Effects of chain unsaturation on the equation of state for lipid monolayers at the air-water interface.

An equation of state for lipid monolayers at the air-water interface is presented, which takes into account the effects of the conformation and the number and position of double bonds of the hydrocarbon chains. The total Hamiltonian of the monolayer is assumed to consist of three terms. Two of them are calculated exactly within the limitations of the formulation. These are the two-dimensional entropy of mixing of the lipid and water molecules at the surface and the conformational entropy of the chains using a model available from the literature. These two terms give rise to positive surface pressure. The third term, which includes all energies that are not amenable to calculation, was obtained as the difference between the sum of the two calculated terms and experimental data and is found to represent an approximately area-independent tension. The effects of chain unsaturation on the equation of state were modeled in the present theory in two ways; the chain bend caused by cis double bonds increases the minimal molecular area, and the double bond linkage on a chain decreases the degrees of freedom of the chain. Calculations revealed that the former is highly significant whereas the latter is negligible. The deduced equation of state reproduces experimental data with appropriate values for three parameters, which represent the collapse area, the overlap of adjacent chains, and the combined effects of the intra- and intermolecular interactions other than the surface mixing entropy and the chain conformational energy.     

Two-dimensional diffusion of amphiphiles in phospholipid monolayers at the air-water interface.

Steady-state and time-resolved fluorescence spectroscopy has been used to examine lateral diffusion in dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) and dimyristoyl-L-alpha-phosphatidylcholine (DMPC) monolayers at the air-water interface, by studying the fluorescence quenching of a pyrene-labeled phospholipid (pyrene-DPPE) by two amphiphilic quenchers. Steady-state fluorescence measurements revealed pyrene-DPPE to be homogeneously distributed in the DMPC lipid matrix for all measured surface pressures and only in the liquid-expanded (LE) phase of the DPPC monolayer. Time-resolved fluorescence decays for pyrene-DPPE in DMPC and DPPC (LE phase) in the absence of quencher were best described by a single-exponential function, also suggesting a homogeneous distribution of pyrene-DPPE within the monolayer films. Addition of quencher to the monolayer film produced nonexponential decay behavior, which is adequately described by the continuum theory of diffusion-controlled quenching in a two-dimensional environment. Steady-state fluorescence measurements yielded lateral diffusion coefficients significantly larger than those obtained from time-resolved data. The difference in these values was ascribed to the influence of static quenching in the case of the steady-state measurements. The lateral diffusion coefficients obtained in the DMPC monolayers were found to decrease with increasing surface pressure, reflecting a decrease in monolayer fluidity with compression.     

Interaction of nominally soluble proteins with phospholipid monolayers at the air-water interface

The interactions of carbonmonoxyhemoglobin (HbCO), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and polyhistidine with phospholipid monolayers at the air-water interface were studied at physiological pH and ionic strength. HbCO and GAPDH both interact more strongly with monolayers containing negatively charged lipids. The interaction of HbCO and GAPDH with lipid monolayers decreases with increasing pH. Both the HbCO-monolayer and the GAPDH-monolayer interactions can be modeled as diffusion-limited processes, with kinetic data fit to a stretched exponential equation. The significance of these kinetics are discussed. Polyhistidine interacts only with monolayers containing lipids with negatively charged headgroups. In total, the results presented are consistent with an HbCO-lipid interaction with a large electrostatic component, a GAPDH-lipid interaction with comparable electrostatic and hydrophobic components, and a polyhistidine-lipid interaction that is solely electrostatic.     

