Partition of antimicrobial D-L-α-cyclic peptides into bacterial model membranes.

Fluorescence spectroscopy is used to characterize the partition of three second-generation D,L-α-cyclic peptides to two lipid model membranes. The peptides have proven antimicrobial activity, particularly against Gram positive bacteria, and the model membranes are formed of either with 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG) or its mixture with 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), at a molar ratio of (1:1). The peptide's intrinsic fluorescence was used in the Steady State and/or Time Resolved Fluorescence Spectroscopy experiments, showing that the peptides bind to the membranes, and the extent of their partition is thereof quantified. The peptide-induced membrane leakage was followed using an encapsulated fluorescent dye. Overall, the partition is mainly driven by electrostatics, but also involves hydrophobic interactions. The introduction of a hydrocarbon tail in one of the residues of the parent peptide, CPR, adjacent to the tryptophan (Trp) residue, significantly improves the partition of the modified peptides, CPRT10 and CPRT14, to both membrane systems. Further, we show that the length of the tail is the main distinguishing factor for the extension of the partition process. The parent peptide induces very limited leakage, at odds with the peptides with tail, that promote fast leakage, increasing in most cases with peptide concentration, and being almost complete for the highest peptide concentration and negatively charged membranes. Overall, the results help the unravelling of the antimicrobial action of these peptides and are well in line with their proven high antimicrobial activity.

Solubilisation of model membrane by DDAO surfactant - partitioning, permeabilisation and liposome-micelle transition.

Solubilisation of model membranes of dioleoylphosphatidylcholine (DOPC) and DOPCcholesterol (CHOL) induced by surfactant N,N-dimethyl-1-dodecanamine-N-oxide (DDAO) was studied. At the maintained pH ~ 7.5, the DDAO molecules are in their neutral state with respect to the pK ~ 5. Pore formation in lipid bilayer was studied by fluorescence probe leakage method. The changes in the size of lipid aggregates upon increasing DDAO concentration were followed turbidimetrically. Effective ratio Re at different steps of the solubilisation process was determined. The molar partition coefficient of DDAO in case of the DOPC membrane is Kp = 2262 ± 379, for DOPC-CHOL membrane Kp = 2092 ± 594. Within the experimental error, the partition coefficient, as well as effective ratios Re, are not considerably influenced when one third of DOPC molecules is substituted with CHOL (DOPC:CHOL = 2:1). Constituents of buffer (50 mmol/dm3 PBS, 150 mmol/dm3 NaCl) caused aggregation of DOPC and DOPC-CHOL unilamellar liposomes at zero and low DDAO concentration, as was shown by SANS, turbidimetry and DIC microscopy. After solubilisation of bilayer structures by surfactant, mixed DOPC-DDAO and DOPC-CHOL-DDAO micelles with the shape of cylinders with elliptical cross section were detected.

The membrane structure and function affected by water.

Various experimental data reveal intriguing peculiarities in structural properties of biomimetic membranes. Interestingly, one of the common alterations that is observed at the membrane-water interface underlines the important role of membrane hydration properties. A plausible mechanism of action in the case of many membrane additives seems to be in shifting the water encroachment the way that bilayers absorb more or less water molecules - one of the smallest and often neglected biomolecule. The difference in water interactions with different lipids and cholesterol has been noted at the interface and up to the bilayer center, the ion depending interplay between lipid-water and ion-water hydrations has been shown, and the anaesthetic effect also appears to link tightly to hydration, to discuss but a few examples. Although a complete understanding of the physicochemical processes taking place in biomembranes is not established fully, the understanding of lipid bilayer structural changes as a result of different properties of environment outside and/or inside the membrane provides a foundation for better insights into the structure-function relationships that most certainly take place in complex biomembrane systems.

Alcohol Interactions with Lipid Bilayers.

We investigate the structural changes to lipid membrane that ensue from the addition of aliphatic alcohols with various alkyl tail lengths. Small angle neutron diffraction from flat lipid bilayers that are hydrated through water vapor has been employed to eliminate possible artefacts of the membrane curvature and the alcohol's membrane-water partitioning. We have observed clear changes to membrane structure in both transversal and lateral directions. Most importantly, our results suggest the alteration of the membrane-water interface. The water encroachment has shifted in the way that alcohol loaded bilayers absorbed more water molecules when compared to the neat lipid bilayers. The experimental results have been corroborated by molecular dynamics simulations to reveal further details. Namely, the order parameter profiles have been fruitful in correlating the mechanical model of structural changes to the effect of anesthesia.

Cholesterol protects phosphatidylcholine liposomes from N,N-dimethyl-1-dodekanamine N-oxide influence.

