A new method for investigation of lipid assemblies with a lipoid pH indicator in monomolecular films (original) (raw)
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Charge determination of membrane molecules in polymer-supported lipid layers
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1995
A method for two-dimensional micro-electrophoresis and charge determination of fluorescence-labeled membrane molecules in lipid layers is presented. Therefore, the labeled molecules are dissolved in a lipid monolayer which acts as a fluid matrix. The essential part of the sample preparation is an aqueous polymer film composed of agarose onto which the layer is transferred by Langmuir-Blodgett technique. The induced force of an applied electric field leads to a drift of the charged fluorescent molecules. The mobility is determined by an analysis of the steady-state bleach profile which is produced by continuous fluorescence micro-photolysis of a rectangular area of the monolayer. Testing a variety of amphiphilic molecules, measurements yielded values of zero, plus or minus one elementary net charge within a margin of error. The experimental setup described here can be used for lateral separation, enrichment and isoelectric focusing of membrane components.
Lipoid pH indicators as probes of electrical potential and polarity in micelles
The Journal of Physical Chemistry, 1977
Two types of fluorescent pH indicators, a hydroxycoumarin and an aminocoumarin dye, are incorporated by means of long paraffinic chain substituents to neutral, anionic, and cationic micelles. The apparent pK values for the dyes in the micelles are compared to the pK values in aqueous solution. Shifts of the apparent pK are found for charged as well as for uncharged micelles, being different for the two indicators. A comparison of the pK shifts of the indicators in neutral micelles to pK shifts in a series of mixed solvents of different dielectric constant (e) leads us to attribute the shifts in the neutral system to a reduced polarity at the micelle surface for which a value of e 32 is estimated. With respect to charged micelles this polarity effect is responsible for part of the apparent pK shift. It may be revealed by comparing the results for the two indicator types. The value of the effective dielectric constant is found to be similar to that in the neutral micelle. The remaining part of the apparent pK shift may be taken as a measure of the electrical potential at the surface of the charged micelles. Some examples of potential measurements for various counterions are presented. A thermodynamical section attempts to present clear definitions of (and relations between) terms as "apparent pK", "interfacial pK", "interfacial pH", and "acidity function".
Electrochimica Acta, 2018
The interactions of cationic lipopeptide C 15 H 31 CO-Trp-Lys-D-Leu-Lys with model lipid bilayers supported on gold surface were investigated using electrochemical methods combined with atomic force microscopy imaging and quartz crystal microbalance measurements. It has been found that the mode of the lipopeptide action strongly depends on the net charge of the lipid membrane. In case of the negatively charged bilayers composed of L-a-phosphatidylethanolamines and L-a-phosphatidylglycerols extracted from E. coli bacteria, C 15 H 31 CO-Trp-Lys-D-Leu-Lys molecules initially aggregate on the top of the membrane as a result of the electrostatic attraction between cationic peptide moiety and anionic polar heads of phosphatidylglycerols. Further fusion of the aggregates leads to the swelling and partial disruption of the membrane, while its permeability is substantially increased. Interestingly, the changes in the molecular organization of negatively charged membrane were much more pronounced in the upper leaflet of the bilayer. The effect of C 15 H 31 CO-Trp-Lys-D-Leu-Lys on zwitterionic bilayer composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and cholesterol was definitely different. In this case, the disturbance of the membrane structure was rather subtle. It has been observed that a small fraction of lipopeptide molecules rapidly inserts into the bilayer and perturbation of the assembly is homogenous throughout the film. This was followed by a noticeable decrease in membrane permeability and stiffening of the lipid bilayer. Such sealing effect indicates that the insertion of lipopeptide did not result either in pore formation or membrane rupture.
International journal of biomedical imaging, 2010
Lipid lateral organization in binary-constituent monolayers consisting of fluorescent and nonfluorescent lipids has been investigated by acquiring multiple emission spectra during measurement of each force-area isotherm. The emission spectra reflect BODIPY-labeled lipid surface concentration and lateral mixing with different nonfluorescent lipid species. Using principal component analysis (PCA) each spectrum could be approximated as the linear combination of only two principal vectors. One point on a plane could be associated with each spectrum, where the coordinates of the point are the coefficients of the linear combination. Points belonging to the same lipid constituents and experimental conditions form a curve on the plane, where each point belongs to a different mole fraction. The location and shape of the curve reflects the lateral organization of the fluorescent lipid mixed with a specific nonfluorescent lipid. The method provides massive data compression that preserves and e...