Behavior of a GPI-anchored protein in phospholipid monolayers at the air-water interface

The interaction between alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein (AP-GPI), and phospholipids was monitored using Langmuir isotherms and PM-IRRAS spectroscopy. AP-GPI was injected under C16 phospholipid monolayers with either a neutral polar head (1,2-dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (DPPC)) or an anionic polar head (1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS)). The increase in molecular area due to the injection of protein depended on the surface pressure and the type of phospholipid. At all surface pressures, it was highest in the case of DPPS monolayers. The surface elasticity coefficient E, determined from the pi-A diagrams, allowed to deduct that the AP-GPI-phospholipid mixtures presented a molecular arrangement less condensed than the corresponding pure phospholipid films. PM-IRRAS spectra suggested different protein-lipid interactions as a function of the nature of the lipids. AP-GPI modified the organization of the DPPS deuterated chains whereas AP-GPI affected only the polar group of DPPC at low surface pressure (8 mN/m). Different protein hydration layers between the DPPC and DPPS monolayers were suggested to explain these results. PM-IRRAS spectra of AP-GPI in the presence of lipids showed a shape similar to those collected for pure AP-GPI, indicating a similar orientation of AP-GPI in the presence or absence of phospholipids, where the active sites of the enzyme are turned outside of the membrane.     

Properties of cholesteryl oleate and triolein in mixed monolayers at the air-water interface

The properties of cholesteryl oleate and triolein in mixed monolayers at the air-water interface have been measured between 24 and 37 degrees C. Analysis of force-area curves obtained as a function of the mol fraction of cholesteryl oleate indicates that at relatively low surface pressures these compounds are miscible in two dimensions up to a limit of about 0.5 mol fraction. At higher pressures either cholesteryl oleate or both lipids are expelled from the monolayer to form a bulk phase which is in rapid equilibrium with the surface phase. In the monolayer phase, orientation of the ester function of cholesteryl oleate is toward the aqueous phase, interaction with triolein is minimal, and packing is uniform over the solubility range. This together with the susceptibility of the cholesteryl oleate to enzymatic hydrolysis, suggests the applicability of monolayer systems to the study of cholesterol esterase activity. Comparison of our results with the bulk properties of these lipids suggests that the expelled cholesteryl oleate exists as a smectic mesophase and thus the system may provide a model for studying the transfer of molecules between the interior and surface of lipid deposits of the type found in atherosclerotic lesions.     

Compositional domain immiscibility in whole myelin monolayers at the air-water interface and Langmuir-Blodgett films

Monomolecular layers of whole myelin membrane can be formed at the air-water interface from vesicles or from solvent solution of myelin. The films appear microheterogeneous as seen by epifluorescence and Brewster angle microscopy. The pattern consists mainly of two coexisting liquid phases over the whole compression isotherm. The liquid nature of the phases is apparent from the fluorescent probe behavior, domain mobility, deformability and boundary relaxation due to the line tension of the surface domains. The monolayers were transferred to alkylated glass and fluorescently labeled against myelin components. The immunolabeling of two major proteins of myelin (myelin basic protein, proteolipid-DM20) and of 2′,3′-cyclic nucleotide 3′-phosphodiesterase shows colocalization with probes partitioning preferentially in liquid-expanded lipid domains also containing ganglioside G_M1. A different phase showing an enrichment in cholesterol, galactocerebroside and phosphatidylserine markers is also found. The distribution of components is qualitatively independent of the lateral surface pressure and is generally constituted by one phase enriched in charged components in an expanded state coexisting with another phase enriched in non-charged constituents of lower compressibility. The domain immiscibility provides a physical basis for the microheterogeneity found in this membrane model system.     

Compositional domain immiscibility in whole myelin monolayers at the air-water interface and Langmuir-Blodgett films

Monomolecular layers of whole myelin membrane can be formed at the air-water interface from vesicles or from solvent solution of myelin. The films appear microheterogeneous as seen by epifluorescence and Brewster angle microscopy. The pattern consists mainly of two coexisting liquid phases over the whole compression isotherm. The liquid nature of the phases is apparent from the fluorescent probe behavior, domain mobility, deformability and boundary relaxation due to the line tension of the surface domains. The monolayers were transferred to alkylated glass and fluorescently labeled against myelin components. The immunolabeling of two major proteins of myelin (myelin basic protein, proteolipid-DM20) and of 2',3'-cyclic nucleotide 3'-phosphodiesterase shows colocalization with probes partitioning preferentially in liquid-expanded lipid domains also containing ganglioside G(M1). A different phase showing an enrichment in cholesterol, galactocerebroside and phosphatidylserine markers is also found. The distribution of components is qualitatively independent of the lateral surface pressure and is generally constituted by one phase enriched in charged components in an expanded state coexisting with another phase enriched in non-charged constituents of lower compressibility. The domain immiscibility provides a physical basis for the microheterogeneity found in this membrane model system.     

