Interaction of fluorescently labeled pardaxin and its analogues with lipid bilayers.

Fluorescence measurements were used to monitor the interaction of the neurotoxin pardaxin and its analogues with membranes. Eight peptides were selectively labeled with the fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl, either at their N-terminal or at their C-terminal. No detectable changes in membrane permeability or hemolytic activity were observed upon modification. Upon the titration of solutions containing the different peptides with small unilamellar vesicles, the fluorescent emission spectra of 7-nitrobenz-2-oxa-1,3-diazole-4-yl-labeled pardaxin and its analogues, but not those of control peptides, displayed blue shifts in addition to enhanced intensities upon relocation of the probe to a more apolar environment. The results revealed that the N terminus of pardaxin is buried within the lipid bilayer while the C terminus is located at the bilayer's surface. Binding isotherms were obtained from the observed increases in the fluorescence emission yields, from which surface partition constants, in the range of 10(4) M-1, were in turn derived. The existence of an aggregation process was suggested by the shape of the binding isotherms. Furthermore, the results show good correlation between the incidence of aggregation and the ability of the different analogues to induce the release of relatively large molecules from vesicles. As such, our results suggest that the mechanism of pore formation employed by pardaxin and its analogues could be described by the "barrel stave" model.     

The antimicrobial peptide trichogin and its interaction with phospholipid membranes.

The interaction of the antimicrobial peptide trichogin GA IV with phospholipid bilayers has been studied. A series of analogs of trichogin was synthesized in which the nitroxide spin label, 4-amino-4-carboxy-2,2,6,6-tetramethylpiperidino-1-oxyl (TOAC), replaced one of the three alpha-aminoisobutyric acid (Aib) residues in the sequence. These modified peptides were used to assess the location of different residues of the peptide in a phospholipid bilayer composed of egg phosphatidylcholine containing 0.4 mol% of a fluorescently labelled phospholipid. We demonstrate that the substitution of Aib residues with TOAC does not alter the manner in which the peptide affects membrane curvature or induces vesicle leakage. The proximity of the nitroxide group on the peptide to the 4,4-difluoro-4-bora-3a,4a-diaza-S-indacene (BODIPY) fluorophore attached to the phospholipid was estimated from the extent of quenching of the fluorescence. By this criterion it was concluded that the peptide penetrates into the bilayer and that Aib4 is the most deeply inserted of the Aib residues. The results suggest that the helix axis of the peptide is oriented along the plane of the membrane. All of the peptides were shown to raise the bilayer to the hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine, indicating that they promote positive membrane curvature. This is a property observed with peptides that do not penetrate deeply into the bilayer or are oriented along the bilayer normal. We also demonstrate trichogin-promoted leakage of the aqueous contents of liposomes. These results indicate that the peptides cause bilayer destabilization. The extent of leakage induced by trichogin is very sensitive to the peptide to lipid ratio over a narrow range.     

Synthesis and antimicrobial activity of dermaseptin S1 analogues

Dermaseptins are peptides found in the skin secretions of Phyllomedusinae frogs. These peptides exert lytic action on some microorganisms without substantial haemolysis. In an attempt to understand the antimicrobial activity of these peptides we designed several dermaseptin S1 (ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ) (DS1) analogues. All peptides were tested on the growth of prokaryotic (Gram-positive and Gram-negative bacteria) and eukaryotic (the yeast Candida albicans and the protozoon Leishmania major) organisms. Our data showed a dose-dependent killing effect by most DS1 derivatives. Maximal antibacterial activity was displayed by a 16-mer peptide that was more active than native DS1.     

Interaction of fluorescently labeled myosin subfragment 1 with nucleotides and actin

