduli from the distributions of microscopic tilts and splays of the lipid components. We present here an open source implementation of the method as a set of Python modules using the computational framework OpenStructure. These modules offer diverse algorithms typically used in the calculatation the elastic moduli, including routines to align MD trajectories of complex lipidic systems, to determine the water/lipid interface, to calculate lipid tilts and splays, as well as to fit the corresponding distributions to extract the elastic properties. We detail the implementation of the method and give several examples of how to use the modules in specific cases. The method presented here is, to our knowledge, the only available computational approach allowing to quantify the elastic properties of lipidic assemblies of arbitrary shape and composition (including lipid mixtures). The implementation as python modules offers flexibility, which has already allowed the method to be applied to diverse lipid assembly types, ranging from bilayers in the liquid ordered and disordered phases to a study of the inverted-hexagonal phase, and with different force-fields (both all-atom and coarse grained representations). The modules are freely available through GitHub at https://github.com/njohner/ost_pymodules/ while OpenStructure can be obtained at http://www.openstructure.org .

Implementation of a methodology for determining elastic properties of lipid assemblies from molecular dynamics simulations.

BackgroundThe importance of the material properties of membranes for diverse cellular processes is well established. Notably, the elastic properties of the membrane, which depend on its composition, can directly influence membrane reshaping and fusion processes as well as the organisation and function of membrane proteins. Determining these properties is therefore key for a mechanistic understanding of how the cell functions.ResultsWe have developed a method to determine the bending rigidity and tilt modulus, for lipidic assemblies of arbitrary lipid composition and shape, from molecular dynamics simulations. The method extracts the elastic moduli from the distributions of microscopic tilts and splays of the lipid components. We present here an open source implementation of the method as a set of Python modules using the computational framework OpenStructure. These modules offer diverse algorithms typically used in the calculatation the elastic moduli, including routines to align MD trajectories of complex lipidic systems, to determine the water/lipid interface, to calculate lipid tilts and splays, as well as to fit the corresponding distributions to extract the elastic properties. We detail the implementation of the method and give several examples of how to use the modules in specific cases.ConclusionsThe method presented here is, to our knowledge, the only available computational approach allowing to quantify the elastic properties of lipidic assemblies of arbitrary shape and composition (including lipid mixtures). The implementation as python modules offers flexibility, which has already allowed the method to be applied to diverse lipid assembly types, ranging from bilayers in the liquid ordered and disordered phases to a study of the inverted-hexagonal phase, and with different force-fields (both all-atom and coarse grained representations). The modules are freely available through GitHub at https://github.com/njohner/ost_pymodules/ while OpenStructure can be obtained at http://www.openstructure.org.

Molecular Modeling of the Lamellar to Inverse Hexagonal Phase Transition in Dope-Dopc Lipid Systems

In the proper physiological state, myelin sheaths consist of tightly packed lipids and proteins and insulate neurons in both the central and peripheral nervous systems. However, defective myelin sheaths in multiple sclerosis (MS), and other demyelinating diseases, ultimately cause vesiculation of the lipids contained in the sheaths. Increasing the proportion of phosphatidylethanolamine in myelin was shown experimentally to cause structural instabilities due to an abnormal packing structure. The unstable form favors inverted hexagonal phases as a result of lipid headgroup size and geometry, whereas normal myelin composition favors a smooth lamellar phase. Through a molecular mean field theory approach, we produce phase diagrams of lipid systems with varying concentrations of phosphatidylethanolamine and phosphatidylcholine to capture the transitions between inverted hexagonal and lamellar phases. Analyzing structural instabilities in myelin sheaths may provide us with a clearer understanding of multiple sclerosis and other demyelinating diseases.

Characterization of thylakoid lipid membranes from cyanobacteria and higher plants by molecular dynamics simulations

The thylakoid membrane is mainly composed of non-common lipids, so called galactolipids. Despite the importance of these lipids for the function of the photosynthetic reaction centers, the molecular organization of these membranes is largely unexplored. Here we use multiscale molecular dynamics simulations to characterize the thylakoid membrane of both cyanobacteria and higher plants. We consider mixtures of up to five different galactolipids plus phosphatidylglycerol to represent these complex membranes. We find that the different lipids generally mix well, although nanoscale heterogeneities are observed especially in case of the plant membrane. The fluidity of the cyanobacterial membrane is markedly reduced compared to the plant membrane, even considering elevated temperatures at which thermophilic cyanobacteria are found. We also find that the plant membrane more readily undergoes a phase transformation to an inverted hexagonal phase. We furthermore characterized the conformation and dynamics of the cofactors plastoquinone and plastoquinol, revealing of the fast flip-flop rates for the non-reduced form. Together, our results provide a molecular view on the dynamical organization of the thylakoid membrane.

