Solid supported lipid membranes: new concepts for the biomimetic functionalization of solid surfaces.

Surface-layer (S-layer) supported lipid membranes on solid substrates are interfacial architectures mimicking the supramolecular principle of cell envelopes which have been optimized for billions of years of evolution in most extreme habitats. The authors implement this biological construction principle in a variety of layered supramolecular architectures consisting of a stabilizing protein monolayer and a functional phospholipid bilayer for the design and development of new types of solid-supported biomimetic membranes with a considerably extended stability and lifetime-compared to existing platforms-as required for novel types of bioanalytical sensors. First, Langmuir monolayers of lipids at the water/air interface are used as test beds for the characterization of different types of molecules which all interact with the lipid layers in various ways and, hence, are relevant for the control of the structure, stability, and function of supported membranes. As an example, the interaction of S-layer proteins from the bulk phase with a monolayer of a phospholipid synthetically conjugated with a secondary cell wall polymer (SCWP) was studied as a function of the packing density of the lipids in the monolayer. Furthermore, SCWPs were used as a new molecular construction element. The exploitation of a specific lectin-type bond between the N-terminal part of selected S-layer proteins and a variety of glycans allowed for the buildup of supramolecular assemblies and thus functional membranes with a further increased stability. Next, S-layer proteins were self-assembled and characterized by the surface-sensitive techniques, surface plasmon resonance spectroscopy and quartz crystal microbalance with dissipation monitoring. The substrates were either planar gold or silicon dioxide sensor surfaces. The assembly of S-layer proteins from solution to solid substrates could nicely be followed in-situ and in real time. As a next step toward S-layer supported bilayer membranes, the authors characterized various architectures based on lipid molecules that were modified by a flexible spacer separating the amphiphiles from the anchor group that allows for a covalent coupling of the lipid to a solid support, e.g., using thiols for Au substrates. Impedance spectroscopy confirmed the excellent charge barrier properties of these constructs with a high electrical resistance. Structural details of various types of these tethered bimolecular lipid membranes were studied by using neutron reflectometry. Finally, first attempts are reported to develop a code based on a SPICE network analysis program which is suitable for the quantitative analysis of the transient and steady-state currents passing through these membranes upon the application of a potential gradient.

Charge inversion at minute electrolyte concentrations.

Anionic dimyristoylphosphatidic acid monolayers spread on LaCl3 solutions reveal strong cation adsorption and a sharp transition to surface overcharging at unexpectedly low bulk salt concentrations. We determine the surface accumulation of La3+ with anomalous x-ray reflectivity and find that La3+ compensates the lipid surface charge by forming a Stern layer with approximately 1 La3+ ion per 3 lipids below a critical bulk concentration, ct approximately 500 nM. Above ct, the surface concentration of La3+ increases to a saturation level with approximately 1 La3+ per lipid, thus implying that the total electric charge of the La3+ exceeds the surface charge. This overcharge is observed at approximately 4 orders of magnitude lower concentration than predicted in ion-ion correlation theories. We suggest that transverse electrostatic correlations between mobile ions and surface charges (interfacial Bjerrum pairing) may contribute to the charge inversion.

Structure and elastic properties of smectic liquid crystalline elastomer films.

Mechanical measurements, x-ray investigations, and optical microscopy are employed to characterize the interplay of chemical composition, network topology, and elastic response of smectic liquid crystalline elastomers (LCEs) in various mesophases. Macroscopically ordered elastomer films of submicrometer thicknesses were prepared by cross linking freely suspended smectic polymer films. The cross-linked material preserves the mesomorphism and phase transitions of the precursor polymer. The elastic response of the smectic LCE is entropic, and the corresponding elastic moduli are of the order of MPa. In the tilted ferroelectric smectic-C* phase, the network structure plays an important role. Due to the coupling of elastic network deformations to the orientation of the mesogenic groups in interlayer cross-linked materials (mesogenic cross-linker units), the stress-strain characteristics is found to differ qualitatively from that in the other phases.