Isolated myosin heads (subfragment 1) were modified by covalent attachment of 5-(iodoacetamido)fluorescein or 5-(iodoacetamido)salicylic acid to the essential sulfhydryl group SH1. The extrinsic fluorescence of the modified proteins was sensitive to binding of nucleotides and F-actin. With the fluorescein derivative [subfragment 1 (S1) modified with 5-(iodoacetamido)fluorescein (IAF) at SH1 (S1-AF)], association with MgADP decreased the probe fluorescence by 30%, whereas binding to actin increased the emission by a factor of 2. In the ternary complex acto-S1-AF X MgADP, the effect of nucleotide on the intensity of the attached fluorescein canceled the effect of actin. The fluorescence state of this ternary complex was similar to that of S1-AF X MgADP. The emission of S1-AF was resolved into two components with lifetimes of 4.3 and 0.6 ns and relative contributions of 33% and 67%, respectively. Interaction of S1-AF with nucleotides and actin did not alter the lifetimes but significantly shifted their fractional contributions. Quenching studies showed that the short lifetime likely arose from the fluorescein moiety statically quenched by internal groups. Binding of MgADP to the salicylate derivative [S1 modified with 5-(iodoacetamido)salicylic acid at SH1 (S1-SAL)] induced a 25% enhancement of the probe fluorescence, whereas formation of acto-S1-SAL decreased the emission by 10% regardless of whether MgADP was bound to the protein. Both labeled S1 species bound MgADP with a similar affinity, comparable to that of unmodified S1 previously reported by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of RNA with phospholipid membranes

RNAs binding with liposomes under near physiological conditions were obtained by molecular selection. Structural analysis showed that the RNAs could form complexes owing to complementary sequences located in loops. Oligomerization of the RNAs selected was experimentally confirmed. The results and published data testified that formation of high-molecular-weight complexes is a major mechanism increasing the RNA affinity for phospholipid membranes. The role of RNA-membrane interactions in early evolution is discussed in terms of the RNA world hypothesis.     

Interaction of chlorpromazine with phospholipid membranes

The interaction of chlorpromazine (CPZ) with artificial membranes (egg-yolk phosphatidylcholine liposomes) has been studied. Measurements of the surface electric potential, which is modified in the presence of the ionized form of the drug, were obtained by electron paramagnetic resonance spectroscopy (EPR) using a positively charged amphiphilic spin-probe. This probe partitions between the aqueous and lipidic phases depending on the surface potential and on the structural state of the membrane. The surface potential was measured as a function of drug concentration in the range where the spectral line-shapes are not affected by the incorporation of the drug. From these experimental results and through an appropriate formalism we obtain information on the binding of the drug to the lipid bilayer and on the ionization of the drug in the lipidic phase. Correspondence to: C. Anteneodo     

Interaction of synthetic glycophospholipids with phospholipid bilayer membranes.

A series of glycophospholipids synthesized by coupling mono-, di-, or tri-saccharides to dioleoylphosphatidylethanolamine (DOPE) by reductive amination was used to investigate the interaction of glycophospholipids with phospholipid bilayer membranes. These synthetic glycophospholipids functioned as a stabilizer for the formation of DOPE bilayer vesicles. The minimal mol% of glycophospholipid needed to stabilize the DOPE vesicles were as follows: 8% N-neuraminlactosyl-DOPE (NANL-DOPE), 20% N-maltotriosyl-DOPE (MAT-DOPE), 30% N-lactosyl-DOPE (Lac-DOPE), and 42% N-galactosyl-DOPE (Gal-DOPE). The estimated hydration number of glycophospholipid in reverse micelles was 87, 73, 46, and 14 for NANL-DOPE, MAT-DOPE, Lac-DOPE, and Gal-DOPE, respectively. Thus, the hydration intensity of the glycophospholipid was directly related to the ability to stabilize the DOPE bilayer phase for vesicle formation. Glycophospholipids also reduced the transition temperature from gel to liquid-crystalline phase (Tm) of dipalmitoylphosphatidylcholine (DPPC) bilayers. Interestingly, incorporation of NANL-DOPE induced a decrease of membrane fluidity of DPPC bilayers in the gel phase while other glycophospholipids had no effect. Also, low level of NANL-DOPE but not other glycophospholipids increased the transition temperature (TH) from liquid-crystalline to hexagonal phase of dielaidoylphosphatidylethanolamine bilayers. These results showed that NANL-DOPE with a highly hydratable headgroup which provides a strong stabilization activity for the L alpha phase of phospholipid membranes, may also be involved in specific interactions with neighboring phospholipids via its saccharide moiety.     

Interaction of the P-type cardiotoxin with phospholipid membranes.

