Molecular organization of the human serotonin transporter at the air/water interface

The serotonin transporter (SERT) is the target of several important antidepressant and psychostimulant drugs. It has been shown that under defined conditions, the transporter spread at the air/water interface was able to bind its specific ligands. In this paper, the interfacial organization of the protein has been assessed from dynamic surface pressure and ellipsometric measurements. For areas comprising between 10,400 and 7,100 A(2)/molecule, ellipsometric measurements reveal an important change in the thickness of the SERT film. This change was attributed to the reorientation of the transporter molecules from a horizontal to their natural predictive transmembrane orientation. The thickness of the SERT film at 7,100 A(2)/molecule was found to be approximately equal to 84 A and coincided well with the theoretical value estimated from the calculations based on the dimensions of alpha-helices containing membrane proteins. These data suggest that the three-dimensional arrangement of the SERT may be represented as a box with lengths d(z)=83--85 A and d(y) or d(x)=41--47 A.     

Secondary structure of spiralin in solution, at the air/water interface, and in interaction with lipid monolayers

The surface of spiroplasmas, helically shaped pathogenic bacteria related to the mycoplasmas, is crowded with the membrane-anchored lipoprotein spiralin whose structure and function are unknown. In this work, the secondary structure of spiralin under the form of detergent-free micelles (average Stokes radius, 87.5 A) in water and at the air/water interface, alone or in interaction with lipid monolayers was analyzed. FT-IR and circular dichroism (CD) spectroscopic data indicate that spiralin in solution contains about 25+/-3% of helices and 38+/-2% of beta sheets. These measurements are consistent with a consensus predictive analysis of the protein sequence suggesting about 28% of helices, 32% of beta sheets and 40% of irregular structure. Brewster angle microscopy (BAM) revealed that, in water, the micelles slowly disaggregate to form a stable and homogeneous layer at the air/water interface, exhibiting a surface pressure up to 10 mN/m. Polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) spectra of interfacial spiralin display a complex amide I band characteristic of a mixture of beta sheets and alpha helices, and an intense amide II band. Spectral simulations indicate a flat orientation for the beta sheets and a vertical orientation for the alpha helices with respect to the interface. The combination of tensiometric and PMIRRAS measurements show that, when spiroplasma lipids are used to form a monolayer at the air/water interface, spiralin is adsorbed under this monolayer and its antiparallel beta sheets are mainly parallel to the polar-head layer of the lipids without deep perturbation of the fatty acid chains organization. Based upon these results, we propose a 'carpet model' for spiralin organization at the spiroplasma cell surface. In this model, spiralin molecules anchored into the outer leaflet of the lipid bilayer by their N-terminal lipid moiety are composed of two colinear domains (instead of a single globular domain) situated at the lipid/water interface. Owing to the very high amount of spiralin in the membrane, such carpets would cover most if not all the lipids present in the outer leaflet of the bilayer.     

Infrared reflection-absorption of melittin interaction with phospholipid monolayers at the air/water interface.

The interaction of melittin with monolayers of 1,2-dipalmitoylphosphatidylcholine and 1,2-dipalmitoylphosphatidylserine has been investigated with infrared external reflection-absorption spectroscopy. Improved instrumentation permits determination of acyl chain conformation and peptide secondary structure in situ at the air/water interface. The IR frequency of the 1,2-dipalmitoylphosphatidylcholine antisymmetric acyl chain CH2 stretching vibration decreases by 1.3 cm-1 upon melittin insertion, consistent with acyl chain ordering, whereas the same vibrational mode increases by 0.5 cm-1 upon peptide interaction with the 1,2-dipalmitoylphosphatidylserine monolayer, indicative of chain disordering. Thus the peptide interacts quite differently with zwitterionic compared with negatively charged monolayer surfaces. Melittin in the monolayer adopted a secondary structure with an amide l(l') frequency (1635 cm-1) dramatically different from the alpha-helical motif (amide l frequency 1656 cm-1 in a dry or H2O hydrated environment, amide l' frequency 1645 cm-1 in an H-->D exchanged alpha-helix) assumed in bilayer or multibilayer environments. This work represents the first direct in situ spectroscopic indication that peptide secondary structure in lipid monolayers may differ from that in bilayers.     

