Polar Headgroup Region Research Articles

Interactions of the AT1 antagonist valsartan with dipalmitoyl-phosphatidylcholine bilayers

Valsartan is a marketed drug with high affinity to the type 1 angiotensin (AT1) receptor. It has been reported that AT1 antagonists may reach the receptor site by diffusion through the plasma membrane. For this reason we have applied a combination of differential scanning calorimetry (DSC), Raman spectroscopy and small and wide angle X-ray scattering (SAXS and WAXS) to investigate the interactions of valsartan with the model membrane of dipalmitoyl-phosphatidylcholine (DPPC). Hence, the thermal, dynamic and structural effects in bulk as well as local dynamic properties in the bilayers were studied with different valsartan concentrations ranging from 0 to 20mol%. The DSC experimental results showed that valsartan causes a lowering and broadening of the phase transition. A splitting of the main transition is observed at high drug concentrations. In addition, valsartan causes an increase in enthalpy change of the main transition, which can be related to the induction of interdigitation of the lipid bilayers in the gel phase. Raman spectroscopy revealed distinct interactions between valsartan with the lipid interface localizing it in the polar head group region and in the upper part of the hydrophobic core. This localization of the drug molecule in the lipid bilayers supports the interdigitation view. SAXS measurements confirm a monotonous bilayer thinning in the fluid phase, associated with a steady increase of the root mean square fluctuation of the bilayers as the valsartan concentration is increased. At high drug concentrations these fluctuations are mainly governed by the electrostatic repulsion of neighboring membranes. Finally, valsartans' complex thermal and structural effects on DPPC bilayers are illustrated and discussed on a molecular level.

Structural Dynamics of the S4 Voltage Sensor Helix in Bilayers Lacking Lipid Phosphates

Voltage-dependent K+ (Kv) channels require lipid phosphate groups for proper functioning. The S4 helix, which carries the gating charges in the voltage-sensing domain, can be inserted into biological membranes while being stabilized by an hydrogen-bonding network in which lipid phosphates play an essential role. To identify protein-lipid dynamics and interactions in the absence of lipid phosphates, we performed molecular dynamics (MD) simulations of a variant of the KvAP S4 sensor (S4mut) in bilayers with and without lipid phosphates. We find that in bilayers lacking lipid phosphates (DOTAP), the snorkeling Arg residues are not anchored to the polar headgroup region and hence display more structural flexibility along the bilayer normal compared to phosphate-containing lipid bilayers (POPC). The increased flexibility of the Arg resi-dues and their close interaction with water molecules leads to a collapsed hydrophobic core and a water-permeable DOTAP bilayer. These results show that lipid phosphates are required for stabilizing the S4mut-containing bilayer.

The Influence of Cholesterol on the Properties and Permeability of Hypericin Derivatives in Lipid Membranes.

The promising photosensitizing properties of hypericin, a natural quinine substituted with hydroxyl and alkyl groups, have led to the proposal that it can be utilized in photodynamic therapy. Neither the detailed mechanism behind the powerful action of hypericin, arising as a result of light excitation, nor the intracellular localization and transportation of the molecule is yet fully understood. The behavior of hypericin derivatives in a pure dipalmitoylphosphatidylcholine (DPPC) lipid membrane has recently been studied theoretically by means of molecular dynamics simulations. Natural membranes however contain many important constituents-cholesterol being one of the most essential-that influence the function and structure of the membrane, and thereby also the behavior of drug molecules therein. In the present study, we investigated hypericin and its brominated derivatives in membranes containing 9 and 25 mol % cholesterol. The results show that the presence of cholesterol in the membrane affects the permeability of the hypericin molecules and does so differently for the various molecules in the two membranes. Hypericin containing one bromine was found to exhibit the lowest free energy profile for the transport process into the lipids, and also the highest permeability coefficients, indicating that this molecule displays the fastest and easiest diffusion in the membranes. All three molecules were found to accumulate most preferably close to the polar headgroup region in both membranes.