Cooperativity of phospholipid reorganization upon interaction of dipyridamole with surface monolayers on water.

Results from various surface sensitive characterization techniques suggest a model for the interaction of the piperidinopyrimidine dipyridamole (DIP)--known as a vasodilator and inhibitor of P-glycoprotein associated multidrug resistance of tumor cells--with phospholipid monolayers in which the drug is peripherally associated with the membrane, binding (up to) five phospholipids at a time. These multiple interactions are responsible for a very strong association of the drug with the lipid monolayer even at exceedingly low concentrations (approximately 0.2 mol%). Electrostatic interactions and hydrogen bonding are likely involved in the binding of DIP to DPPC. Cooperative effects among the lipids are invoked to explain the macroscopically measurable changes of lipid monolayer properties even when only one out of 100 DPPC molecules is directly associated with a DIP molecule. A reversal of the observed changes upon drug association with the membrane as the DIP concentration surpasses a threshold concentration (c(crit)approximately 0.5 mol%) may be explained by cooperativity in a different context, the self-aggregation of drug molecules. With its implications for the interaction of DIP with phospholipid films, this work provides a first approach to the explanation of the high sensitivity of cell membranes to piperidinopyrimidine drugs on a molecular level.

Giant lateral electrostriction in ferroelectric liquid-crystalline elastomers.

Mechanisms for converting electrical energy into mechanical energy are essential for the design of nanoscale transducers, sensors, actuators, motors, pumps, artificial muscles, and medical microrobots. Nanometre-scale actuation has to date been mainly achieved by using the (linear) piezoelectric effect in certain classes of crystals (for example, quartz), and 'smart' ceramics such as lead zirconate titanate. But the strains achievable in these materials are small--less than 0.1 per cent--so several alternative materials and approaches have been considered. These include grafted polyglutamates (which have a performance comparable to quartz), silicone elastomers (passive material--the constriction results from the Coulomb attraction of the capacitor electrodes between which the material is sandwiched) and carbon nanotubes (which are slow). High and fast strains of up to 4 per cent within an electric field of 150 MV x m(-1) have been achieved by electrostriction (this means that the strain is proportional to the square of the applied electric field) in an electron-irradiated poly(vinylidene fluoride-trifluoroethylene) copolymer. Here we report a material that shows a further increase in electrostriction by two orders of magnitude: ultrathin (less than 100 nanometres) ferroelectric liquid-crystalline elastomer films that exhibit 4 per cent strain at only 1.5 MV x m(-1). This giant electrostriction was obtained by combining the properties of ferroelectric liquid crystals with those of a polymer network. We expect that these results, which can be completely understood on a molecular level, will open new perspectives for applications.

Self-organized lateral patterning of a rare earth complex and stearic acid in Langmuir films.

Metal supercomplexes form on compression of a film of the individual complexes mixed with a fatty acid: A suite of techniques--Langmuir film balance, and optical and scanning force microscopy--show the transformation of [Eu(tta)3 (phen)] into a thin film of strongly luminescent tetrameric supercomplexes. When the area per molecule is low, a phase-separated meshwork structure forms. The cartoon shows the surface arrangement, in which the supercomplexes sit on a layer of the single europium complexes and corralled by a fatty acid.

Molecular chirality and domain shapes in lipid monolayers on aqueous surfaces.