The cardiotoxin (cytotoxin II, or CTII) isolated from cobra snake (Naja oxiana) venom is a 60-residue basic membrane-active protein featuring three-finger beta sheet fold. To assess possible modes of CTII/membrane interaction 31P- and 1H-NMR spectroscopy was used to study binding of the toxin and its effect onto multilamellar vesicles (MLV) composed of either zwitterionic or anionic phospholipid, dipalmitoylglycerophosphocholine (Pam2Gro-PCho) or dipalmitoylglycerophosphoglycerol (Pam2Gro-PGro), respectively. The analysis of 1H-NMR linewidths of the toxin and 31P-NMR spectral lineshapes of the phospholipid as a function of temperature, lipid-to-protein ratios, and pH values showed that at least three distinct modes of CTII interaction with membranes exist: (a) nonpenetrating mode; in the gel state of the negatively charged MLV the toxin is bound to the surface electrostatically; the binding to Pam2Gro-PCho membranes was not observed; (b) penetrating mode; hydrophobic interactions develop due to penetration of the toxin into Pam2Gro-PGro membranes in the liquid-crystalline state; it is presumed that in this mode CTII is located at the membrane/water interface deepening the side-chains of hydrophobic residues at the tips of the loops 1-3 down to the boundary between the glycerol and acyl regions of the bilayer; (c) the penetrating mode gives way to isotropic phase, stoichiometrically well-defined CTII/phospholipid complexes at CTII/lipid ratio exceeding a threshold value which was found to depend at physiological pH values upon ionization of the imidazole ring of His31. Biological implications of the observed modes of the toxin-membrane interactions are discussed.     

Interaction of gramicidin S and its aromatic amino-acid analog with phospholipid membranes

To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological membranes, a series of five decameric cyclic cationic β-sheet-β-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar ≡ Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic—exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (K_b). The K_b1 and K_b2 values for endothermic two-step binding processes corresponded to high and low binding affinities (K_b1 ≥ 100 K_b2). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis.     

Interaction of dopamine with artificial phospholipid membranes

The interaction of dopamine (DA) with phospholipid membranes has been investigated. The membrane current in planar bilipid membrane (BLM) modified by amphotericin B in voltage clamp conditions under alternating polarity was shown to symmetrically increase 1.2 times when DA was added outside the BLM. This implies a uniform change of charge on each membrane surface and hence the diffusion of DA within the BLM and its exposure on the internal side. The appearance of single threads and bundles of filaments within the internal liposomal cavities was observed in the ultrastructure of suspended thin-walled liposomes filled with globular actin after the introduction of DA into external solution. This reshaped liposomes into rod-like, spindle-shaped or angular structures. Actin serves as a marker for DA due to its property to polymerize itself under the influence of DA. Thus, the structural reorganization of liposomes manifests the presence of DA inside them and the induction of actin polymerization.     

Combinations of fluorescently labeled pulmonary surfactant proteins SP-B and SP-C in phospholipid films

Hydrophobic pulmonary surfactant (PS) proteins B (SP-B) and C (SP-C) modulate the surface properties of PS lipids. Epifluorescence microscopy was performed on solvent-spread monolayers of fluorescently labeled porcine SP-B (R-SP-B, labeled with Texas Red) and SP-C (F-SP-C, labeled with fluorescein) in dipalmitoylphosphatidylcholine (DPPC) (at protein concentrations of 10 and 20 wt%, and 10 wt% of both) under conditions of cyclic compression and expansion. Matrix-assisted laser desorption/ionization (MALDI) spectroscopy of R-SP-B and F-SP-C indicated that the proteins were intact and labeled with the appropriate fluorescent probe. The monolayers were compressed and expanded for four cycles at an initial rate of 0.64 A2 x mol(-1) x s(-1) (333 mm2 x s x [-1]) up to a surface pressure pi approximately 65 mN/m, and pi-area per residue (pi-A) isotherms at 22 +/- 1 degrees C were obtained. The monolayers were microscopically observed for the fluorescence emission of the individual proteins present in the film lipid matrix, and their visual features were video recorded for image analysis. The pi-A isotherms of the DPPC/protein monolayers showed characteristic "squeeze out" effects at pi approximately 43 mN/m for R-SP-B and 55 mN/m for F-SP-C, as had previously been observed for monolayers of the native proteins in DPPC. Both proteins associated with the expanded (fluid) phase of DPPC monolayers remained in or associated with the monolayers at high pi (approximately 65 mN/m) and redispersed in the monolayer upon its reexpansion. At comparable pi and area/molecule of the lipid, the proteins reduced the amounts of condensed (gel-like) phase of DPPC monolayers, with F-SP-C having a greater effect on a weight basis than did R-SP-B. In any one of the lipid/protein monolayers the amounts of the DPPC in condensed phase were the same at equivalent pi during compression and expansion and from cycle to cycle. This indicated that only minor loss of components from these systems occurred between compression-expansion cycles. This study indicates that hydrophobic PS proteins associate with the fluid phase of DPPC in films, some proteins remain at high surface pressures in the films, and such lipid-protein films can still attain high pi during compression.     