Interaction of metalloporphyrins with lipid bilayers, a calorimetric study

Interaction of metalloporphyrins with lipid bilayers, a calorimetric studyThe interaction of three metalloporphyrins, containing manganese, iron and cobalt atoms, with lipid bilayers composed of neutral (DPPC) or charged (DMPG) phospholipids were studied by means of scanning differential calorimetry. We found only minute effects exerted by studied compounds on DPPC, while phase transitions of charged DMPG were seriously affected by porphyrins. Analysis of experimental data revealed that due to the electrostatic interactions DMPG bilayers are perturbed not only in the polar head group region. Putative rearrangements of the polar heads packing affects also the acyl chain region of this lipid bilayer. Perturbation of DMPG polar heads induced by porphyrin in complex with manganese atoms is bigger than that induced by other porphyrins.

Distribution of BODIPY-labelled phosphatidylethanolamines in lipid bilayers exhibiting different curvatures

In this paper we have investigated the behaviour of newly synthesised mono-palmitoyl- and dipalmitoyl-phosphatidylethanolamine probes (abbreviated as mPE and dPE, respectively) labelled in the polar headgroup region by either the FL-BODIPY or the 564/570-BODIPY fluorophore and solubilised in lipid systems that exhibit different curvatures. Because of the bulky BODIPY-groups, the monoacyl-form derivatives have a conic-like shape, whereas that for the diacyl derivatives is rather cylindrical. A careful analysis of time-resolved resonance energy transfer experiments by means of analytical models as well as Monte Carlo simulations shows that the mPE derivatives have a comparable affinity to highly curved bilayer regions (torroidal pores formed by magainin-2 in lipid bilayers, or the rims of discoid bicelles) and to planar bilayer regions (i.e. the flat region of lipid bilayers and bicelles). Furthermore, the monoacyl-probes are as compared to the diacyl-probes effectively closer to each other in a lipid bilayer, while none of these probes seems to be randomly distributed. Self-aggregation is most efficiently induced by the larger aromatic 564/570-BODIPY chromophore, but it is suppressed when using the diacyl instead of the monoacyl-form, and/or by attaching BODIPY-groups to the acyl-chain.

Interactions of phosphorus-containing dendrimers with liposomes

The influence of cationic phosphorus-containing dendrimers generation 3 and 4 on model DMPC or DPPC lipid membranes was studied. Measurements of fluorescence anisotropy and differential scanning calorimetry (DSC) were applied to assess changes in lipid bilayer parameters, including fluidity, anisotropy, and phase-transition temperature. Interaction with both hydrophobic and hydrophilic regions of the bilayer was followed by these methods. Dendrimers of both generations influence lipid bilayers by decreasing membrane fluidity. The results suggest that dendrimers can interact both with the hydrophobic part and the polar head-group region of the phospholipid bilayer. Higher generation dendrimers interact more strongly with model membranes, and the concentration, as well as the generation, is of similar importance.

Effects of peptide hydrophobicity on its incorporation in phospholipid membranes — an NMR and ellipsometry study

Effects of peptide hydrophobicity on lipid membrane binding, incorporation, and defect formation was investigated for variants of the complement-derived antimicrobial peptide CNY21 (CNYITELRRQHARASHLGLAR), in anionic 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE)/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) and zwitterionic 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes. Using a method combination of, e.g., ellipsometry, CD, and fluorescence spectroscopy, it was shown that peptide adsorption, as well as peptide-induced liposome leakage and bactericidal potency against Escherichia coli and Pseudomonas aeruginosa, was promoted by increasing the hydrophobicity of CNY21 through either substituting the two histidines (H) in CNY21 with more hydrophobic leucine (L) residues, or end-tagging with tritryptophan (WWW). Fluorescence spectroscopy revealed that both CNY21WWW and the WWW tripeptide localized to the polar headgroup region of these phospholipid membranes. Deuterium NMR experiments on macroscopically oriented membranes containing fully (palmitoyl) deuterated POPC (POPC-d31) demonstrated that both CNY21L and CNY21WWW induced disordering of the lipid membrane. In contrast, for cholesterol-supplemented POPC-d31 bilayers, peptide-induced disordering was less pronounced in the case of CNY21L, indicating that the peptide is unable to partition to the interior of the lipid membrane in the presence of cholesterol. CNY21WWW, on the other hand, retained its membrane-disordering effect also for cholesterol-supplemented POPC-d31. These findings were supported by pulsed field gradient NMR experiments where the lateral lipid diffusion was determined in the absence and presence of peptides. Overall, the results provide some mechanistic understanding to previously observed effects of peptide hydrophobization through point mutations and end-tagging, particularly so for complement-based antimicrobial peptides.

Therapeutic and cosmeceutical potential of ethosomes: An overview.