The shapes of domain boundaries in the mesoscopic phase separation of phospholipids in aqueous surface monolayers are analyzed with particular attention to the influence of molecular chirality. We have calculated equilibrium shapes of such boundaries, and show that the concept of spontaneous curvature-derived from an effective pair potential between the chiral molecules-yields an adequate description of the contribution of chirality to the total energy of the system. For enantiomeric dipalmitoylphosphatidylcholine in pure monolayers, and in mixtures with impurities that adsorb preferentially at the (one-dimensional) boundary line between the isotropic and anisotropic fluid phases, such as cyanobiphenyl (5CB), a total energy term that includes line tension, electrostatic dipole-dipole interaction, and spontaneous curvature is sufficient to describe the shapes of well-separated domain boundaries in full detail. As soon as interdomain distances fall below the domain sizes upon compression of a monolayer, fluctuations take over in determining its detailed structural morphology. Using Minkowski measures for the well-studied dimyristoyl phosphatidic acid (DMPA)/cholesterol system, we show that calculations accounting for line tension, electrostatic repulsion, and molecular chirality yield boundary shapes that are of the same topology as the experimentally observed structures. At a fixed molecular area in the phase coexistence region, the DMPA/cholesterol system undergoes an exponential decay of the line tension lambda with decreasing subphase temperature T.

Submolecular organization of DMPA in surface monolayers: beyond the two-layer model.

A new approach to the data refinement of X-ray reflection measurements from lipid surface monolayers, applied to DMPA on pure water, reveals the structural organization of the lipid in unprecedented detail and provides new insights into headgroup conformation and hydration as a function of lateral pressure. While conventional box models are incapable of modeling the experimental data at high momentum transfer satisfactorily, a quasimolecular composition-space refinement approach using distribution functions to map the spatial organization of submolecular headgroup fragments yields a much better description and overcomes inherent difficulties of box models. Upon going from the fluid liquid-expanded (LE) phase to the hexatic liquid-condensed (LC) phase, the orientation of the headgroup is tightly coupled to the ordering of the acyl chains. Headgroups tilt toward the surface normal to accommodate for the large reduction in available area per lipid molecule. The spread of the headgroup fragment distribution is considerably larger than the global interface roughness and increases slightly with compression. In distinction to earlier work on DMPE using the two-box approach, we find that the phosphate hydration stays essentially constant across the whole isotherm. The discrepancy between the results observed with the different models is attributed to intrinsic deficiencies of the box model.

Structural models of lipid surface monolayers from X-ray and neutron reflectivity measurements.

Structural investigations of phospholipid monolayers on aqueous subphases on the submolecular level using X-ray and neutron reflectivity measurements are reviewed. While such investigations have been limited in the past by a relatively restricted accessible momentum transfer range, recent developments in synchrotron technology--almost doubling this range--have considerably improved the capabilities of the technique. Until recently, data interpretation has entirely relied on 'box models' which describe the structures as molecularly homogeneous slabs--one hydrophobic and one hydrophilic. It is shown that box models of phospholipid monolayers are rather inadequate to model data at the high momentum transfer available nowadays in X-ray measurements. As an alternative, a hybrid data inversion strategy is proposed that treats the hydrophobic alkane phase as a homogeneous slab and describes the position of submolecular fragments of the lipid headgroups by means of distribution functions along the interface. Within this approach, composition-space refinement--enabling the coupling of data sets from various X-ray and neutron contrasts--in connection with volumetric constraints enables structural characterization of lipid monolayers in unprecedented detail. Extending a recent characterization of dimyristoylphosphatidic acid (DMPA) monolayers on pure water [Schalke et al., Biochim. Biophys. Acta 1464 (2000) 113-126] it is shown that stoichiometric binding of the divalent cations--DMPA-:Cat2+= 2:1--occurs only at exceedingly low areas per molecule, A lipid. At low surface pressure pi, both cations and anions are incorporated into the headgroup in significant amounts, approximately 0.68 Ba2+ and approximately 0.35 Cl- per PA molecule at pi = 2 mN m(-1). They are continuously squeezed out upon compression, until upon approaching Alipid = 41 A2, the stoichiometric ratio between bound cations and acidic headgroups is observed. The average inclination angle alpha of the headgroups as well as their water content is constant along the whole isotherm. The intrinsic contribution to the distribution width--i.e. the spread that is due to a distribution of the fragments within the headgroup without the action of capillary waves--increases with compression up to pi approximately 30 mN m(-1) and drops sharply thereafter in a regime of the isotherm where Alipid approaches its limiting value. The same general picture is observed for DMPA on subphases with 10 mM Ca2+, although the lower electron density of that cation limits the precision of the results.