An analysis of the interaction of protein with lipid monolayers at the air/water interface

1. Measurements have been made of the interaction of cytochrome c, bovine serum albumin and synthetic oxytocin with low-pressure (2dyn/cm) monolayers of stearic acid, phosphatidylcholine and phosphatidylethanolamine. 2. [(14)C]Carboxymethylation of the cytochrome c and albumin followed by surface-radioactivity determinations have shown that only a proportion of the protein added to the subphase is bound to the monolayers and that initially the degree of binding is dependent on the protein concentration. The binding is irreversible in the sense that the adsorbed protein cannot be removed by transferring the film containing the interacted protein to a fresh subphase containing no protein. 3. Three successive types of interaction can usually be recognized. (a) Initially, whole molecules of protein penetrate the lipid film and occupy the same area as those of the protein spread at the air/water interface. (b) Above certain film pressures a part of each protein molecule, probably hydrophobic side chains, penetrates the film. The change in surface pressure per unit of bound protein is much smaller than in (a). (c) At higher film pressures, adsorption without penetration occurs. With cytochrome c this is initially dependent on a favourable electrostatic interaction.     

Bacterial activity at the air/water interface

Microbial Ecology - By using substrate molecules of varying degrees of surface activity, we were able to measure some features of bacterial activity in the surface microlayers (SM) and in the...     

Spreading of membranes at the air/water interface

This article presents an original technique for spreading membranes at the air/water interface. We have characterized enzymatically lipoprotein films d     

Spreading of liposomes at the air/water interface

Two types of film structure are formed when liposomes are spread at the air/water interface. At zero surface pressure, there is a slow transformation of the closed bilayered structure into a lipid monolayer. The internal content of the liposomes is released into the aqueous subphase. In contrast, when multilamellar liposomes are spread against a surface pressure, they retain their internal content at the air/water interface by forming multilayered structures. Among the liposomes which dipped through the interface an important fraction loses its internal content. During the spreading process at zero surface pressure, it seems that the outer layer of the liposome spreads with a better yield as compared with the inner layer. It is possible to use this spreading technique to determine the asymmetrical distribution of lipids across bilayers.     

Coniferyl alcohol reactivity at the air/water interface

In order to investigate the sensitivity of the lignin monomer coupling reactions to the environment physicochemical conditions, coniferyl alcohol (CA) was polymerised at the air/water interface. Characterisation of the interface during the reaction by surface pressure measurement and ellipsometry demonstrates that the reaction occurs near or at the interface. Coupling products were analysed by HPLC and compared to reaction products obtained in the case of polymerisation in solution. Relative proportions of beta-beta and beta-O-4 dehydrodimers were found to increase in air/water interface experiment.     

Spreading of biomembranes at the air/water interface.

This paper presents the compression isotherms obtained by spreading membranes of intestinal brush border, human erythrocyte and Escherichia coli (cytoplasmic) at the air/water interface. Unilamellar membrane films were formed, with a good yield, at zero surface pressure, whereas multilamellar structures were formed at high surface pressure. Once formed, the films were particularly stable and could be manipulated without any detectable loss. With doubly-labelled E. coli cytoplasmic membrane, we could show that phospholipids and proteins spread, with the same yield, as a single unit. Moreover, we studied the influence of hydrolytic enzymes, chemical agents and cations on the compression isotherm of biomembranes. The resultant changes in architecture of membrane films can provide a very simple method of studying the influence of membrane packing on catalytic activity and protein conformation of membrane-bound proteins.     

Surface balance study of the interaction between microorganisms and lipid monolayer at the air/water interface.

Using the surface balance technique, we have compared the interaction between Acholeplasma laidlawii and some marine bacteria towards different types of monolayered lipid films. Cells from A. laidlawii and Serratia marinorubra penetrate the film, whereas cells from Psuedomonas fluorescens form a layer underneath the film. The forces that bind microorganisms to the air/water interface are not strong enough to scatter a condensed monolayer but increase the strength of loosely packed monolayers.     

Surface balance study of the interaction between microorganisms and lipid monolayer at the air/water interface.