The main disadvantage of transdermal drug delivery is the poor penetration of most compounds into the human skin. The main barrier of the skin is located within its uppermost layer, the stratum corneum (SC). Several approaches have been developed to weaken this skin barrier. One of the approaches for increasing the skin penetration of drugs and many cosmetic chemicals is the use of vesicular systems, such as, liposomes and ethosomes. Ethosomes are phospholipid-based elastic nanovesicles containing a high content of ethanol (20–45%). Ethanol is known as an efficient permeation enhancer and has been added in the vesicular systems to prepare elastic nanovesicles. It can interact with the polar head group region of the lipid molecules, resulting in the reduction of the melting point of the stratum corneum lipid, thereby increasing lipid fluidity and cell membrane permeability. The high flexibility of vesicular membranes from the added ethanol permits the elastic vesicles to squeeze themselves through the pores, which are much smaller than their diameters. Ethosomal systems are much more efficient in delivering substances to the skin in the terms of quantity and depth, than either conventional liposomes or hydroalcoholic solutions. The scope of this small review is to introduce the novel concept of ethosomes and to describe some approaches and mechanisms of stimulating topical and transdermal products with ethosomes.

NBD-cholesterol probes to track cholesterol distribution in model membranes

A series of cholesterol (Chol) probes with NBD and Dansyl fluorophores attached to the 3-hydroxyl position via carbamate linkers has been designed and synthesized and their ability to mimic the behavior of natural cholesterol in bilayer membranes has been examined. Fluorescence spectroscopy data indicate that the NBD-labeled lipids are located in the polar headgroup region of the bilayer with their position varying with the method of fluorophore attachment and the linker length. The partitioning of the Chol probes between liquid-ordered (L o) and liquid-disordered (L o) phases in supported bilayers prepared from ternary lipid mixtures of DOPC, Chol and either egg sphingomyelin or DPPC was examined by fluorescence microscopy. The carbamate-linked NBD-Chols show a stronger preference for partitioning into L o domains than does a structurally similar probe with an ester linkage, indicating the importance of careful optimization of probe and linker to provide the best Chol mimic. Comparison of the partitioning of NBD probes to literature data for native Chol indicates that the probes reproduce well the modest enrichment of Chol in L o domains as well as the ceramide-induced displacement of Chol. One NBD probe was used to follow the dynamic redistribution of Chol in phase separated membranes in response to in situ ceramide generation. This provides the first direct optical visualization of Chol redistribution during enzymatic ceramide generation and allows the assignment of new bilayer regions that exclude dye and have high lateral adhesion to ceramide-rich regions.

Relationship between the Antioxidant Capacity and Effect of Rosemary (Rosmarinus officinalis L.) Polyphenols on Membrane Phospholipid Order

The antioxidant activity of rosemary (Rosmarinus officinalis L.) extracts is mainly due to phenolic abietane diterpenes and phenolic acids such as rosmarinic acid. In this study a comprehensive characterization of non-water-soluble and water-soluble extracts from rosemary was achieved by liquid chromatography coupled to electrospray and mass spectrometry. The antioxidant activity of these extracts and their respective major compounds (carnosic acid, carnosol, rosmadial, genkwanin, and rosmarinic acid) was analyzed and compared by using different in vitro systems. Whereas rosmarinic acid, carnosic acid, and carnosol exhibited similar antioxidant activity in a phospholipid membrane-free assay, carnosol behaved as an extremely potent antioxidant in a membrane-based assay (4-6 times stronger than the rest of the compounds). This differential antioxidant behavior suggests that factors other than the radical scavenging capability may be involved. All of the diterpenes induced severe effects on lipid order and packing of phospholipid model membranes. Rosmadial and carnosol decreased the number and/or mobility of water molecules located at the polar head group region of the membrane phospholipids as seen by Laurdan fluorescence spectroscopy. Carnosol also strongly enhanced lipid order at the hydrophobic core of the membrane. These effects throughout the bilayer correlated to the stronger antioxidant capacity of carnosol to inhibit lipid peroxidation. On the contrary, carnosic acid decreased membrane fluidity at deeper regions of the bilayer as measured by bilayer-to-micelle transition assay and self-quenching measurements by using octadecylrhodamine B. These effects may contribute to membrane stabilization and hindrance of radical propagation, which may cooperate with the electron donor ability of rosemary diterpenes in protecting the membranes against oxidative damage.