Effect of hydrophobic surfactant peptides SP-B and SP-C on binary phospholipid monolayers. I. Fluorescence and dark-field microscopy.

The influence of the hydrophobic proteins SP-B and SP-C, isolated from pulmonary surfactant, on the morphology of binary monomolecular lipid films containing phosphocholine and phosphoglycerol (DPPC and DPPG) at the air-water interface has been studied using epifluorescence and dark-field microscopy. In contrast to previously published studies, the monolayer experiments used the entire hydrophobic surfactant protein fraction (containing both the SP-B and SP-C peptides) at physiologically relevant concentrations (approximately 1 wt %). Even at such low levels, the SP-B/C peptides induce the formation of a new phase in the surface monolayer that is of lower intrinsic order than the liquid condensed (LC) phase that forms in the pure lipid mixture. This presumably leads to a higher structural flexibility of the surface monolayer at high lateral pressure. Variation of the subphase pH indicates that electrostatic interaction dominates the association of the SP-B/C peptides with the lipid monolayer. As evidenced from dark-field microscopy, monolayer material is excluded from the DPPC/DPPG surface film on compression and forms three-dimensional, surface-associated structures of micron dimensions. Such exclusion bodies formed only with SP-B/C peptides. This observation provides the first direct optical evidence for the squeeze-out of pulmonary surfactant material in situ at the air-water interface upon increasing monolayer surface pressures.

X-ray diffraction from a single layer of purple membrane at the air/water interface.

X-ray diffraction patterns have been recorded from a single layer of purple membrane ( approximately 50 A thickness) at the air/water interface in a Langmuir trough. Grazing-incidence X-ray diffraction is demonstrated to be a promising method for obtaining structural information on membrane proteins under physiological conditions. The method is so sensitive that diffraction can be measured from samples with only 10(13) protein molecules in the beam. Diffraction from hexagonal crystals of purple membrane with a lattice constant of 61. 3 A was observed up to the order {h,k}={4,3}, corresponding to a resolution of approximately 9 A. The work reported here is a first step towards a new way of protein crystallography using grazing-incidence X-ray diffraction at the air/water interface.

Bacterial S-layer protein coupling to lipids: x-ray reflectivity and grazing incidence diffraction studies.

The coupling of bacterial surface (S)-layer proteins to lipid membranes is studied in molecular detail for proteins from Bacillus sphaericus CCM2177 and B. coagulans E38-66 recrystallized at dipalmitoylphosphatidylethanolamine (DPPE) monolayers on aqueous buffer. A comparison of the monolayer structure before and after protein recrystallization shows minimal reorganization of the lipid chains. By contrast, the lipid headgroups show major rearrangements. For the B. sphaericus CCM2177 protein underneath DPPE monolayers, x-ray reflectivity data suggest that amino acid side chains intercalate the lipid headgroups at least to the phosphate moieties, and probably further beyond. The number of electrons in the headgroup region increases by more than four per lipid. Analysis of the changes of the deduced electron density profiles in terms of a molecular interpretation shows that the phosphatidylethanolamine headgroups must reorient toward the surface normal to accommodate such changes. In terms of the protein structure (which is as yet unknown in three dimensions), the electron density profile reveals a thickness lz approximately 90 A of the recrystallized S-layer and shows water-filled cavities near its center. The protein volume fraction reaches maxima of >60% in two horizontal sections of the S-layer, close to the lipid monolayer and close to the free subphase. In between it drops to approximately 20%. Four S-layer protein monomers are located within the unit cell of a square lattice with a spacing of approximately 131 A.

Novel biosensoric devices based on molecular protein hetero-multilayer films.