Using the surface balance technique, we have compared the interaction between Acholeplasma laidlawii and some marine bacteria towards different types of monolayered lipid films. Cells from A. laidlawii and Serratia marinorubra penetrate the film, whereas cells from Psuedomonas fluorescens form a layer underneath the film. The forces that bind microorganisms to the air/water interface are not strong enough to scatter a condensed monolayer but increase the strength of loosely packed monolayers.     

Miscibility of DPPC and DPPA in monolayers at the air/water interface

Monolayers of mixtures of 1,2-dipalmitoylphosphatidylcholine (DPPC) as the substrate and 1,2-dipalmitoylphosphatidic acid (DPPA) as the product of the hydrolysis reaction catalyzed by phospholipase D (PLD) were investigated in the presence of Ca2+. The miscibility behavior and the microstructure of mixed domains have been studied by grazing incidence X-ray diffraction (GIXD), Brewster angle microscopy and film balance measurements. The phase diagram reveals partial miscibility on both sides and a wide miscibility gap, which becomes narrower at high pressure. At low pressure, the segregation of condensed DPPA-rich domains in a fluid-like DPPC matrix was detected already at small DPPA concentrations and their structure was determined. A small amount of DPPC mixed into the segregated DPPA domains induces the transformation from rectangular to an oblique unit cell and increases the tilt angle in the condensed domains. At high pressure, two types of condensed phase domains were found: DPPC-rich and DPPA-rich. A drastic reduction of the tilt angle in the DPPC-rich domains with increasing amount of DPPA was observed. The decrease of the tilt angle must be connected with a change of the head group conformation of DPPC in such mixed domains.     

Adsorption and desorption studies of phospholipid at the air/water interface

The desorption and adsorption properties of phosphatidylserine (extracted from beef brain) at the air/water interface were studied through surface pressure measurements. The rate of dissolution of phosphatidylserine monolayer into the underlying water of natural pH is extremely slow at room temperature but increases rather suddenly around 40°C. This sudden increase of dissolution rate might be explained as the Kraft point phenomena analogous to the ionic surfactants.     

Influence of Ca2+ and temperature on the interaction of gangliosides with valinomycin in mixed monolayers at the air/water interface

The effects of Ca2+ and temperature on mixed ganglioside-valinomycin-monolayers at the air/water interface were studied. Surface pressure-area isotherms of the pure gangliosides (GM1, GD1a) exhibited the typical monolayer characteristics. Pressure-area isotherms of the cyclodepsipeptide, valinomycin, were determined. In mixed monolayers, positive and negative deviation from the mean molecular area indicated the two components were miscible. Especially in GD1a mixtures, the addition of 0.01 mM calcium exhibited, with low molar fractions of valinomycin, a demixing effect in the direction of the phase separation of the components.     

The surface organization of diacylglycerol pyrophosphate and its interaction with phosphatidic acid at the air–water interface

Diacylglycerol pyrophosphate (DGPP), a phosphorylated form of phosphatidic acid (PA), gained attention recently due to its role as signaling lipid. However, little is known about its surface organization and potential impact on membrane-mediated function. In this work we investigated the interfacial behavior of Langmuir monolayers formed with pure DGPP and of its mixtures with PA. We found that changes of the subphase pH affect the surface behavior of DGPP. At pH 8, DGPP forms liquid expanded monolayers with a compressibility modulus of about 60 mN m^?1 at collapse. On acidic solutions, the compressibility modulus increases to 90 mN m^?1 and the average molecular area is smaller. At pH 8, DGPP and its precursor PA form thermodynamically favored topographically homogeneous non-ideal mixtures. The interaction among these lipids leads to a non-ideal diminution of the mean molecular area and consequently, to an increase of the compressibility modulus, with variations of the surface electrostatics. The favorable interaction of PA and DGPP, leading to changes of the film packing suggest that DGPP may act as a structural signal transducer in membrane-mediated cellular processes.     

Interfacial tension of phosphatidylcholine-cholesterol system in monolayers at the air/water interface

Interfacial tension of an egg lecithin-cholesterol system was measured across the whole concentration range. Surface pressure-area isotherm measurements were carried out in a Langmuir trough at the air/water interface at room temperature (22 degrees C). The interfacial tension of the air/water interface was divided into contributions of components. The interfacial tension of a 1:1 complex between phosphatidylcholine and cholesterol was calculated. Its value equals 18 mN/m. The difference between the stability constant of 1:1 complex in the bilayer and the monolayer at the air/water interface is discussed.     

