Characterization of Langmuir-Blodgett films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphat idylcholine by FTIR-ATR

Monomolecular films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatidylc holine (PPDPC) were transferred from an air/water interface onto a germanium attenuated total reflection crystal by the Langmuir-Blodgett (LB) technique. The assemblies were thereafter investigated by Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy. To determine the molecular organization in the deposited layers we monitored the CH2 and C = O stretching and the CH2 bending regions of the infrared spectra of these lipids in detail. Using Fourier self-deconvolution technique, the carbonyl stretching mode was resolved into two models corresponding to the conformational differences in the ester linkages of the phospholipid sn-1 and sn-2 acyl chains. By varying the temperature of the subphase and using different surface pressures, we were able to transfer different conformational states of DPPC onto a germanium ATR crystal. Deposition of DPPC at 40 mN m-1 and at 15 degrees C or at 20 mN m-1 and at 35 degrees C results in LB-assemblies in ordered or disordered states, respectively, as judged by the IR spectra. These structures in LB films correspond to the state of DPPC in liposomes below and above the temperature of the order-disorder phase transition. Irrespective of the surface pressure and subphase temperature used during the deposition, an ordering process was found in DPPC films when the number of the transferred layers was increased from one to five. The pyrene-labelled phosphatidylcholine analogue, PPDPC, behaved differently from DPPC. In the case where one to three layers of PPDPC transferred at 35 mN m-1 and at 20 degrees C only conformational structures resembling those in fully hydrated liposomes above the main transition temperature were observed.     

SpA蛋白与磷脂单分子膜相互作用的研究

利用石英表面沉积ＬＢ膜的紫外吸收特性与ＣＤ谱特性研究了磷脂单分子膜与ＳｐＡ的相互作用,实验结果表明,单分子膜的疏密状态,蛋白质与膜表面的电荷特性及亚相Ｃａ＾２＋离子浓度均对该蛋白质与磷脂的相互作用有显著的影响,蛋白质在磷膜中的镶嵌与吸附作用为生物膜的重组提供了新的途径。     

Self-assembly effect during the adsorption of polynucleotides on stearic acid langmuir-blodgett monolayer

Interaction of polyadenylic acid, poly(A), with stearic acid Langmuir-Blodgett (LB) monolayer was studied in different electrolyte surroundings. For this purpose LB films of stearic acid, transferred on the mica substrate from poly(A) containing subphase, were analyzed with atomic force microscopy (AFM). The density of polynucleotides surface coverage is ruled by the monovalent electrolyte concentration in the subphase that is in good agreement with previous results. Divalent cations in the subphase are needed to stabilize poly(A) molecules on the surface through formation of "salt bridges". At the very low divalent electrolyte concentration polynucleotides adsorb on the LB film to domains in which the effect of self-assembly is observed. Increase of divalent electrolyte concentration leads to the loss of this orientation effect. The explanation of this effect is proposed.     

The surface properties of apolipoproteins A-I and A-II at the lipid/water interface

The monolayer system was employed to investigate the relative affinities of apolipoproteins A-I and A-II for the lipid/water interface. The adsorption of reductively 14C-methylated apolipoproteins to phospholipid monolayers spread at the air/water interface was determined by monitoring the surface pressure of the mixed monolayer and the surface concentration of the apoprotein. ApoA-II has a higher affinity than apoA-I for lipid monolayers; for a given initial surface pressure, apoA-II adsorbs more than apoA-I to monolayers of egg phosphatidylcholine (PC), distearoyl-PC and human high-density lipoprotein (HDL3) surface lipids. Comparison of the molecular packing of apolipoproteins A-I and A-II suggests that apoA-II adopts a more condensed conformation at the lipid/water interface compared to apoA-I. The ability of apoA-II to displace apoA-I from egg PC and HDL3 surface lipid monolayers was studied by following the adsorption and desorption of the reductively 14C-methylated apolipoproteins. At saturating subphase concentrations of the apoproteins (3.10(-5) g/100 ml), two molecules of apoA-II absorbed for each molecule of apoA-I displaced. This displacement was accompanied by an increase in surface pressure. An identical stoichiometry for the displacement of apoA-I from HDL particles by apoA-II has been reported by others. At low subphase concentrations of apoproteins (5.10(-6) g/100 ml), the apoA-I/lipid monolayer was not fully compressed and could accommodate the adsorbing apoA-II molecules without displacement of apoA-I molecules. ApoA-I molecules were unable to displace apoA-II from the lipid/water interface. The average residue hydrophobicity of apoA-II is higher than that of apoA-I; this may contribute to the higher affinity of apoA-II for lipids compared to apoA-I. The probable helical regions in apolipoproteins A-I and A-II were located using a secondary structure prediction algorithm. The analysis suggests that the amphiphilic properties of the alpha-helical regions of apoA-I and apoA-II are probably not significantly different. Further understanding of the differences in surface activity of these apolipoproteins will require more knowledge of their secondary and tertiary structures.     

