Vectorially oriented membrane protein monolayers: profile structures via x-ray interferometry/holography.

X-ray interferometry/holography was applied to meridional x-ray diffraction data to determine uniquely the profile structures of a single monolayer of an integral membrane protein and a peripheral membrane protein, each tethered to the surface of a solid inorganic substrate. Bifunctional, organic self-assembled monolayers (SAMs) were utilized to tether the proteins to the surface of Ge/Si multilayer substrates, fabricated by molecular beam epitaxy, to facilitate the interferometric/holographic x-ray structure determination. The peripheral membrane protein yeast cytochrome c was covalently tethered to the surface of a sulfhydryl-terminated 11-siloxyundecanethiol SAM via a disulfide linkage with residue 102. The detergent-solubilized, photosynthetic reaction center integral membrane protein was electrostatically tethered to the surface of an analogous amine-terminated SAM. Optical absorption measurements performed on these two tethered protein monolayer systems were consistent with the x-ray diffraction results indicating the reversible formation of densely packed single monolayers of each fully functional membrane protein on the surface of the respective SAM. The importance of utilizing the organic self-assembled monolayers (as opposed to Langmuir-Blodgett) lies in their ability to tether specifically both soluble peripheral membrane proteins and detergent-solubilized integral membrane proteins. The vectorial orientations of the cytochrome c and the reaction center molecules were readily distinguishable in the profile structure of each monolayer at a spatial resolution of 7 A.     

Orientation distributions for cytochrome c on polar and nonpolar interfaces by total internal reflection fluorescence.

The formation of chemisorbed monolayers of yeast cytochrome c on both uncharged polar and nonpolar soft surfaces of organic self-assembled monolayers (SAM) on solid inorganic substrates was followed in situ by polarized total internal reflection fluorescence. Two types of nonpolar surfaces and one type of uncharged polar surface were used. The first type of nonpolar surface contained only thiol endgroups, while the other was composed of a mixture of thiol and methyl endgroups. The uncharged polar surface was provided by the mixture of thiol and hydroxyl endgroups. The thiol endgroups were used to form a covalent disulfide bond with the unique surface-exposed cysteine residue 102 of the protein. The mean tilt angle of the protein's zinc-substituted porphyrin was found to be 41 degrees and 50 degrees for the adsorption onto the nonpolar and uncharged polar surfaces, respectively. The distribution widths for the pure thiol and the thiol/methyl and thiol/hydroxyl mixtures were 9 degrees, 1 degrees, and 18 degrees, respectively. The high degree of the orientational order and good stability achieved for the protein monolayer on the mixed thiol/methyl endgroup SAM makes this system very attractive for studies of both intramolecular and intermolecular electron transfer processes.     

Characterization and redox properties of cytochrome c552 from Thermus thermophilus adsorbed on different self-assembled thiol monolayers, used to model the chemical environment of the redox partner

The structure of cytochrome c552 (Cyt-c552) from Thermus thermophilus shows many differences to other c-type cytochromes. The rich lysine domain close to the heme does not exist in this cytochrome, allowing us to postulate that the interaction with its redox partner must be different to the cytochrome c/cytochrome c oxidase interaction. We report a study of Cyt-c552 adsorbed on self-assembled monolayers (SAMs) of functionalized alkanethiols used to mimic the chemical properties of its redox partner (ba3-oxydase). Hydrophilic (-COOH), polar (-OH), hydrophobic (-CH3), and mixed (-OH/-CH3) SAMs grafted on roughened silver electrodes were characterized by X-ray photoelectron spectroscopy. Surface enhanced resonance Raman spectroscopy (SERRS) was employed to determine the structure and the redox properties (E degrees and number of transferred electron) of the heme of Cyt-c552 adsorbed on roughened silver electrodes coated by the different SAMs. The surface that most closely models the environment of the ba3-oxidase is a mixed SAM formed by 50% polar [Ag-(CH2)5-CH2OH] and 50% hydrophobic [Ag-(CH2)5-CH3] alkanethiols. Only the native form B1(6cLS) of Cyt-c552 is detected by SERRS when the protein is adsorbed on such a surface that promotes a protein orientation favorable for the electron transfer (number of transferred electron = 1). We shall discuss the differences and similarities of the electron-transfer mechanism of Cyt-c552 compared to cyt-c.     