Interaction of bundled Ser-rich amphiphilic peptides with phospholipid membranes.

To investigate properties of hydrophilic bundled peptides and their interactions with phospholipid membranes, bundled peptides named [Trp2]- and [Trp12]-4alpha-46S9, which are composed of four fragments of amphiphilic 24-mer peptide, were designed and synthesized. Tryptophan (Trp) was introduced at the 2nd position from the N-terminal or at the centre (12th) of the helix to monitor the peptide-lipid interaction. Circular dichroism measurements indicated that the peptides had low alpha-helicities in a buffer solution (pH 7.4) and also in the presence of dipalmitoyl-DL-3-phosphatidylcholine (DPPC) vesicles. In the presence of DPPC/dipalmitoyl-DL-3-phosphatidylglycerol (DPPG) (3:1) vesicles, the measurement could not be taken because of turbidity induced by vesicle aggregation. Both peptides had moderate perturbation activity for both the neutral and acidic vesicles at 25 degrees C. The perturbation patterns at 50 degrees C were much different from those at 25 degrees C and the maximum activity reached 100% at a low peptide concentration. The results of the measurement of membrane fusion activity of peptides showed a similar tendency to that found in the perturbation experiment. A quenching experiment indicated that the Trp2 and Trp12 residues in [Trp2]- and [Trp12]-4alpha-46S9 were scarcely embedded in neutral lipid membranes.     

Interaction of antimycobacterial and anti-pneumocystis drugs with phospholipid membranes

Liposomes can be used as carriers of drugs in the treatment of viral, bacterial and protozoal infections. The potential for liposome-mediated therapy of Mycobacterium avium-intracellulare complex infections, one of the most common opportunistic infections in AIDS, is currently under study. Here, we have investigated the effect of the lipid-soluble antimycobacterial drugs ansamycin, clofazimine and CGP7040 on the thermotropic behavior of liposomes composed of dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) using differential scanning calorimetry (DSC). In the presence of ansamycin (rifabutine), the peak gel-liquid crystalline phase transition temperature (Tm) of DPPG was reduced, as was the sub-transition temperature (Ts), whereas the Tm of DPPC was reduced only slightly. The temperature of the pre-transition (Tp) of DPPC was lowered, while the pre-transition of DPPG was abolished. Ansamycin also caused the broadening of the transition endotherm of both lipids. Equilibration of the drug/lipid complex for 1 or 5 days produced different thermotropic behavior. In the presence of clofazimine, the cooperativity of the phase transition of DPPG decreased. Above 10 mol% clofazimine formed two complexes with DPPG, as indicated by two distinguishable peaks in DSC thermograms. The Tm of both peaks were lowered as the mole fraction increased. Clofazimine had minimal interaction with DPPC. In contrast, CGP7040 interacted more effectively with DPPC than with DPPG, causing a reduction of the size of the cooperative unit of DPPC even at 2 mol%. The main transition of DPPC split into 3 peaks at 5 mol% drug. The pre-transition was abolished at all drug concentrations and the sub-transition disappeared at 10 mol% CGP7040. These studies suggest that maximal encapsulation of clofazimine in liposomes would require a highly negatively charged membrane, while that of CGP7040 would necessitate a zwitterionic membrane. We have also investigated the interaction of the water-soluble antibiotic pentamidine, which has been used against Pneumocystis carinii, the most lethal of AIDS-related opportunistic pathogens. Aerosol administration of this drug leads to long-term sequestration of the drug in the lungs. The DPPG/pentamidine complex exhibited a pre-transition at 3.5 degrees C, an endothermic peak at 42 degrees C, and an exothermic peak at 44.5 degrees C, followed by another endothermic peak at 55 degrees C. The exotherm depended on the history of the sample, requiring pre-incubation for several minutes below the 42 degrees C transition. These observations suggest that upon melting of the DPPG chains at 42 degrees C, the DPPG crystallizes as a DPPG/pentamidine complex that melts at 55 degrees C.     