Stimuli responsive polymorphism of C12NO/DOPE/DNA complexes: Effect of pH, temperature and composition

N,N-dimethyldodecylamine-N-oxide (C12NO) is a surfactant that may exist either in a neutral or cationic protonated form depending on the pH of aqueous solutions. Using small angle X-ray diffraction (SAXD) we observe the rich structural polymorphism of pH responsive complexes prepared due to DNA interaction with C12NO/dioleoylphosphatidylethanolamine (DOPE) vesicles and discuss it in view of utilizing the surfactant for the gene delivery vector of a pH sensitive system. In neutral solutions, the DNA uptake is low, and a lamellar Lα phase formed by C12NO/DOPE is prevailing in the complexes at 0.2≤C12NO/DOPE<0.6mol/mol. A maximum of ~30% of the total DNA volume in the sample is bound in a condensed lamellar phase LαC at C12NO/DOPE=1mol/mol and pH7.2. In acidic conditions, a condensed inverted hexagonal phase HIIC was observed at C12NO/DOPE=0.2mol/mol. Commensurate lattice parameters, aHC≈dLC, were detected at 0.3≤C12NO/DOPE≤0.4mol/mol and pH=4.9–6.4 suggesting that LαC and HIIC phases were epitaxially related. While at the same composition but pH~7, the mixture forms a cubic phase (Pn3m) when the complexes were heated to 80°C and cooled down to 20°C. Finally, a large portion of the surfactant (C12NO/DOPE>0.5) stabilizes the LαC phase in C12NO/DOPE/DNA complexes and the distance between DNA strands (dDNA) is modulated by the pH value. Both the composition and pH affect the DNA binding in the complexes reaching up to ~95% of the DNA total amount at acidic conditions.

A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes.

The lipid cubic phase or in meso method is a robust approach for crystallizing membrane proteins for structure determination. The uptake of the method is such that it is experiencing what can only be described as explosive growth. This timely, comprehensive and up-to-date review introduces the reader to the practice of in meso crystallogenesis, to the associated challenges and to their solutions. A model of how crystallization comes about mechanistically is presented for a more rational approach to crystallization. The possible involvement of the lamellar and inverted hexagonal phases in crystallogenesis and the application of the method to water-soluble, monotopic and lipid-anchored proteins are addressed. How to set up trials manually and automatically with a robot is introduced with reference to open-access online videos that provide a practical guide to all aspects of the method. These range from protein reconstitution to crystal harvesting from the hosting mesophase, which is noted for its viscosity and stickiness. The sponge phase, as an alternative medium in which to perform crystallization, is described. The compatibility of the method with additive lipids, detergents, precipitant-screen components and materials carried along with the protein such as denaturants and reducing agents is considered. The powerful host and additive lipid-screening strategies are described along with how samples that have low protein concentration and cell-free expressed protein can be used. Assaying the protein reconstituted in the bilayer of the cubic phase for function is an important element of quality control and is detailed. Host lipid design for crystallization at low temperatures and for large proteins and complexes is outlined. Experimental phasing by heavy-atom derivatization, soaking or co-crystallization is routine and the approaches that have been implemented to date are described. An overview and a breakdown by family and function of the close to 200 published structures that have been obtained using in meso-grown crystals are given. Recommendations for conducting the screening process to give a more productive outcome are summarized. The fact that the in meso method also works with soluble proteins should not be overlooked. Recent applications of the method for in situ serial crystallography at X-ray free-electron lasers and synchrotrons are described. The review ends with a view to the future and to the bright prospects for the method, which continues to contribute to our understanding of the molecular mechanisms of some of nature's most valued proteinaceous robots.

Cooperativity between Water and Lipids in Lamellar to Inverted-Hexagonal Phase Transition

It has been unclear whether the role of water in the self-assembly of soft materials and biomolecules is influential or water is just a background medium. Here we investigate the correlation betwee...

Conformation and Lipid Interaction of the Fusion Peptide of the Paramyxovirus PIV5 in Anionic and Negative-Curvature Membranes from Solid-State NMR

Viral fusion proteins catalyze the merger of the virus envelope and the target cell membrane through multiple steps of protein conformational changes. The fusion peptide domain of these proteins is important for membrane fusion, but how it causes membrane curvature and dehydration is still poorly understood. We now use solid-state NMR spectroscopy to investigate the conformation, topology, and lipid and water interactions of the fusion peptide of the PIV5 virus F protein in three lipid membranes, POPC/POPG, DOPC/DOPG, and DOPE. These membranes allow us to investigate the effects of lipid chain disorder, membrane surface charge, and intrinsic negative curvature on the fusion peptide structure. Chemical shifts and spin diffusion data indicate that the PIV5 fusion peptide is inserted into all three membranes but adopts distinct conformations: it is fully α-helical in the POPC/POPG membrane, adopts a mixed strand/helix conformation in the DOPC/DOPG membrane, and is primarily a β-strand in the DOPE membrane. 31P NMR spectra show that the peptide retains the lamellar structure and hydration of the two anionic membranes. However, it dehydrates the DOPE membrane, destabilizes its inverted hexagonal phase, and creates an isotropic phase that is most likely a cubic phase. The ability of the β-strand conformation of the fusion peptide to generate negative Gaussian curvature and to dehydrate the membrane may be important for the formation of hemifusion intermediates in the membrane fusion pathway.