Aggregation behaviors of gelatin with cationic gemini surfactant at air/water interface

The dilational rheological properties of gelatin with cationic gemini surfactant 1,2-ethane bis(dimethyl dodecyl ammonium bromide) (C(12)C(2)C(12)) at air/water interface were investigated using oscillating barriers method at low frequency (0.005-0.1 Hz), which was compared with single-chain surfactant dodecyltrimethyl ammonium bromide (DTAB). The results indicate that the maximum dilational modulus and the film stability of gelatin-C(12)C(2)C(12) are higher than those of gelatin-DTAB. At high concentration of C(12)C(2)C(12) or DTAB, the dilational modulus of gelatin-surfactant system becomes close to that corresponding to pure surfactant, suggesting gelatin at interface is replaced by surfactant. This replacement is also observed by surface tension measurement. However, it is found that gelatin-C(12)C(2)C(12) system has two obvious breaks but gelatin-DTAB has not in surface tension isotherms. These phenomena are ascribed to the double charges and strong hydrophobicity of C(12)C(2)C(12). Based on these experimental results, a mechanism of gelatin-surfactant interaction at air/water interface is proposed.     

Second harmonic generation of glucose oxidase at the air/water interface

We present a study of the adsorption of the glucose oxidase enzyme (GOx) at the air/water interface, using the nonlinear optical technique of surface second harmonic generation (SSHG). Resonant SSHG experiments were achieved by probing the pi-pi* transition of the flavin adenine dinucleotide (FAD) chromophores embedded in the GOx protein. Because of the subsequent resonance enhancement of the signal, the second harmonic (SH) wave arising from the GOx entities adsorbed at the interface was detectable for protein bulk aqueous concentrations as low as 70 nM. The protein adsorption was followed, and, at high GOx coverage, a change in the orientation of the FAD chromophore was observed, indicating either a rearrangement or a reorientation of the protein at the interface. Inasmuch as GOx is negatively charged at the biological pH of 7, its interactions with charged surfactants were also investigated. As expected, spreading positively charged surfactants onto a partial protein monolayer was found to increase the GOx surface concentration, whereas in the case of negatively charged surfactants, the GOx surface concentration decreased until the SH signal went back to the pure buffer solution response level. With the increasing GOx surface concentration, the rearrangement or reorientation of the protein was also observed.     

Conformational changes of the lecithin headgroup in monolayers at the air/water interface

The headgroup conformation of the phospholipid dipalmitoyl-glycero-phosphocholine (DPPC) in monolayers at the air/water interface has been studied by neutron reflection in the fluid like liquid-expanded (LE) and in the crystal like solid (S) phase. Information on the headgroup conformation in the two phases has been obtained by scattering contrast variation of the lipid monolayer using four differently deuterated species of DPPC: perdeuterated, chain perdeuterated, choline group perdeuterated and selectively headgroup deuterated. Since the measurements were done mainly on a subphase of null reflecting water (i.e. water scattering contrast matched to the air) there is no subphase contribution to reflectivity and the simplest one layer model can be employed for the data analysis, thus minimising the number of free parameters. A remarkable change of the headgroup orientation was observed between the LE and the S phase. We found that the phosphate-nitrogen dipole of the DPPC headgroup exhibits an in-plane orientation with respect to the monolayer in the LE phase but it assumes a more parallel orientation to the surface normal at lateral pressures above 30 mN/m (S phase). Moreover, this conformational change is accompanied by a significant alteration of the headgroup hydration.Abbreviations DPPC Dipalmitoyl-Phosphatidylcholine - DMPC Dimyristoyl-Phosphatidylcholine - DPPE Dipalmitoyl-Phosphatidylethanolamine - DMPE Dimyristoyl-Phosphatidylethanolamine - DMPA Dimyristoyl-Phosphatic Acid - DMPG Dimyristoyl-Phosphatidylglycerol Correspondence to: T M. Bayed