Interaction of cytochromec with phospholipid monolayers and bilayer membranes

Summary For the study of the interaction between oxidized cytochromec and phosphatidylinositide, two different model systems were used: (1) monolayers which were deposited after the method of Langmuir and Blodgett onto glass plates, and (2) bimolecular (“black”) membranes in aqueous phase. The amount of bound protein was determined with a sensitive spectrophotometer. It was found that at low ionic strength about 10¹³ cytochromec molecules per cm² are bound to the lipid surface, which nearly corresponds to a densely packed monolayer. At high ionic strength (∼ 0.1m) or low pH (pH<3), the adsorbed protein layer becomes unstable. This result indicates that the interaction is mainly electrostatic. In accordance with this conclusion is the observation that the rate of adsorption is diffusion controlled; i.e., almost every protein molecule hitting the surface is bound. The cytochromec monolayer can be reduced by ascorbate. In contrast to ferrocytochromec in solution, the bound ferrocytochrome was found to be autoxidable.     

Interaction of Zn2+ with phospholipid membranes

To characterize the specificity of zinc binding to phospholipid membranes in terms of headgroup structure, hydration and phase behavior we studied the zwitterionic lipid 1-palmitoyl-2-oleoyl-phosphatidylcholine as a function of hydration at 30 degreesC in the presence and absence of ZnCl2. Zinc forms a 2:1-1:1 complex with the lipid, and in particular with the negatively charged phosphate groups. Zn2(+)-bridges between neighboring lipid molecules stabilize the gel phase of the lipid relative to the liquid-crystalline state. Upon Zn2+ binding the C-O-P-O-C- backbone of the lipid headgroup changes from a gauche/gauche into the trans/trans conformation and it loses roughly 50% of the hydration shell. The ability of the Zn2(+)-bound phosphate groups to take up water is distinctly reduced, meaning that the headgroups have become less hydrophilic. The energetic cost (on the scale of Gibbs free energy) for completely dehydrating the lipid headgroups is decreased by approximately 10 kJ/mole in the presence of Zn2+. The interaction of phospholipid headgroups with Zn2+ is conveniently described by a hydrated zinc-phosphate complex the key energy contribution of which is more covalent than electrostatic in nature. Dehydration of phospholipid headgroups due to complexation with zinc cations is suggested to increase fusogenic potency of lipid membranes. Zinc appears to be one of the most potent divalent cation in inducing membrane fusion.     

Dynamic quenchers in fluorescently labeled membranes. Theory for quenching in a three-phase system.

The theory for quenching of fluorescently labeled membranes by dynamic quenchers is described for a three-phase system: a fluorescently labeled membrane, a nonlabeled membrane, and an aqueous phase. Two different experimental protocols are possible to determine quenching parameters. Using the first protocol, partition coefficients and bimolecular quenching constants were determined for a hydrophobic quencher in carbazole-labeled membranes in the presence of an unlabeled reference membrane. These parameters determined for 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) using this three-phase analysis were in good agreement with values determined by a two-phase analysis without the reference lipid. Hence, the theory was verified. In the second protocol, the quencher partition coefficient was determined for unlabeled membranes in the presence of a carbazole-labeled reference membrane. Partition coefficients for DDE determined by this method were the same as partition coefficients determined for carbazole-labeled membranes using the two-phase analysis. The greater ease in determining partition coefficients and bimolecular quenching constants by the three-phase analysis and, in particular, the ability to determine the partition coefficient in unlabeled membranes make the three-phase analysis especially useful. This method was used to study the effect varying the membrane lipid composition has on the partition coefficient. The data indicate that partition coefficients of DDE in fluid membranes are not dramatically dependent upon polar head group composition, fatty acid composition, or cholesterol content. However, partitioning into gel-phase lipids is at least 100-fold less than fluid-phase lipids.