Interfacial behaviour of bovine testis hyaluronidase

The interfacial properties of bovine testicular hyaluronidase were investigated by demonstrating the association of hyaluronidase activity with membranes prepared from bovine testis. Protein adsorption to the air/water interface was investigated using surface pressure-area isotherms. In whichever way the interfacial films were obtained (protein injection or deposition), the hyaluronidase exhibited a significant affinity for the air/water interface. The isotherm obtained 180 min after protein injection into a pH 5.3 subphase was similar to the isotherm obtained after spreading the same amount of protein onto the same subphase, indicating that bovine testicular hyaluronidase molecules adopted a similar arrangement and/or conformation at the interface. Increasing the subphase pH from 5.3 to 8 resulted in changes of the protein isotherms. These modifications, which could correspond to the small pH-induced conformational changes observed by Fourier-transform IR spectroscopy, were discussed in relation to the pH influence on the hyaluronidase activity. Adding hyaluronic acid, the enzyme substrate, to the subphase tested the stability of the interfacial properties of hyaluronidase. The presence of hyaluronic acid in the subphase did not modify the protein adsorption and allowed substrate binding to a preformed film of hyaluronidase at pH 5.3, the optimal pH for the enzyme activity. Such effects of hyaluronic acid were not observed when the subphase was constituted of pure water, a medium where the enzyme activity was negligible. These influences of hyaluronic acid were discussed in relation to the modelled structure of bovine testis hyaluronidase where a hydrophobic region was proposed to be opposite of the catalytic site.     

The interaction of cytochrome c with monolayers of phosphatidylethanolamine

1. The interaction between [(14)C]carboxymethylated cytochrome c and monolayers of egg phosphatidylethanolamine at the air/water interface has been investigated by measurements of surface radioactivity, pressure and potential. 2. On adding (14)C-labelled cytochrome c to the subphase under monolayers with a surface pressure below 24dynes/cm. there was an initial surface pressure increment as the protein penetrated, followed by an adsorption that could be detected only by a continued increase in the surface radioactivity. 3. Above film pressures of 24dynes/cm. only adsorption was observed, i.e. an increment in surface radioactivity with none in surface pressure. 4. The changes in surface parameters with penetration of cytochrome c added to the subphase were indirectly proportional to the initial pressure of the monolayer. With hydrogenated phosphatidylethanolamine the constant of proportionality was increased but penetration again ceased at 24dynes/cm. 5. On compressing a phosphatidylethanolamine film containing penetrated cytochrome c to 40dynes/cm. only a proportion of the protein was ejected on a subphase of 10mm-sodium chloride, whereas on a subphase of m-sodium chloride nearly all the protein was lost. 6. With both penetration and adsorption only a small proportion of the added cytochrome c interacted with the phospholipid films, and initially the amount bound was proportional to the added protein concentration. There was no evidence of a stoicheiometric relationship between the protein and phospholipid or the build-up of multilayers. The bonded protein was not released by removing cytochrome c from the subphase. 7. The addition of m-sodium chloride to the subphase delays the rate of protein penetration into low-pressure films, but the final surface-pressure increment is not appreciably decreased. In contrast, m-sodium chloride almost completely stops adsorption on to films at all pressures. 8. When sodium chloride is added to the subphase below cytochrome c adsorbed to monolayers at high pressures, so that the final concentration is 1m, only a proportion of the protein is desorbed and this decreases as the time of the interaction increases. This indicates that adsorption is initially electrostatic, followed by the formation of non-ionic bonds. 9. Alteration of the subphase pH under a high-pressure film leads to a steady increase in adsorption from pH3 to 8.5 followed by a rapid fall to zero adsorption at pH11. 10. The penetration into phospholipid monolayers at 10dynes/cm. shows a rate that is consistent with the relative electrostatic status of the two components of the interaction as the subphase pH is varied between 3 and 10.5. The final equilibrium penetration shows a pronounced peak in the increments of surface pressure at pH9.0 although a similar peak is not observed in the surface radioactivity. This indicates that more residues of the protein are penetrating into the film at about this pH. 11. Determinations were made of the electrophoretic mobilities of phosphatidylethanolamine particles both alone and after interaction with cytochrome c. 12. The electrophoretic mobilities of cytochrome c adsorbed on lipid particles showed an isoelectric point below that of cytochrome c. This and the observations on the monolayers suggest that, with cytochrome c, protein-protein interactions are weak compared with other proteins.     