Synthesis and characterization of bioconjugates of S-layer proteins

The self-assembling proteins that form crystalline surface layers (S-layers) on many microbial species have found numerous applications due to their nanostructured nature. To devise a new method to construct surface displays that exploit S-layer self-assembly activity and nanostructural properties, we have constructed polymer bioconjugates of S-layer proteins. The conjugates formed are similar in function to the monomer alkanethiols that form self-assembled monolayers (SAMs) on gold surfaces. However, the self-assembly is driven by the protein "headgroup" that positions polymer-tethered endgroups on a surface. This paper examines the integration of protein purification, conjugation, and surface assembly that has led to the development of this new method for the formation of nanostructured surfaces. Purified S-layer proteins from Lactobacillus brevis were conjugated with small molecule probes and polymers using amine-based reactions. To keep multiple labeling of protein amine groups to acceptable levels, the conjugations were performed at pH 6.5, allowing for limited yields (24-39%) as determined by mass spectrometry and SDS-polyacrylamide gel electrophoresis. As the presence of high levels of unlabeled S-layer proteins is undesired, we have developed a protocol for further purification that employs monomeric avidin affinity chromatography. The surface self-assembly of the polymer bioconjugates onto amine-terminated microspheres was studied using epi-fluorescence, confocal, and scanning electron microscopy. The surfaces obtained exhibited homogeneous distributions of tethered molecules. Also, in cases where the modular assembly of two distinct types of tethered endgroups was accomplished, there was no evidence for phase separation in the surfaces. The modular assembly method will provide a potential route to controlling surface display density as the starting assembly conditions guide displayed endgroup concentrations in mixed molecular monolayers.     

Vectorially oriented monolayers of detergent-solubilized Ca(2+) -ATPase from sarcoplasmic reticulum.

A method for tethering proteins to solid surfaces has been utilized to form vectorially oriented monolayers of the detergent-solubilized integral membrane protein Ca(2+) -ATPase from the sarcoplasmic reticulum (SR). Bifunctional, organic self-assembled monolayers (SAMs) possessing "headgroup" binding specificity for the substrate and "endgroup" binding specificity for the enzyme were utilized to tether the enzyme to the substrate. Specifically, an amine-terminated 11-siloxyundecaneamine SAM was found to bind the Ca(2+)-ATPase primarily electrostatically. The Ca(2+)-ATPase was labeled with the fluorescent probe 5-(2-[(iodoacetyl)amino]ethyl)aminonaphthalene-1-sulfonic acid before monolayer formation. Consequently, fluorescence measurements performed on amine-terminated SAM/enzyme monolayers formed on quartz substrates served to establish the nature of protein binding. Formation of the monolayers on inorganic multilayer substrates fabricated by molecular beam epitaxy made it possible to use x-ray interferometry to determine the profile structure for the system, which was proved correct by x-ray holography. The profile structures established the vectorial orientation of the Ca(2+)-ATPase within these monolayers, to a spatial resolution of approximately 12 A. Such vectorially oriented monolayers of detergent-solubilized Ca(2+)-ATPase from SR make possible a wide variety of correlative structure/function studies, which would serve to elucidate the mechanism of Ca(2+) transport by this enzyme.     

Vectorially oriented monolayers of the cytochrome c/cytochrome oxidase bimolecular complex.

Vectorially oriented monolayers of yeast cytochrome c and its bimolecular complex with bovine heart cytochrome c oxidase have been formed by self-assembly from solution. Both quartz and Ge/Si multilayer substrates were chemical vapor deposited with an amine-terminated alkylsiloxane monolayer that was then reacted with a hetero-bifunctional cross-linking reagent, and the resulting maleimide endgroup surface then provided for covalent interactions with the naturally occurring single surface cysteine 102 of the yeast cytochrome c. The bimolecular complex was formed by further incubating these cytochrome c monolayers in detergent-solubilized cytochrome oxidase. The sequential formation of such monolayers and the vectorially oriented nature of the cytochrome oxidase was studied via meridional x-ray diffraction, which directly provided electron density profiles of the protein(s) along the axis normal to the substrate plane. The nature of these profiles is consistent with previous work performed on vectorially oriented monolayers of either cytochrome c or cytochrome oxidase alone. Furthermore, optical spectroscopy has indicated that the rate of binding of cytochrome oxidase to the cytochrome c monolayer is an order of magnitude faster than the binding of cytochrome oxidase to an amine-terminated surface that was meant to mimic the ring of lysine residues around the heme edge of cytochrome c, which are known to be involved in the binding of this protein to cytochrome oxidase.     