Optimizing Drug Release: Bilayer to Inverted Hexagonal Phase Transition of Cationic XTC2 and Anionic DSPS Lipid System is Influenced by pH, Temperature, and Salt Concentration

The effectiveness of a macromolecular drug delivered in lipid nanoparticles (LNP) depends upon the biophysical properties of the delivery vehicle. Recent research has shown that the design of the cationic lipid component of LNPs improves the intracellular delivery of therapeutic siRNA[1]. Even in these optimized LNPs, only 1% of the siRNA taken up by the cell via endocytosis is actually released into the cell cytosol. We proposed a mechanism of endosome disruption that relies on the formation of non-bilayer phases in the presence of anionic endosomal lipid and synthetic cationic lipids. A model system using prototypical anionic lipid 1,2-distearoyl(d70)-sn-glycero-3-[phospho-L-serine] (DSPS-d70) in 1:1 molar ratio to the cationic lipid DLin-KC2-DMA (XTC2) (pKa∼6.7) was characterized by 2H and 31P NMR spectroscopy. Through spectral analysis, we determined that at physiological pH (∼7.4) the XTC2/DSPS system exhibits a stable gel phase for temperatures below 45oC while an isotropic signal emerges at higher temperatures - no inverted hexagonal (HII) phase is observed. At low pH (∼4.75), the XTC2/DSPS system is principally in a bilayer gel phase at low temperatures with a non-bilayer HII phase predominating at higher temperatures. The transition from gel to HII phase is dependent on salt concentration and is most evident in the range of 15-25oC for 0.25M [Na+], 20-30oC for 0.5M [Na+] and 35-45oC for 1M [Na+]. Through depaking the spectra, order parameter profiles SCD have been obtained and compared for DSPS-d70 chains in bilayer and HII phases. These will be useful for computational simulation and eventually to design in vivo animal model experiments.[1] Semple, S.C., et al., Rational design of cationic lipids for siRNA delivery. Nat Biotechnol, 2010. 28(2): p. 172-6.

Lipid Nature and Their Influence on Opening of Redox-Active Liposomes

The pathway for content release from reduction-sensitive liposomes based on a quinone-dioleoylphosphatidylethanolamine lipid conjugate (Q-DOPE) is outlined using results from fluorescent dye content release assays as well as single- and multiple-angle light scattering. Experimental observations are consistent with a shape/size change of the reduced liposomes prior to their aggregation, with subsequent near-quantitative content release achieved only when the lipid membrane experiences conditions favorable to a lamellar to an inverted hexagonal phase transition. Addition of poly(ethyleneglycol)-modified DOPE (PEG-DOPE) to the Q-DOPE liposomal formulation results in stabilization of the lipid bilayer, whereas incorporation of DOPE yields faster content release. At high DOPE concentrations, DOPE/PEG-DOPE/Q-DOPE liposomes exhibit larger content release, indicating a change in pathway for content release. The outcomes here provide a better understanding of the underlying principles of triggered liposomal content release and the potential utility of specific lipid properties for the rational design of drug delivery systems based on the novel Q-DOPE lipid.

Structural organization and phase behavior of phospholipid fractions of actinobacteria in relation to storage conditions

X-ray diffraction analysis provided information on structural organization, phase behavior, and stability of the major parameters of phospholipid fractions of the cell membranes of actinobacteria strains Streptomyces hygroscopicus RIA 1433, Nonomuraea roseoviolacea subsp. carminata INA 4281, and Nonomuraea sp. INA 34-06 depending on the storage conditions and hydration level. Phospholipids of S. hygroscopicus were shown to form densely packed multilamellar layers. The phospholipid fraction of this microorganism was notable for homogeneity and stability of its structural organization upon storage at 4°C during 10 months. On the contrary, lipids of the phospholipid fractions of N. roseoviolacea subsp. carminata INA 4281 and Nonomuraea sp. INA 34-06 formed lamellar and inverted hexagonal (HII) phases. The phase depended on hydration level and changed in the course of storage. We assume that the revealed differences in phase structural organization of actinobacterial phospholipid fractions may indicate long-term stability of their membrane structures.