Interaction of C-phycocyanin with lipid monolayers under nitrogen and in the presence of air

The surface interaction of C-phycocyanin with lipids was studied using the monolayer technique. The surface activity of the protein was found to be higher at the lipid-water interface than at the nitrogen-water interface, particularly at high surface pressures of the lipid monolayer. The maximum initial surface pressures beyond which phycocyanin could not penetrate the dipalmitoylphosphatidylcholine and monogalactosyldiglycerol monolayers were 27 and 30 mN m-1, respectively. Below these values the protein demonstrated preferential interaction with the monogalactosyldiglycerol monolayer. The surface properties of the unfolded protein at pH 2.5 at the lipid-water interface were compared with those of the protein at pH 7.0. Higher affinity of the three-dimensional structure of the protein to lipid monolayers was observed, in particular by high subphase protein concentration. When the lipid films were subjected to oxidation stress by exposure to air, the surface properties of C-phycocyanin and dipalmitoylphosphatidylcholine were not greatly affected but the surface activity of monogalactosyldiacylglycerol was reduced dramatically by autoxidation. The oxidation of monogalactosyldiacylglycerol could not be prevented by the introduction of C-phycocyanin molecules at the lipid-water interface.     

Mixed monolayers of amphiphile-modified nucleic bases and diynoic acids. I. Phase states at air-water interface and in Langmuir-Blodgett films

Monolayers of amphiphile-modified nucleic bases with diynoic acid were obtained and characterized. The synthesized nucleic bases contained in the monolayer complementarily bind the nucleotide molecules contained in the aqueous subphase, and the structure of the resulting monolayers can be fixed by the photopolymerization of diynoic acid. The resulting monolayer exemplifies a novel type of model systems for investigating molecular recognition at the surface of biological membranes. Procedures for the transfer of the monolayers onto solid substrates and photopolymerization of the diynoic acid in mixtures with the derivatives of nucleic bases were developed. The films obtained were structurally characterized using atomic force microscopy. Compression isotherms of the mixed monolayers as well as individual components of monolayers at the air-water interface allowed one to determine the concentration range at which the diynoic acid form true mixtures or domain structures with the derivatives of nucleic base. A study of the films transferred to the solid substrate by atomic force microscopy indicated that this concentration dependence of miscibility behavior was conserved in the transferred films.     