Surface enhanced Raman scattering of a lipid Langmuir monolayer at the air-water interface

Surface enhanced Raman spectra were recorded from a phospholipid monolayer directly at the air-water interface. We used an organized monolayer of negatively charged tetramyristoyl cardiolipins as a template for the electrochemical generation of silver deposits. This two-dimensional electrodeposition of silver under potentiostatic control was the substrate for enhancement of Raman spectra. We report the optimized conditions for the Raman enhancement, the microscopic observations of the deposits, and their characterization by atomic force microscopy. Laser excitation at 514.5 nm leads to intense and reproducible surface enhanced Raman scattering spectra recorded in situ from one monolayer of cardiolipin, using 0.5 mol % of 10N nonyl acridine orange or 5 mol % of acridine in the film, and demonstrates the possibility of estimating the pH at the metal/phospholipidic film interface.     

Amyloid-beta-sheet formation at the air-water interface

An amyloid(1-40) solution rich in coil, turn, and alpha-helix, but poor in beta-sheet, develops monolayers with a high beta-sheet content when spread at the air-water interface. These monolayers are resistant to repeated compression-dilatation cycles and interaction with trifluoroethanol. The secondary structure motifs were detected by circular dichroism (CD) in solution and with infrared reflection-absorption spectroscopy (IRRAS) at the interface. Hydrophobic influences are discussed for the structure conversion in an effort to understand the completely unknown reason for the natural change of the normal prion protein cellular (PrP(C)) into the abnormal prion protein scrapie (PrP(Sc)).     

Molecular recognition of concanavalin A on mannoside diacetylene lipid monolayer at the air-water interface.

The interaction of p-10,12-pentacosadiyne-1-n-phenylamide alpha-D-mannopyranoside (MPDA) with protein concanavalin A (Con A) was studied at the air/water interface. The expansion of molecular area of PDA (10,12-pentacosadiynoic acid)/MPDA mixed monolayer after injection of Con A in subphase shows strong interaction between Con A and the monolayer. The maximum expansion of molecular area decreases as the molar ratio of MPDA increases due to the steric hindrance effect. By using enzyme mannosidase to cut-off the mannoside headgroup of MPDA, expansion of molecular area was greatly reduced, indicating that the binding of Con A is specific to the mannoside headgroup. The kinetics of the binding fits to the first order bimolecular reaction model. Fluorescence quenching of fluorescein isothiocyanate labeled Con A after injection into the subphase gives a direct proof of the molecular recognition.     

Structure of rhodopsin in monolayers at the air-water interface: a PM-IRRAS and X-ray reflectivity study

Monomolecular films of the membrane protein rhodopsin have been investigated in situ at the air-water interface by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and X-ray reflectivity in order to find conditions that retain the protein secondary structure. The spreading of rhodopsin at 0 or 5 mN m(-1) followed by a 30 min incubation time at 21 degrees C resulted in the unfolding of rhodopsin, as evidenced from the large increase of its molecular area, its small monolayer thickness, and the extensive formation of beta-sheets at the expense of the alpha-helices originally present in rhodopsin. In contrast, when spreading is performed at 5 or 10 mN m(-1) followed by an immediate compression at, respectively, 4 or 21 degrees C, the secondary structure of rhodopsin is retained, and the thickness of these films is in good agreement with the size of rhodopsin determined from its crystal structure. The amide I/amide II ratio also allowed to determine that the orientation of rhodopsin only slightly changes with surface pressure and it remains almost unchanged when the film is maintained at 20 mN m(-1) for 120 min at 4 degrees C. In addition, the PM-IRRAS spectra of rod outer segment disk membranes in monolayers suggest that rhodopsin also retained its secondary structure in these films.