Heme structure and orientation in single monolayers of cytochrome c on polar and nonpolar soft surfaces

Polarized x-ray absorption fine structure (XAFS) spectroscopy has been performed in fluorescence mode under total external reflection conditions on frozen hydrated single monolayers of yeast cytochrome c (YCC). The protein molecules were vectorially oriented within the monolayer by tethering their naturally occurring and unique surface cysteine residues to the sulfhydryl-endgroups at the surface of a mixed organic self-assembled monolayer, itself covalently attached to an ultrapure silicon wafer. The sulfhydryl-endgroups were isolated by dilution with either methyl- or hydroxyl-endgroups, producing macroscopically nonpolar or uncharged-polar soft surfaces, respectively. Independent information on the heme-plane orientation relative to the monolayer plane was obtained experimentally via optical linear dichroism. The polarized XAFS data have been analyzed both qualitatively and by a global mapping approach limited to systematically altering the various iron-ligand distances within a model for the local atomic environment of the heme prosthetic group, and comparing the theoretically generated XAFS spectra with those obtained experimentally. A similar analysis of unpolarized XAFS data from a frozen solution of YCC was performed using either the heme environment from the NMR solution or the x-ray crystallographic data for YCC as the model structure. All resulting iron-ligand distances were then used in molecular dynamics (MD) computer simulations of YCC in these three systems to investigate the possible effects of anisotropic ligand motions on the fits of the calculated to the experimental XAFS spectra.     

Formation of oriented polypeptides on Au(111) surface depends on the secondary structure controlled by peptide length.

We synthesized three different lengths of poly(L-lysine) containing an -SH group at the terminal (PLL(n)-SH, n (polymerization degree) = 4, 10, 30) and adsorbed them on an Au(111) surface. To analyze the formation process and the structure of self-assembled monolayers (SAMs), we used atomic force microscopy (AFM) and Fourier transform infrared reflection absorption spectra (FT-IR RAS). At the initial stage of SAM growth, formation of nanosize domains was confirmed by AFM imaging. The alpha-helical PLL(30)-SH exhibited a well-defined SAM structure after adsorption reached equilibrium. The alpha-helical PLL(30)-SH was almost perpendicular to the gold surface and exhibited interesting molecular packing due to the secondary structure of PLL(30)-SH and the underlying Au(111) array. The tilt angle of the helix axis from the substrate normal was estimated to be about 50 degrees (AFM) and 44 degrees (FT-IR RAS) respectively. On the other hand, PLL(4)-SH and PLL(10)-SH formed beta-sheet-type SAMs on the Au(111) surface based on the structure determined by FT-IR RAS spectrum.     

Structural investigation of the covalent and electrostatic binding of yeast cytochrome c to the surface of various ultrathin lipid multilayers using x-ray diffraction.

X-Ray diffraction was used to characterize the profile structures of ultrathin lipid multilayers having a bound surface layer of cytochrome c. The lipid multilayers were formed on an alkylated glass surface, using the Langmuir-Blodgett method. The ultrathin lipid multilayers of this study were: five monolayers of arachidic acid, four monolayers of arachidic acid with a surface monolayer of dimyristoyl phosphatidylserine, and four monolayers of arachidic acid acid with a surface monolayer of thioethyl stearate. Both the phosphatidylserine and the thioethyl stearate surfaces were found previously to covalently bind yeast cytochrome c, while the arachidic acid surface electrostatically binds yeast cytochrome c. Meridional x-ray diffraction data were collected from these lipid multilayer films with and without a bound yeast cytochrome c surface layer. A box refinement technique, previously shown to be effective in deriving the profile structures of ultrathin multilayer lipid films with and without electrostatically bound cytochrome c, was used to determine the multilayer electron density profiles. The surface monolayer of bound cytochrome c was readily apparent upon comparison of the multilayer electron density profiles for the various pairs of ultrathin multilayer films plus/minus cytochrome c for all cases. In addition, cytochrome c binding to the multilayer surface significantly perturbs the underlying lipid monolayers.     