Stability of unimolecular films of 32P-labelled lecithin

1. The stability of monolayers of a highly unsaturated yeast lecithin labelled with (32)P has been investigated by a surface radioactivity technique. 2. Lecithin films on distilled water at all surface pressures between 6 and 48dynes/cm. were completely stable on rapid perfusion of the subphase and on addition of ionic amphipathic substances to the film. 3. Ultrasonically treated lecithin added to the subphase caused a slow loss of surface radioactivity but little pressure change. 4. The addition of proteins to the subphase caused negligible changes in the film even when conditions were favourable for electrostatic heterocoagulation and penetration. 5. Lecithin films were not hydrolysed by a strongly acid subphase at room temperature. The very low rate of hydrolysis produced by alkali was proportional to the subphase OH(-)ion concentration: the apparent activation energy and temperature coefficient (Q(10)) of the reaction were 14250 cal. and 2.37 respectively. 6. Alkaline hydrolysis of lecithin monolayers was markedly stimulated by adding methanol (10-20%, v/v) to the subphase. The addition of ionic amphipaths to the monolayer had the expected type of effect on the hydrolysis rate, but its magnitude was far less than that suggested by an application of the Poisson-Boltzmann equation for ion distribution at a charged interface (Davies & Rideal, 1963).     

UV-induced reaction kinetics of dilinoleoylphosphatidylethanolamine monolayers

The UV-induced reactivity of dilinoleoylphosphatidylethanolamine (DLiPE) Langmuir and Langmuir-Blodgett films has been studied by in situ measurements of the changes in the mean molecular area, UV-vis and Fourier transform infrared spectroscopy, and atomic force microscopy (AFM). Optimum orientation and packing density of the DLiPE molecules in the monolayer were achieved by adding uranyl acetate to the subphase. A first-order reaction kinetic model was successfully fitted to the experimental reaction kinetics data obtained at a surface pressure of 30 mN/m. Topographical studies of LB films by AFM were performed on bilayer structures as a function of subphase composition and UV irradiation time. The orientational effect of the uranyl ions on the monolayer molecules was observed as an enhanced homogeneity of the freshly prepared monomeric LB films. However, the long-term stability of these films proved to be bad; clear reorganization and loss of a true monolayer structure were evidenced by the AFM images. This instability was inhibited for the UV-irradiated films, indicating that the UV irradiation gave rise to a cross-linked structure.     

The role of surfactant proteins in DPPC enrichment of surface films

A pressure-driven captive bubble surfactometer was used to determine the role of surfactant proteins in refinement of the surface film. The advantage of this apparatus is that surface films can be spread at the interface of an air bubble with a different lipid/protein composition than the subphase vesicles. Using different combinations of subphase vesicles and spread surface films a clear correlation between dipalmitoylphosphatidylcholine (DPPC) content and minimum surface tension was observed. Spread phospholipid films containing 50% DPPC over a subphase containing 50% DPPC vesicles did not form stable surface films with a low minimum surface tension. Addition of surfactant protein B (SP-B) to the surface film led to a progressive decrease in minimum surface tension toward 1 mN/m upon cycling, indicating an enrichment in DPPC. Surfactant protein C (SP-C) had no such detectable refining effect on the film. Surfactant protein A (SP-A) had a positive effect on refinement when it was present in the subphase. However, this effect was only observed when SP-A was combined with SP-B and incubated with subphase vesicles before addition to the air bubble containing sample chamber. Comparison of spread films with adsorbed films indicated that refinement induced by SP-B occurs by selective removal of non-DPPC lipids upon cycling. SP-A, combined with SP-B, induces a selective adsorption of DPPC from subphase vesicles into the surface film. This is achieved by formation of large lipid structures which might resemble tubular myelin.     

A comparison of the surface activities of human apolipoproteins A-I and A-II at the air/water interface

Surface pressure (pi) and adsorption isotherms for human apolipoproteins A-I and A-II at the air/water interface have been determined and used to deduce the probable molecular structures of the monomolecular films. The surface concentrations were measured using the surface radioactivity method to monitor the adsorption of reductively [14C]methylated apoproteins. Apolipoprotein A-I and apolipoprotein A-II are extremely surface-active proteins and adsorb to exert maximal pi values of 22 and 24 mN.m-1 respectively, at a steady-state subphase concentration of about 3.10(-5) g/100 ml (equivalent to 11 and 17 nM for apolipoprotein A-I and apolipoprotein A-II, respectively). At saturation monolayer coverage, the average molecular areas for apolipoprotein A-I and apolipoprotein A-II are 15 and 13 A2/residue, respectively. These packing densities are consistent with monolayers consisting largely of alpha-helical protein molecules lying with the long axes of the helical segments in the plane of the interface. Comparison of the molecular packings of spread and adsorbed monolayers of these proteins indicates that at low pi values, the adsorbed films are more expanded, but at high pi values, the molecular packing in both types of film is the same.     