We have developed a novel concept for the modification of technical surfaces with molecularly well-organized layers of bioorganic components. A molecular construction set has been used to implement this concept which is based on molecularly stratified polyelectrolyte films as a structure decoupling protein layers from solid substrates. Utilizing this technology, one can start from a number of different substrates to obtain the same surface structures, on which protein hetero-multilayer films can be prepared to functionalize the interface for (potentially very different) purposes. We have demonstrated the viability of this concept by constructing a biosensor surface that was characterized by x-ray, spectroscopic, and immunological techniques. For the preparation of this functionalized interface, a cascade of biospecific recognition processes has been used, in which a layer of SpA was supplanted on a dense SA monolayer (binding stoichiometry, approximately 0.2 SpA/SA). On the SpA, IgG has been immobilized (binding stoichiometry, approximately 0.5 IgG/ SpA). The resultant biosensor displays extremely low unspecific background reactivity, high sensitivity, and good response linearity over more than 3 orders of magnitude in antigen concentration.

Influence of surface chemistry on the structural organization of monomolecular protein layers adsorbed to functionalized aqueous interfaces.

The molecular organization of streptavidin (SA) bound to aqueous surface monolayers of biotin-functionalized lipids and binary lipid mixtures has been investigated with neutron reflectivity and electron and fluorescence microscopy. The substitution of deuterons (2H) for protons (1H), both in subphase water molecules and in the alkyl chains of the lipid surface monolayer, was utilized to determine the interface structure on the molecular length scale. In all cases studied, the protein forms monomolecular layers underneath the interface with thickness values of approximately 40 A. A systematic dependence of the structural properties of such self-assembled SA monolayers on the surface chemistry was observed: the lateral protein density depends on the length of the spacer connecting the biotin moiety and its hydrophobic anchor. The hydration of the lipid head groups in the protein-bound state depends on the dipole moment density at the interface.

Recognition processes at a functionalized lipid surface observed with molecular resolution.

The specific binding of proteins to functionalized lipid monolayers on aqueous subphases was characterized by neutron reflectivity and fluorescence microscopy measurements. Due to the high affinity and high specificity of their noncovalent interaction, streptavidin (SA) and biotin (vitamin H) were chosen as a model system to investigate the structural characteristics of a recognition process on a molecular length scale. Changes in the neutron reflection from the surfaces of NaCl aqueous (H2O or D2O) protein solutions (10(-8) M SA) were used to monitor the interaction of the protein with a monolayer of a biotinylated lipid in situ. Refinement of the reflectivity data and independent fluorescence microscopic observation of the interface using FITC-labeled SA showed that the protein forms macroscopically homogeneous (and presumably crystalline) domains covering a large portion of the surface. Moreover, the neutron reflection experiments clearly showed the formation of a monomolecular protein layer with an effective thickness, dp = 43.7 +/- 2 A. The area per protein molecule occupied in the film was A0 = 2860 +/- 200 A2 and nw = 260 +/- 100 water molecules were associated with each protein molecule. Quantitative binding was found to occur at biotin surface concentrations as low as 1 molecule/1,250 A2 (compared with approximately 1 molecule/40 A2 for dense packing). This study demonstrates the application of a promising new tool for the systematic investigation of molecular recognition processes in protein/lipid model systems.

Structural properties of phosphatidylcholine in a monolayer at the air/water interface: Neutron reflection study and reexamination of x-ray reflection measurements.

Neutron reflectivities of phosphatidylcholine monolayers in the liquid condensed (LC) phase on ultrapure H(2)O and D(2)O subphases have been measured on a Langmuir film balance. Using a dedicated liquid surface reflectometer, reflectivities down to R = 10(-6) in the momentum transfer range Q(z) = 0-0.4 A(-1) were accessed.In a new approach, by refining neutron reflectivity data from chain-perdeuterated DPPC-d(62) in combination with x-ray measurements on the same monolayer under similar conditions it is shown that the two techniques mutually complement one another. This analysis leads to a detailed conception of the interface structure. It is found that in the LC phase (which is analogous to the L(beta), phase in vesicle dispersions) the head group is interpenetrated with subphase water (4 +/- 2.5 molecules per lipid) and the average tilt angle of the hydrophobic chains from the surface normal is 33 +/- 3 degrees.