Molecular packing of high-density and low-density lipoprotein surface lipids and apolipoprotein A-I binding

The surface pressure (pi)-molecular area (A) isotherms for monolayers of human high-density lipoprotein (HDL3) and low-density lipoprotein (LDL) phospholipids and of mixed monolayers of these phospholipids with cholesterol spread at the air-water interface were used to deduce the likely molecular packing at the surfaces of HDL3 and LDL particles. LDL phospholipids form more condensed monolayers than HDL3 phospholipids; for example, the molecular areas of LDL and HDL3 phospholipids at pi = 10 dyn/cm are 88 and 75 A2/molecule, respectively. The closer packing in the LDL phospholipids monolayer can be attributed to the higher contents of saturated phosphatidylcholines and sphingomyelin relative to HDL3. Cholesterol condenses both HDL3 and LDL phospholipid monolayers but has a greater condensing effect on the LDL phospholipid monolayer. The pi-A isotherms for mixed monolayer of HDL3 phospholipid/cholesterol and LDL phospholipid/cholesterol at stoichiometries similar to those at the surfaces of lipoprotein particles suggest that the monolayer at the surface of the LDL particle is significantly more condensed than that at the surface of the HDL3 particle. The closer lateral packing in LDL is due to at least three factors: (1) the difference in phospholipid composition; (2) the higher unesterified cholesterol content in LDL; and (3) a stronger interaction between cholesterol and LDL phospholipids relative to HDL3 phospholipids. The influence of lipid molecular packing on the affinity of human apolipoprotein A-I (apo A-I) for HDL3 and LDL surface lipids was evaluated by monitoring the adsorption of 14C-methylated apo A-I to monolayers of these lipids spread at various initial surface pressures (pi i).(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular dynamics simulations of a protein on hydrophobic and hydrophilic surfaces.

Molecular dynamics simulations have been used to investigate the behavior of the peripheral membrane protein, cytochrome c, covalently tethered to hydrophobic (methyl-terminated) and hydrophilic (thiol-terminated) self-assembled monolayers (SAMs). The simulations predict that the protein will undergo minor structural changes when it is tethered to either surface, and the structures differ qualitatively on the two surfaces: the protein is less spherical on the hydrophilic SAM where the polar surface residues reach out to interact with the SAM surface. The protein is completely excluded from the hydrophobic SAM but partially dissolves in the hydrophilic SAM. Consequently, the surface of the thiol-terminated SAM is considerably less ordered than that of the methyl-terminated SAM, although a comparable, high degree of order is maintained in the bulk of both SAMs: the chains exhibit collective tilts in the nearest-neighbor direction at angles of 20 degrees and 17 degrees with respect to the surface normal in the hydrophobic and the hydrophilic SAMs, respectively. On the hydrophobic SAM the protein is oriented so that the heme plane is more nearly parallel to the surface, whereas on the hydrophilic surface it is more nearly perpendicular. The secondary structure of the protein, dominated by alpha helices, is not significantly affected, but the structure of the loops as well as the helix packing is slightly modified by the surfaces.     

Enzyme-catalyzed oxidation of cholesterol in mixed phospholipid monolayers reveals the stoichiometry at which free cholesterol clusters disappear.