Interactions of cytochrome c and [14C]-carboxymethylated cytochrome c with monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin

1. The interactions between cytochrome c (native and [(14)C]carboxymethylated) and monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin at the air/water interface was investigated by measurements of surface radioactivity, pressure and potential. 2. On a subphase of 10mm-or m-sodium chloride, penetration of cytochrome c into egg phosphatidylcholine monolayers, as measured by an increase of surface pressure, and the number of molecules penetrating, as judged by surface radioactivity, were inversely proportional to the initial pressure of the monolayer and became zero at 20dynes/cm. The constant of proportionality was increased when the cytochrome c was carboxymethylated or decreased when the phospholipid was hydrogenated, but the cut-off point remained at 20dynes/cm. 3. Penetrated cytochrome c could be removed almost entirely by compression of the phosphatidylcholine monolayer above 20dynes/cm. 4. With phosphatidic acid and cardiolipin monolayers on 10mm-sodium chloride the binding of cytochrome c was much stronger and cytochrome c penetrated into films nearing the collapse pressure (>40dynes/cm.). The penetration was partly electrostatically facilitated, since it was decreased by carrying out the reaction on a subphase of m-sodium chloride, and the relationship between the surface pressure increment and the initial film pressure moved nearer to that observed with phosphatidylcholine. 5. Surface radioactivity determinations showed that [(14)C]carboxymethylated cytochrome c was still adsorbed on phosphatidic acid and cardiolipin monolayers after the cessation of penetration. This adsorption was primarily electrostatic in nature because it could be prevented and substantially reversed by adding m-sodium chloride to the subphase and there was no similar adsorption on phosphatidylcholine films. 6. The penetration into and adsorption on the three phospholipid monolayers was examined as a function of the pH of the subphase and compared with the state of ionization of both the phospholipid and the protein, and the area occupied by the latter at an air/water interface. 7. It is concluded that the binding of cytochrome c to phospholipids can only be partially understood by a consideration of the ionic interaction between the components and that subtle conformational changes in the protein must affect the magnitude and stability of the complex. 8. If cytochrome c is associated with a phospholipid in mitochondria then cardiolipin would fulfil the characteristics of the binding most adequately.     

Study of P-glycoprotein effect on the lipid monolayer properties by the Langmuir-Blodgett technique

Expression of the P-glycoprotein (Pgp) is proved to be one of the main reasons for the development of the multidrug resistance (MDR) phenotype by cancer cells. The effect of Pgp on the properties of lipid monolayers was studied using membrane fractions of sensitive and Pgp over-expressing multidrug resistance cancer cells containing 11, 24 or 32% of Pgp relative to the total content of membrane proteins. The effect of the Pgp membrane concentration on the properties of monolayers prepared from the membrane fractions was analyzed by the Langmuir-Blodgett method. The subphase composition was found to play a critical role in the stability of monolayers at any Pgp concentration. The optimal subphase comprised 10 mM tris-HCl buffer, pH 6.5, which made it possible to create very stable monolayer films with the pressure of collapse of about 30-40 mN/m. Monolayers prepared from membrane fractions of sensitive cells and cells containing the maximum (32%) amount of Pgp were found to be much more stable compared with fractions comprising 11 or 24% of Pgp. The analysis of monolayer compression dynamics revealed three distinct stages: (1) the self-organization of lipid molecules, which is characterized by an abrupt change of surface potential; (2) the compression of Pgp molecules at the constant potential of monolayers; and (3) the compression of lipid molecules, which is characterized by a quasilinear increase of both pressure and surface potential. It was shown that the specific surface areas of monolayers formed from sensitive and Pgp-enriched membranes containing 11 or 24% of Pgp are very similar, whereas the surface area of the monolayer formed from membranes containing 32% of Pgp is nearly 1.5-fold greater. This fact may reflect the effect of the threshold rearrangement of the structure of lipid molecules or cooperative modifications of lipid-Pgp interactions induced by the increase in the Pgp content from 24 to 32%. The effect of verapamil, a well-known Pgp modulator, on the properties of monolayers was studied. It was show that verapamil is able to induce changes of the surface of Pgp-containing monolayers, and these modifications are maximal at the Pgp:verapamil 1:1 molar ratio. The data present the first experimental evidence for the possible intervention of Pgp modulator into the processes of lipid-lipid or lipid-Pgp cooperative interactions within Pgp-enriched membranes.     