The interaction of N,N-dimethyl-1-dodekanamine N-oxide (C12(CH3)2NO) with egg yolk phosphatidylcholine (EYPC) liposomes containing cholesterol (CHOL) was studied. The perturbation of CHOL-EYPC bilayers in unilamellar liposomes (ULL) was observed by the leakage of fluorescent probe calcein. Weak leakage is observed at low surfactant concentration cC12NO (minimal perturbation of the bilayer) followed by an intensive leakage at a middle cC12NO (creation of pores). No change in fluorescence intensity was measured at high cC12NO (calcein totally released from liposomes). The higher CHOL amount in the bilayer, the more surfactant is needed to create pores in the bilayer. Solubiliazation of CHOL-EYPC ULL induced by C12NO was studied turbidimetrically. The solubilization curve consists of three parts: saturation of bilayer at low cC12NO (liposomes are preserved), followed by solubilization (liposome - mixed micelle transition) and post-solubilization. The c12NO concentration needed for the onset of the soubilization raises with the increase of nCHOL:nEYPC. The structure of liposomes is still preserved at total calcein release for all nCHOL:nEYPC.

Influence of cholesterol and ß-sitosterol on the structure of EYPC bilayers.

The influence of cholesterol and ß-sitosterol on egg yolk phosphatidylcholine (EYPC) bilayers is compared. Different interactions of these sterols with EYPC bilayers were observed using X-ray diffraction. Cholesterol was miscible with EYPC in the studied concentration range (0-50 mol%), but crystallization of ß-sitosterol in EYPC bilayers was observed at X ≥ 41 mol% as detected by X-ray diffraction. Moreover, the repeat distance (d) of the lamellar phase was similar upon addition of the two sterols up to mole fraction 17%, while for X ≥ 17 mol% it became higher in the presence of ß-sitosterol compared to cholesterol. SANS data on suspensions of unilamellar vesicles showed that both cholesterol and ß-sitosterol similarly increase the EYPC bilayer thickness. Cholesterol in amounts above 33 mol% decreased the interlamellar water layer thickness, probably due to "stiffening" of the bilayer. This effect was not manifested by ß-sitosterol, in particular due to the lower solubility of ß-sitosterol in EYPC bilayers. Applying the formalism of partial molecular areas, it is shown that the condensing effect of both sterols on the EYPC area at the lipid-water interface is small, if any. The parameters of ESR spectra of spin labels localized in different regions of the EYPC bilayer did not reveal any differences between the effects of cholesterol and ß-sitosterol in the range of full miscibility.

The effects of cholesterol and ß-sitosterol on the structure of saturated diacylphosphatidylcholine bilayers.

The structures of DMPC and DPPC bilayers in unilamellar liposomes, in the presence of 33.3 mol% cholesterol or the plant sterol ß-sitosterol, have been studied by small-angle neutron scattering. The bilayer thickness d(L) increases in a similar way for both sterols. The repeat distance in multilamellar liposomes, as determined by small-angle X-ray diffraction, is larger in the presence of ß-sitosterol than in the presence of cholesterol. We observe that each sterol modifies the interlamellar water layer differently, cholesterol reducing its thickness more efficiently than ß-sitosterol, and conclude that cholesterol suppresses bilayer undulations more effectively than ß-sitosterol.

Areas of monounsaturated diacylphosphatidylcholines.

We have studied the structural properties of monounsaturated diacylphosphatidylcholine lipid bilayers (i.e., diCn:1PC, where n = 14, 16, 18, 20, 22, and 24 is the number of acyl chain carbons). High-resolution x-ray scattering data were analyzed in conjunction with contrast-varied neutron scattering data using a technique we recently developed. Analyses of the data show that the manner by which bilayer thickness increases with increasing n in monounsaturated diacylphosphatidylcholines is dependent on the double bond's position. For commonly available monounsaturated diacylphosphatidylcholines, this results in the nonlinear behavior of both bilayer thickness and lipid area, whereas for diC18:1PC bilayers, lipid area assumes a maximum value. It is worthwhile to note that compared to previous data, our results indicate that lipid areas are smaller by approximately 10%. This observation highlights the need to revisit lipid areas, as they are often used in comparisons with molecular dynamics simulations. Moreover, simulators are encouraged to compare their results not only to x-ray scattering data, but to neutron data as well.

Bilayer thickness in unilamellar extruded 1,2-dimyristoleoyl and 1,2-dierucoyl phosphatidylcholine vesicles: SANS contrast variation study of cholesterol effect.

Small-angle neutron scattering on extruded unilamellar vesicles in water was used to study bilayer thickness when cholesterol (CHOL) was added at 44.4 mol% to 1,2-dimyristoleoylphosphatidylcholine (diC14:1PC) and 1,2-dierucoylphosphatidylcholine (diC22:1PC) bilayers. Using the (1)H(2)O/(2)H(2)O contrast variation and the small-angle form of Kratky-Porod approximation, the bilayer gyration radii at infinite contrast R(g,infinity) and the bilayer thickness parameters d(g,infinity) = 12(0.5)R(g,infinity) were obtained at 30 degrees C. Addition of cholesterol to diC14:1PC increased the d(g,infinity) from 3.72 +/- 0.02 to 4.26 +/- 0.01 nm, while in the diC22:1PC bilayers the d(g,infinity) change observed was within the experimental error: +0.23 +/- 0.23 nm.