Structural and dynamic properties of juxta-membrane segments of caveolin-1 and caveolin-2 at the membrane interface

Caveolins (cav1-3) are essential membrane proteins found in caveolae. The caveolin scaffolding domain of cav-1 includes a short sequence containing a CRAC motif (V94TKYWFYR101) at its C-terminal end. To investigate the role of this motif in the caveolin-membrane interaction at the atomic level, we performed a detailed structural and dynamics characterization of a cav-1(V94-L102) nonapeptide encompassing this motif and including the first residue of cav-1 hydrophobic domain (L102), in dodecylmaltoside (DM) or dodecylphosphocholine (DPC) micelles, as membrane mimics. Cav-1(V94-L102) partitioned better in DPC and in DM/anionic lipid micelles than in DM micelles, as shown by fluorescence titration and CD. NMR data revealed that this peptide folded as an amphipathic helix located in the polar head group region of DPC micelles. The two tyrosine side-chains, flanked by arginine and lysine residues, are situated on one face of this helix, whereas the phenylalanine and tryptophan side-chains are located on the opposite face. Fluorescence studies showed significant Trp subnanosecond rotations, the presence of several rotamers, and a heterogeneous location within the water/micelle interface. NMR studies of the shorter cav-1(V94-R101) peptide and of the homologous sequence of cav-2(I79SKYVMYKF87) allowed the description of the effect of L102 and of the amino acid variations occurring in cav-2 on the structure and localization in DPC micelles. Based on the topological model of caveolins, our results suggest that the cav-1 and cav-2 nonapeptides studied form interfacial alpha-helix membrane anchors in which the K/RhhhYK/Rh motif, also found in cav-3, may play a significant role.

Kinetics of lipid-membrane binding and conformational change of L-BABP

We designed an experimental approach to differentiate the kinetics of protein binding to a lipid membrane from the kinetics of the associated conformational change in the protein. We measured the fluorescence intensity of the single Trp6 in chicken liver bile acid-binding protein (L-BABP) as a function of time after mixing the protein with lipid membranes. We mixed the protein with pure lipid membranes, with lipid membranes in the presence of a soluble quencher, and with lipid membranes containing a fluorescence quencher attached to the lipid polar head group. We fitted simultaneously the experimental curves to a three-state kinetic model. We conclude that in a first step, the binding of L-BABP to the interfacial region of the anionic lipid polar head groups occurred simultaneously with a conformational change to the partly unfolded state. In a second slower step, Trp6 buried within the polar head group region, releasing contacts with the aqueous phase.

Interaction of Antimalarial Drugs with DMPC Model Membranes

Primaquine Diphosphate (PQD) and Chloroquine Diphosphate (CQD) are potent therapeutic agents used in the treatment of malaria. The investigation of drug-lipid interactions is pivotal for understanding their biological activity. Electron Spin Resonance (ESR) and Differential Scanning Calorimetry (DSC) were used to investigate the effects of drug binding on the lipid phase transition and acyl chain dynamics of model membranes made up of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) phospholipids. Labels located at different positions along the lipid chain were used to monitor different membrane regions. ESR results indicated that PQD is more effective in changing the membrane structure than CQD. PQD is effective in perturbing the whole chain of DMPC vesicles, whereas the effect of CQD is more pronounced near the polar headgroup region. Furthermore, the results showed a slight decrease of the membrane packing in DMPC gel phase for both drugs. However, PQD causes a slight increase of the lipid packing close to the membrane center, suggesting a deeper insertion of this molecule into DMPC bilayers. DSC thermograms revealed that PQD interacts with DMPC decreasing the main transition temperature (TM) by ca. 2°C and completely abolishing its pre-transition. On the other hand, CQD effects are mainly noticed as a decrease in the cooperativity of the main transition. Because of its lipophilic character, PQD penetrates into the bilayer hydrocarbon region causing considerable disorganization. The higher polarity of CQD is probably related with its low membrane permeability. These results shed light on the molecular mechanism of drug-lipid interaction, which may be useful for the development of lipid drug delivery systems of antimalarial drugs. Acknowledgments: FAPESP, CNPq.