In this study, we have used cholesterol oxidase as a probe to study cholesterol/phospholipid interactions in mixed monolayers at the air/water interface. Mixed monolayers, containing a single phospholipid class and cholesterol at differing cholesterol/phospholipid molar ratios, were exposed to cholesterol oxidase at a lateral surface pressure of 20 mN/m (at 22 degrees C). At equimolar ratios of cholesterol to phospholipid, the average rate of cholesterol oxidation was fastest in unsaturated phosphatidylcholine mixed monolayers (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and egg yolk phosphatidylcholine), intermediate in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and slowest in sphingomyelin monolayers (egg yolk or bovine brain sphingomyelin). The average oxidation rate in mixed monolayers was not exclusively a function of monolayer packing density, since egg yolk and bovine brain sphingomyelin mixed monolayers occupied similar mean molecular areas even though the measured average oxidation rate was different with these two phospholipids. This suggests that the phospholipid acyl chain composition influenced the oxidation rate. The importance of the phospholipid acyl chain length on influencing the average oxidation rate was further examined in defined phosphatidylcholine mixed monolayers. The average oxidation rate decreased linearly with increasing acyl chain lengths (from di-8:0 to di-18:0). When the average oxidation rate was examined as a function of the cholesterol to phospholipid (C/PL) molar ratio in the monolayer, the otherwise linear function displayed a clear break at a 1:1 stoichiometry with phosphatidylcholine mixed monolayers, and at a 2:1 C/PL stoichiometry with sphingomyelin mixed monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Liquid crystalline/gel state phase separation in docosahexaenoic acid-containing bilayers and monolayers

The phase behavior of lipid mixtures containing 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0, 22:6 PC) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied with bilayers using differential scanning calorimetry (DSC), and with monolayers monitoring pressure/area isotherms and surface elasticity, and lipid domain formation followed by epifluorescence microscopy. From DSC studies it is concluded that DPPC/18:0, 22:6 PC phase separates into DPPC-rich and 18:0, 22:6 PC-rich phases. In monolayers, phase separation is indicated by changes in pressure-area isotherms implying phase separation where 18:0, 22:6 PC is 'squeezed out' of the remaining DPPC monolayer. Phase separation into lipid domains in the mixed PC monolayer is quantified by epifluorescence microscopy using the fluorescently labeled phospholipid membrane probe, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl). These results further describe the ability of docosahexaenoic acid to participate in lipid phase separations in membranes.     

The location of cytochrome c on the surface of ultrathin lipid multilayer films using x-ray diffraction.

X-ray diffraction and spectroscopic techniques were used to characterize ultrathin fatty acid multilayers having a bound surface layer of cytochrome c. Three to six monolayers of arachidic acid were deposited onto an alkylated glass surface, using the Langmuir-Blodgett method. These fatty acid multilayer films were stored either in a 1 mM NaHCO3 pH 7.5 solution or a buffered 10 microM cytochrome c solution, pH 7.5. After washing extensively with buffer, these multilayer films were assayed for bound cytochrome c by optical spectroscopy. It was found that the cytochrome c bound only to the odd-numbered monolayer films (which have hydrophilic surfaces). The theoretical number of cytochrome c molecules bound to the ultrathin multilayer films having three or five monolayers was calculated as N = 1.2 x 10(13)/cm2 (assuming a hexagonally close-packed monolayer of protein), which would produce an optical density of 0.002 at a wavelength of 550 nm; for a three or five monolayer ultrathin film that was incubated with cytochrome c, OD550 approximately equal to 0.002. The protein was released from the film when treated with greater than 100 mM KCl solution, as would be expected for an electrostatic interaction. Meridional x-ray diffraction data were collected from the arachidic acid films with and without a bound cytochrome c layer. A box refinement technique, previously shown to be effective in deriving the profile structures of nonperiodic ultrathin films, was used to determine the multilayer electron density profiles. The electron density profiles and their autocorrelation functions showed that bound cytochrome c resulted in an additional electron dense feature on the multilayer surface, consistent with a bound cytochrome c monolayer. The position of the bound protein relative to the multilayer surface was independent of the number of fatty acid monolayers in the multilayer. Future studies will use these methods to investigate the structures of membrane protein complexes bound directly to the surface of multilayer films.     

Epifluorescence microscopic studies of monolayers containing mixtures of dioleoyl- and dipalmitoylphosphatidylcholines.