Langmuir, 1996
Our previous investigations have shown that the pKi of the interfacial pH and polarity probe 4-heptadecyl-7-hydroxycoumarin (HHC) embedded in neutral monolayers is different at the air-water and the solidwater interfaces. This difference questions the universality of the reference pKi,o value defined for each interfacial pH and polarity probe at a chosen neutral interface. The shift pKi,o s/w -pKi,o a/w was interpreted as due to oriented dipole layers located in the solid substrate at about 6 nm from the monolayer-water boundary. In the present study we check this hypothesis by investigating the effect of the ω-dipoles of heptadecanol and 16-bromohexadecanol monolayers at the air-water interface on the acid-base equilibrium of embedded HHC. Emission and excitation spectra of the amphiphilic dye were recorded at different pH values, and pK i values were evaluated from both emission and excitation titration dependences. The negligible difference between the pKi values of HHC in the two matrices implies that remote dipoles have no significant effect. Emission and excitation spectra of the water soluble 4-methyl-7-hydroxycoumarin (MHC) in the same phosphate solutions applied as liquid substrates for the spread monolayers were recorded and used to obtain the value of the bulk pK b. From the difference pKi -pKb the interfacial dielectric constant at the monolayer-water boundary was evaluated. The values i ) 60 ( 5 for heptadecanol and 55 ( 5 for 16-bromohexadecanol monolayers coincide very well with literature data for i for sugar micelles. Because of the important role the saccharide moieties play in biological processes, this coincidence underlines the importance of further investigation of spread and deposited monolayers of long chain alcohols as the simplest models of the more complex di-or polysaccharides.
Polymer-Supported Lipid Bilayers on Benzophenone-Modified Substrates
Biomacromolecules, 2001
Solid-supported lipid membranes are important for their roles in fundamental biophysical research as well as in applications such as biosensors. In our study, lipopolymers containing alkyl side chains were synthesized and a mixture of the lipopolymer and free lipids was preorganized at the air-water interface and then transferred to a solid substrate using the Langmuir-Blodgett technique. A photochemical reaction between a substrate-functionalized benzophenone and C-H bonds on the lipopolymer was used to attach the lipopolymers to the substrate. The final assembly of the membrane was completed by vesicle fusion. Langmuir film experiments at the air-water interface indicate tighter molecular packing for the lipopolymers with 28 mol % alkyl side chains than for the ones with 22 mol %. Atomic force microscopy images point to phase separation of lipopolymers on the substrates due to their dewetting from hydrophobic surfaces. However, a mixture of lipopolymers and free lipids formed a smooth film on the same substrate. After the addition of the second lipid layer on the lipopolymer/free lipid layer, the fluorescence images of the polymer-supported bilayer suggested that the distal lipid layer is homogeneous on the micrometer scale. The relaxation of the fluorescent probe lipids was analyzed after application of an electric field to determine their diffusion coefficient; the distal lipid layer was mobile with an average diffusion coefficient of ∼0.1 µm 2 /s. Moreover, the immobile fraction of the lipids in the distal layer was estimated to be around 15%.
Scientific reports, 2015
Local heterogeneity in lipid self-assembly is important for executing the cellular membrane functions. In this work, we chemically modified 2-(2'-hydroxyphenyl)benzoxazole (HBO) and attached a C8 alkyl chain in two different locations to probe the microscopic environment of four lipidic phases of dodecyl β-maltoside. The fluorescence change in HBO and the new probes (HBO-1 and HBO-2) shows that in all phases (micellar, hexagonal, cubic and lamellar) three HBO tautomeric species (solvated syn-enol, anionic, and closed syn-keto) are stable. The formation of multi tautomers reflects the heterogeneity of the lipidic phases. The results indicate that HBO and HBO-1 reside in a similar location within the head group region, whereas HBO-2 is slightly pushed away from the sugar-dominated area. The stability of the solvated syn-enol tautomer is due to the formation of a hydrogen bond between the OH group of the HBO moiety and an adjacent oxygen atom of a sugar unit. The detected HBO anion...
Modulation of Activity of Ultrashort Lipopeptides toward Negatively Charged Model Lipid Films
Langmuir, 2017
Because of the increasing resistance of pathogens to commonly used antibiotics, there is an urgent need to find alternative antimicrobial compounds with different mechanisms of action. Among them, lipopeptides are recognized as promising candidates. In this work, the Langmuir technique and atomic force microscopy were employed to investigate the interactions of two novel lipopeptides with negatively charged phospholipid membranes, which served as a simplified model of inner membrane of Gram-negative bacteria. Lipid films contained phosphatidylethanolamine and phosphatidylglycerol extracts from E. coli bacteria. Lipopeptides were composed of palmitoyl chain covalently coupled to N-terminus of peptide with Trp-Lys-Leu-Lys amino acid sequence and the conformation of third residue was either D-Leu or L-Leu. It was found that chirality of leucine strongly affects interfacial behavior of these compounds, which was ascribed to the difference in effective size of the peptide portion of the molecules. Although the lipopeptides were the same in terms of amino acid sequence, charge, and identity of lipophilic chain, the experiments revealed that the barrier for their insertion into the lipid membrane is significantly different. Namely, it was lower for lipopeptide containing D-Leu residue. We have also found that insertion of the lipopeptides into the model membranes strongly alters lateral distribution of the membrane components and leads to its substantial fluidization. The dynamics of reorganization was noticeably faster in the presence of lipopeptide with smaller size of peptide moiety, i.e., containing D-Leu. It proves that effective size of the peptide headgroup is an important factor determining lipopeptide activity toward the lipid membranes.