Effect of Dipole Potential Variations on the Surface Charge Potential of Lipid Membranes

When the dipole potential of dimyristoylphosphatidylcholine (DMPC) monolayers was decreased, either by the insertion of phloretin or by the elimination of carbonyl groups at the interphase, the surface charge potential was displaced to lower negative values. At low ionic strength, the decrease of the negative charge density can be ascribed to a different exposure of the phosphate to water, as there is a good correlation to an increase in the area per lipid. At high ionic strength, the magnitude of the changes in the zeta potential produced by the effects on the dipole potential was found to be dependent on the type of anions present in the subphase. Differences between Cl- and ClO4- were ascribed to the adsorption of anions according to their different hydrations and polarizabilities. The influence of a low dipole potential on the anion adsorption can be ascribed to a less positive image charge at the membrane interior, resulting from an increase in the hydrocarbon core permittivity. This is congruent with the neutralization of interfacial dipoles and the area increase, as well as with the decrease in packing of the hydrocarbon groups. Phloretin did not cause changes in the dipole potential of dimyristoylphosphatidylethanolamine (DMPE), and in consequence, no effects on the zeta potential were measured. It is concluded that changes in the inner water/hydrocarbon plane affect the electrostatic potential measured in the outer plane of the polar headgroup region.

The effect of detergents on trimeric G-protein activity in isolated plasma membranes from rat brain cortex: Correlation with studies of DPH and Laurdan fluorescence

The effect of non-ionic detergents on baclofen (GABA B-R agonist)-stimulated G-protein activity was measured as a [ 35S]GTPγS binding assay in the plasma membranes (PM) isolated from the brain tissue. The effect was clearly biphasic — a decrease in the activity was followed by an activation maximum and finally, at high concentrations, drastic inhibition of the G-protein activity was noticed. Contrarily, specific radioligand binding to GABA B-receptor was inhibited in the whole range of detergent concentrations step by step, i.e. it was strictly monophasic. The magnitude of both detergent effects was decreased in the same order of potency: Brij58 > Triton X-100 > Digitonin. The identical order was found when comparing detergents ability to alter fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene ( r DPH) incorporated into the hydrophobic PM interior. Decrease of r DPH, in the order of Brij58 > Triton X-100 > Digitonin, was reflected as decrease of the S-order parameter and rotation correlation time ϕ paralleled by an increase of diffusion wobbling constant D w (analysis by time-resolved fluorescence according to “ wobble-in-cone” model). The influence of the detergents on the membrane organization at the polar headgroup region was characterized by Laurdan generalized polarization (GP). As before, the effect of detergents on GP parameters proceeded in the order: Brij58 > Triton X-100 > Digitonin.

High Vapor Pressure Perfluorocarbons Cause Vesicle Fusion and Changes in Membrane Packing

Perfluorocarbons (PFCs) hold great promise for biomedical applications. However, relatively little is known about the impact of these chemicals on membranes. We used unilamellar vesicles to explore the effects of PFCs on membrane packing and vesicle stability. Four clinically relevant PFCs with varying vapor pressures (PP1, 294 mbar; PP2, 141 mbar; PP4, 9.6 mbar; and PP9, 2.9 mbar) were examined. Microscopy imaging and spectroscopic measurements suggest that PFCs, especially those with high vapor pressures, lead to vesicle fusion within hours. Upon exposure to PP1 and PP2 for 72 h, vesicles retained a spherical shape, but the size changed from ∼200 nm to ∼20–40 μm. In addition, membrane packing underwent marked changes during this timeframe. A significant decrease in water content in the lipid polar headgroup regions occurred during the first 1–2-h exposure to PFCs, followed by a steady increase in water content over time. Possible mechanisms were proposed to explain these dramatic structural changes. The finding that chemically inert PFCs exhibited fusogenic activity and marked changes in membrane surface packing is novel, and should be considered when using PFCs for biomedical applications.

Ground and excited state proton transfer and antioxidant activity of 7-hydroxyflavone in model membranes: Absorption and fluorescence spectroscopic studies