Monolayers of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and some mixtures of these lipids were investigated using an epifluorescence microscopic surface balance. Monolayers were visualized at 23 +/- 1 degree C through the fluorescence of 1 mol% of two different fluorescent probes, 1-palmitoyl-2-(12-[(7-nitro-2-1,3-benzoxadizole-4- yl)amino]dodecanoyl)phosphatidylcholine (NBD-PC), which partitions into the liquid expanded (LE) or disordered lipid phase and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO-C18), which preferentially associates with the liquid condensed (LC) phase or lipid with ordered chains. LC domains were observed in pure DPPC monolayers at relatively low surface pressures (pi), and these domains grew with increasing surface pressure. Only liquid expanded phase was observed in pure DOPC monolayers up to the point of monolayer collapse. In monolayers containing 29:70:1, 49:50:1, and 69:30:1 (mol/mol/mol) of DPPC:DOPC:probe the domains of LC phase were smaller than those seen in DPPC monolayers at equivalent surface pressures. Quantitative analysis of the visual fields shown by the mixed monolayers showed a distribution of sizes of condensed domains at any given pi. At pi = 30 mN m-1, liquid-expanded, or fluid, regions occupied more than 70% of the total monolayer area in all three mixtures studied, whereas DPPC monolayers were more than 75% condensed or solid at that pressure. For monolayers of DPPC:DOPC:NBD-PC 49:50:1 and 69:30:1 the average domain size and the percentage of the total area covered with LC, or rigid, areas increased to a maximum at pi around 35 mN m-1 followed by a decrease at higher pi.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA microdevice for electrochemical detection of Escherichia coli 0157:H7 molecular markers

An electrochemical DNA sensor based on the hybridization recognition of a single-stranded DNA (ssDNA) probe immobilized onto a gold electrode to its complementary ssDNA is presented. The DNA probe is bound on gold surface electrode by using self-assembled monolayer (SAM) technology. An optimized mixed SAM with a blocking molecule preventing the nonspecific adsorption on the electrode surface has been prepared. In this paper, a DNA biosensor is designed by means of the immobilization of a single stranded DNA probe on an electrochemical transducer surface to recognize specifically Escherichia coli (E. coli) 0157:H7 complementary target DNA sequence via cyclic voltammetry experiments. The 21 mer DNA probe including a C6 alkanethiol group at the 5' phosphate end has been synthesized to form the SAM onto the gold surface through the gold sulfur bond. The goal of this paper has been to design, characterise and optimise an electrochemical DNA sensor. In order to investigate the oligonucleotide probe immobilization and the hybridization detection, experiments with different concentration of DNA and mismatch sequences have been performed. This microdevice has demonstrated the suitability of oligonucleotide Self-assembled monolayers (SAMs) on gold as immobilization method. The DNA probes deposited on gold surface have been functional and able to detect changes in bases sequence in a 21-mer oligonucleotide.     

Visualization of lateral phases in cholesterol and phosphatidylcholine monolayers at the air/water interface — a comparative study with two different reporter molecules

This study has compared two chemically distinct NBD-lipids with regard to their partitioning properties into lateral phases of pure and mixed cholesterol/phosphatidylcholine monolayers. Pure NBD-cholesterol (22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol), which has the NBD-function in the sterol side chain (at carbon 22), gave a liquid-expanded force-area isotherm on water at 22°C (having a compressibility of 0.005 to 0.007 m/mN), although epifluorescence microscopy of the compressed NBD-cholesterol monolayer revealed that it had a solid-like surface texture. When the compressed NBD-cholesterol monolayer was allowed to expand, it fragmented into large flakes (tens to hundreds of μm in width) which eventually dissolved into a liquid state. The force-area isotherm of pure NBD-phosphatidylcholine (1-hexadecanoyl-2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecyl-sn-glycero-3-phosphocholine) was also liquid-expanded. When a compressed (30 mN/m) monolayer of NBD-phosphatidylcholine was examined by microscopy, it displayed many bright crystalline spots (about 50 μm across) which appeared to form when the monolayer was allowed to stabilize at this lateral surface pressure. These bright spots disappeared when the monolayer was expanded. When the surface texture of a pure cholesterol monolayer was examined, both probes (at 1 mol%) partitioned very similarly in the sterol monolayer. At low lateral surface pressures (1 and 5 mN/m) the probes appeared to be excluded from the cholesterol phase, forming very bright liquid-like areas against a uniformly black cholesterol phase. At 30 mN/m, NBD-phosphatidylcholine appeared to distribute increasingly into the cholesterol phase, whereas NBD-cholesterol still did not to mix with cholesterol. The characteristic surface texture of the liquid-expanded to liquid-condensed lateral phase transition of pure dipalmitoyl phosphatidylcholine (DPPC) monolayers could be visualized identically with both probes, indicating that these were similarly excluded from the liquid-condensed solid phase of DPPC. Finally, in mixed monolayers containing cholesterol and DPPC (molar ratio 33:67), both probes (at 1 mol%) revealed a similar surface texture of the monolayers (examined at a lateral surface pressure of 0.5 mN/m), suggesting that these partitioned similarly between the different lateral phases present in the mixed monolayer. In conclusion, although the two NBD-probes differed from each other in chemical and physical properties, both acted like ‘impurities’ when admixed into pure or mixed monolayers, and appeared to be equally excluded from lateral phases in which the packing density was high.     