Increase of catalytic activity of lipase towards olive oil by Langmuir-film immobilization of lipase

Proteins represent versatile building blocks for realization of nanostructured materials to be applied in nanobiotechnology. In the present work, the Langmuir–Blodgett technique was utilized to develop nanobiodevices based on protein molecules. Particularly, lipase thin films were fabricated and characterized, with characterization performed in order to optimize the working parameters. As the first step the protein films were studied at the air–water interface and then transferred onto a solid support for further characterization. The films were characterized by different techniques such as UV–Vis spectroscopy, nanogravimetry, atomic force microscopy, and biochemical assays. Catalytic activity of lipase characterized by the maximal reaction rate found to increase over 10 times as a result of inclusion into LB films, while the substrate binding characterized by the Michaelis constant remain unchanged. Catalytic activity per mole of enzyme was found to increase with the increased number of LB layers up to five, and then decrease at 10, while the surface coverage ranged from 70% to 100% from 1 to 10 layers of lipase. This study exploits the possibility to employ LB based protein structures to use in biocatalysis, exemplified by lipase, which is known as an interfacially-activated enzyme, with olive oil as substrate, when lipase should already be in the maximally active state even without a film. We show, however, that it was possible to form even more active lipase nanostructures by the Langmuir–Blodgett technique at the air–water interface, proving that Langmuir-film provides a better catalytic effect in lipase than a mere oil–water boundary.     

Interactions of phospholipid monolayers with carbohydrates

Surface pressure studies of phospholipid monomolecular films of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) formed at an air/water interface have been made and the effects on the films studied when various carbohydrates are present in the subphase. The results obtained show that at a given temperature, the area per molecule of DPPC increases with increasing concentration of the carbohydrate in the subphase. The carbohydrate which has the greatest expanding effect on the phospholipid monolayer is glycerol, followed in turn by trehalose, sucrose, glucose, raffinose, and inositol. The mechanism of monolayer expansion by glycerol is different from that observed in other carbohydrates, as the following experiments demonstrate. Below the phase transition temperature of DPPC, the area per molecule of DPPC at a pressure of 12.5 dyn/cm is the same with and without glycerol in the subphase. However, when the monolayer is heated to a temperature above the phase transition temperature for DPPC, the area/molecule on glycerol is considerably greater than the area/molecule on water at the same surface pressure. Cooling the monolayer back to the lower temperature produces an area/molecule of DPPC which is identical on both water and glycerol subphases. Glycerol therefore has no effect on the low-temperature (condensed) monolayers but causes expansion of the high-temperature (expanded) monolayers. By contrast with glycerol, both trehalose and sucrose interact with the DPPC monolayer producing an increased area/molecule over that observed on water, both with low-temperature (condensed) monolayers and with the high-temperature (expanded) monolayers. The efficiency of these carbohydrates at expanding the monolayer films (with the exception of glycerol) shows a strong correlation with their ability to stabilize membrane structure and function at low water contents.     