The Stark effect in reaction centers from Rhodobacter sphaeroides R-26 and Rhodopseudomonas viridis.

The effect of an electric field on the optical absorption (Stark effect) of reaction centers (RCs) from Rhodobacter sphaeroides and Rhodopseudomonas viridis embedded in films of poly(vinyl alcohol) was measured. The infrared bands were investigated at 295 K and 77 K. In RCs from Rp. viridis at 77 K six peaks (at 982, 849, 835, 818, 803, and 787 nm), associated with the Qy transitions of the six pigments, were resolved; in addition, a small broad band at 865 nm was resolved. In RCs from Rb. sphaeroides only five bands (at 877, 817, 802, 761, and 754 nm) assigned to the Qy transitions were resolved; in addition, two small bands at 697 and 683 nm were observed. The additional bands have been tentatively assigned to vibrational side bands, although the contribution from charge-transfer states cannot be excluded. The Stark spectra had line shapes similar to the second derivative of the absorption spectra and were interpreted in terms of the interaction between the applied electric field and the dipole moments of the ground and excited states. Analyses of the spectra yielded the apparent change in dipole moment delta mu app = f delta mu (where the factor f corrects for the difference between the local field and the applied field) and delta, the angle between delta mu---- and the transition moment mu trans. At 77 K the values of delta mu----app and delta for the peaks at 877, 802, and 761 nm in Rb. sphaeroides were 6.5 debye, 38 degrees; 2.1 debye, 23 degrees; and 3.5 debye, 8 degrees. In Rp. viridis the debye values for the peaks at 982, 835, 818, and 787 were 8.2, 40 degrees; 1.8, 50 degrees; 3.4, 14 degrees; and 2.7, 0 degrees. The large values of delta mu app associated with the long-wavelength peak of the bacteriochlorophyll dimers are consistent with a significant charge-transfer contribution to the excited state of the primary donor.

Electrostatically induced growth of spiral lipid domains in the presence of cholesterol.

The formation of crystalline domains of the phospholipid L-alpha-dimyristoyl-phosphatidic acid containing 1 mol% cholesterol, was studied as a function of head group charge by fluorescence microscopy with monolayers at the air/water interface. It is shown that the usual dendritic growth occurs at low pH (8), whereas spiral domains are formed at high pH (11), where the head group contains two negative charges. The findings are ascribed to an electrostatically induced chain tilt that, in conjunction with an in-plane dipole moment, causes a ferroelectric state. This allows for domain aggregation and orientation originating in elongated domains that, additionally, are bent because of the chirality of the molecules. The structure is stabilized and further elongated due to the anisotropic edge activity of cholesterol.

Impurity controlled phase transitions of phospholipid monolayers.

The phase diagram of monolayers of L-alpha-dimyristoyl phosphatidic acid has been studied by fluorescence microscopy. For pressures corresponding to the nearly horizontal slope in the pressure area diagram the growth of crystalline platelets can be observed. They are of dendritic nature; their sizes can be controlled via pressure, compression speed, temperature and pH, and increased up to 100 micron. Due to repulsive interaction a hexagonal arrangement of crystalline platelets can be established. It is shown that the textures do not depend on the dye probe for concentrations below 3 mol%. On the other hand via incorporation of impurities in concentrations of about 1 mol% the coexistence of lipid and solid phases can be controlled. Since, for a constant surface pressure, this coexistence can be maintained, these monolayers are suitable model systems to study the interactions of proteins and vesicles with coexisting fluid and solid membrane areas.