Molecular dynamics simulations of a hydrated protein vectorially oriented on polar and nonpolar soft surfaces

We present a collection of molecular dynamics computer simulation studies on a model protein-membrane system, namely a cytochrome c monolayer attached to an organic self-assembled monolayer (SAM). Modifications of the system are explored, including the polarity of the SAM endgroups, the amount of water present for hydration, and the coordination number of the heme iron atom. Various structural parameters are measured, e.g., the protein radius of gyration and eccentricity, the deviation of the protein backbone from the x-ray crystal structure, the orientation of the protein relative to the SAM surface, and the profile structures of the SAM, protein, and water. The polar SAM appears to interact more strongly with the protein than does the nonpolar SAM. Increased hydration of the system tends to reduce the effects of other parameters. The choice of iron coordination model has a significant effect on the protein structure and the heme orientation. The overall protein structure is largely conserved, except at each end of the sequence and in one loop region. The SAM structure is only perturbed in the region of its direct contact with the protein. Our calculations are in reasonably good agreement with experimental measurements (polarized optical absorption/emission spectroscopy, x-ray interferometry, and neutron interferometry).     

Lipid oxidation and signalling in programmed cell death

The pulmonary surfactant lines as a complex monolayer of lipids and proteins the alveolar epithelial surface. The monolayer dynamically adapts the surface tension of this interface to the varying surface areas during inhalation and exhalation. Its presence in the alveoli is thus a prerequisite for a proper lung function. The lipid moiety represents about 90% of the surfactant and contains mainly dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG). The surfactant proteins involved in the surface tension adaption are called SP-A, SP-B and SP-C. The aim of the present investigation is to analyse the properties of monolayer films made from pure SP-C and from mixtures of DPPC, DPPG and SP-C in order to mimic the surfactant monolayer with minimal compositional requirement. Pressure-area diagrams were taken. Ellipsometric measurements at the air-water interface of a Langmuir film balance allowed measurement of the changes in monolayer thickness upon compression. Isotherms of pure SP-C monolayers exhibit a plateau between 22 and 25 mN/m. A further plateau is reached at higher compression. Structures of the monolayer formed during compression are reversible during expansion. Together with ellipsometric data which show a stepwise increase in film thickness (coverage) during compression, we conclude that pure SP-C films rearrange reversibly into multilayers of homogenous thickness.

Lipid monolayers collapse locally and irreversibly if films are compressed to approximately 0–4 nm²/molecule. In contrast, mixed DPPG/SP-C monolayers with less than 5 mol% protein collapse in a controlled and reversible way. The pressure-area diagrams exhibit a plateau at 20 mN/m, indicating partial demixing of SP-C and DPPG. The thickness isotherm obtained by ellipsometry indicates a transformation into multilayer structures. In DPPC/DPPG/SP-C mixtures again a reversible collapse was observed but without a drastic increase in surface layer thickness which may be due to the formation of protrusion under the surface. Thus lipid monolayers containing small amounts of SP-C may mimic the lung surfactant.     

Surface activity of the extracts of the lungs and other organs]

Monomolecular surface films on the border-line of the "liquid-air" phases were observed to be formed in the water-saline extracts of the lungs and of the other organs. The surface films were compressed and extended on Wilhelmy's balance; the isotherm surface tension (ST)-square typical of the solutions of the surface-active materials (SAM) were recorded. Analogous curves with the minimum ST below 12 din/cm were recorded in testing the SAM monolayers placed on the liquid lining of the volatile solvent. Pulmonary surfactants differed from the SAM of other organs by the highest capacity to lower the ST in compression of the monolayer.