Steady state and time resolved fluorescence spectroscopy have been used to probe microenvironments of the therapeutically active intrinsically fluorescent flavonoid, 7-hydroxyflavone (7-HF), in model membranes consisting of multilamellar phosphatidylcholine liposomes. Additionally, the antioxidant effects of 7-HF against lipid peroxidation have been evaluated using spectrophotometric assay. Large Stokes shifted emissions with distinct spectroscopic signatures, are observed from the excited state proton transfer (ESPT) tautomer (which is generated by a solvent mediated mechanism) and the ground state anion of 7-HF. The neutral (7-HFN) and anionic (7-HFA) species' appear to be located in the non-polar acyl chain and the polar head group regions of the lipid vesicles respectively. The partition coefficients of 7-HFN and 7-HFA in these vesicles have also been estimated using their intrinsic fluorescence. Anisotropy ( r) versus temperature (T) measurements reveal the utility of the tautomer fluorescence anisotropy as a sensitive parameter for exploring structural changes in the membranes. Fluorescence decay kinetics studies indicate heterogeneity in the microenvironments of both 7-HFN and 7-HFA. Furthermore, we demonstrate that lipid peroxidation of the model membranes is partially arrested upon 7-HF binding, suggesting its potential usefulness as an inhibitor of peroxidative damage of cell membranes.

Modelling the behavior of 5-aminolevulinic acid and its alkyl esters in a lipid bilayer

5-Aminolevulinic acid (5ALA) and ester derivates thereof are used as prodrugs in photodynamic therapy (PDT). The behavior of 5ALA and three esters of 5ALA in a DPPC lipid bilayer is investigated. In particular, the methyl ester displays a very different free energy profile, where the highest barrier is located in the region with highest lipid density, while the others have their peak in the middle of the membrane, and also displays a considerably lower permeability coefficient than neutral 5ALA and the ethyl ester. The zwitterion of 5ALA has the highest permeability constant, but a significant free energy minimum in the polar head-group region renders an accumulation in this region.

Structural changes in dipalmitoylphosphatidylcholine bilayer promoted by Ca 2+ ions: a small-angle neutron scattering study

Small-angle neutron scattering (SANS) curves of unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles in 1–60 mM CaCl 2 were analyzed using a strip-function model of the phospholipid bilayer. The fraction of Ca 2+ ions bound in the DPPC polar head group region was determined using Langmuir adsorption isotherm. In the gel phase, at 20 °C, the lipid bilayer thickness, d L, goes through a maximum as a function of CaCl 2 concentration ( d L = 54.4 Å at ∼2.5 mM of CaCl 2). Simultaneously, both the area per DPPC molecule A L, and the number of water molecules n W located in the polar head group region decrease (Δ A L = A L(DPPC) − A L(DPPC+Ca) = 2.3 Å 2 and Δ n = n W(DPPC) − n W(DPPC+Ca) = 0.8 mol/mol at ∼2.5 mM of CaCl 2). In the fluid phase, at 60 °C, the structural parameters d L, A L, and n W show evident changes with increasing Ca 2+ up to a concentration c C a 2 + ≤ 10 mM . DPPC bilayers affected by the calcium binding are compared to unilamellar vesicles prepared by extrusion. The structural parameters of DPPC vesicles prepared in 60 mM CaCl 2 (at 20 and 60 °C) are nearly the same as those for unilamellar vesicles without Ca 2+.

Application of Thin Titanium/Titanium Oxide Layers Deposited on Gold for Infrared Reflection Absorption Spectroscopy: Structural Studies of Lipid Bilayers

Ultrathin titanium layers when deposited on the surface of gold can be successfully applied for infrared reflection absorption spectroscopy (IRRAS) investigations. It was shown that the reflectivity, the phase shift, and the mean square electric field of the p- and s-polarized IR radiation in up to 20 nm thick titanium layers covered with a 3-4 nm thick layer of native oxide are comparable to those of the air/gold interface. The surface selection rule is fulfilled. Thus, qualitative and quantitative analysis of 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) bilayers transferred in liquid expanded (LE) and liquid condensed (LC) states can be performed. Differences are found in the hydration state and molecular arrangement of the two investigated bilayers. In the DMPC bilayer in the LE state, the C-N bond in the positively charged choline moiety is inclined by approximately 70 degrees toward the surface of the negatively charged titanium substrate. In the phosphate moiety, the in-plane vector of the O-P-O group makes a small angle of approximately 15 degrees to the surface normal. This open structure of the lipid molecule corresponds to the B crystal structure of the DMPC molecule and provides space for strong hydration of the polar headgroup. In the DMPC bilayer in the LC state, the intermolecular distances are reduced; the C-N bond of the choline group makes a smaller angle to the surface normal, and the in-plane vector of the O-P-O group in the phosphate moiety displays a larger tilt. The degree of hydration is reduced. The arrangement of the polar headgroup region corresponds to the A crystal structure of the DMPC molecule.