Structure of SP-B/DPPC mixed films studied by neutron reflectometry

The structures of films of pulmonary surfactant protein B (SP-B) and mixtures of SP-B and dipalmitoylphosphatidylcholine (DPPC) at the air/water interface have been studied by neutron reflectometry and Langmuir film balance methods. From the film balance studies, we observe that the isotherms of pure DPPC and SP-B/DPPC mixtures very nearly overlay one another at very high pressures, suggesting that the SP-B is being excluded from the film. The use of multiple contrasts with neutron reflectometry at a range of surface pressures has enabled the mixing and squeeze out of the DPPC and SP-B mixtures to be studied. We can identify the SP-B component of the interfacial structure and its position as a function of surface pressure. The mixtures are initially a homogeneous layer at low surface pressures. At higher surface pressures, the SP-B is squeezed out of the lipid layer into the subphase, with the first signs detected at 30 mN m⁻¹. At 50 mN m⁻¹, the subphase is almost completely excluded from the DPPC layer, with the SP-B content significantly reduced. Only a small amount of DPPC appears to be associated with the squeezed out SP-B.     

Monomolecular layer formation of ferritin molecules on an amphiphilic cyclodextrin derivative at the air/water interface

A monomolecular layer of ferritin molecules was formed by adsorption from the subphase onto a Langmuir film of an amphiphilic beta-cyclodextrin (beta-CD) derivative at the air/water interface. The course of the adsorption of ferritin molecules was monitored by measuring the surface pressure and the resulting film was observed by transmission electron microscopy (TEM). These results show the potential of the amphiphilic CD derivative to work as a milder template for protein molecules at the air/water interface.     

Formation,structure, and spectrophotometry of air-water interface films containing rhodopsin

Summary Air-water interface films of cattle rhodopsin and defined lipids are formed without the use of organic solvents by a method in which vesicle membranes consisting of egg phosphatidyl choline and purified rhodopsin are osmotically shocked at the interface. Lipid and protein molecules organize as insoluble films at the interface. The structure of these films varies with the lipid to protein mole ratio of the source vesicle membranes. Electron microscopic observations reveal that films formed with membranes of 1501 mole ratio consist of nonoverlapping, randomly distributed vesicle membrane fragments separated by a lipid monolayer. These membrane fragments exist as single sheets on the water surface and occupy approximately 35% of this surface. Essentially all the rhodopsin molecules at the interface are spectroscopically intact and are contained within the membrane fragments. The visible absorption spectrum of the interface films is identical to that of suspensions of rod disc membranes. Moreover, flash illumination of rhodopsin in air-dried multilayers formed from the interface films results in the formation of a stable MetarhodopsinI intermediate (_max480 nm) which can be fully bleached by increasing the relative humidity of the multilayers or can be photoconverted into rhodopsin and, presumably, isorhodopsin. Furthermore, rhodopsin is chemically regenerable at the air-water interface. Bleached rhodopsin can generate dark rhodopsin at the interface in the presence of 11-cis retinal in the aqueous subphase. Thus, the spectroscopic structure and the chemical regenerability function of rhodopsin in these interface films are indistinguishable from those exhibited by the protein in intact rod disc membranes.     

Partially induced transition from horizontal to vertical orientation of helical peptides at the air–water interface and the structure of their monolayers transferred on the solid substrates

To apply the Langmuir–Blodgett (LB) technique as a platform for investigating the fundamental properties of amphiphilic peptides (APs), we have investigated the structure of LB films using the APs. To vertically orient the helical APs like transmembrane proteins in the membrane, the primary structure of the APs was designed to have two domains: a hydrophilic domain (three amino acids) and a hydrophobic domain (ca. 20 amino acids). However, we are still far from having full control of their orientation. This study reports the contribution of the subphase temperature to the change in the orientation of helical APs. When the surface pressure–area isotherm of AP was observed at the subphase temperature at 41.5 °C, the isotherm exhibited a plateau, implying that a phase transition of the monolayer at the air–water interface occurred. Circular dichroism (CD) spectra of the monolayers transferred on the solid substrates revealed that the orientation of the helices changed at the pressure, where the plateau of the isotherm was observed. This change was not observed at 21.5 °C, i.e., the horizontal alignment of helixes was maintained. Atomic force microscopy (AFM) was used to systematically investigate the surface structure of the monolayers transferred at different surface pressures. A structural model of the monolayer that did not contradict with the results obtained by the three different techniques (the isotherm, CD spectroscopy, and AFM) was derived, and it was concluded that the horizontally oriented helices partially changed their orientation to vertical upon compression in the plateau region of the isotherm.