Interaction of delta sleep-inducing peptide and its analogues with cellular membranes: a structure-function analysis

The possibility of a correlation between the membrane properties of the delta sleep-inducing peptide (DSIP) and its analogues and their biological activity in vivo was examined by a comparative study of the membrane effects of these peptides. The peptides exhibiting biological activity in vivo were shown to cause a statistically reliable disordering of lipids in thrombocyte plasma membranes similar to the effect of DSIP. The membrane effect of the D-Val2, D-Tyr2, and Tyr1, Pro2 analogues of DSIP had the same bimodal dose dependence characteristic of natural DSIP. Only a slight nonspecific lipid disordering was registered for Trp-Asp-Ala-Ser-Gly-Glu, a biologically inactive hexapeptide analogue. These results indicate a correlation between the biological activity of the peptides during in vivo tests and their membrane properties in vitro. The structure-function relationship was studied within the group of DSIP analogues examined in vitro. The DSIP modeling effect, especially pronounced under the action of stress factors, was suggested to be directly associated with the ability of DSIP to change the dynamic structure of biological membranes.     

Ion channels of various types induced in lipid membranes by gramicidin a derivatives carrying a cationic sequence at their C-termini

The channel-forming activity of gramicidin A derivatives carrying positively charged amino acid sequences at their C-termini was studied on planar bilayer lipid membranes and liposomes. We showed previously (FEBS Lett., 2005, vol. 579, pp. 5247–5252) that, at low concentrations, these peptides form classical cation-selective pores typical of gramicidin A, whereas, at high concentrations, they form large nonselective pores. The ability of the peptides to form nonselective pores, which was determined by the efflux of carboxyfluorescein, an organic dye, from liposomes, decreased substantially as the length of the gramicidin fragment in the series of cationic analogues was truncated. CD spectra showed that large pores are formed by peptides having both β^6.3 single-stranded and β^5.6 double-stranded helical conformations of the gramicidin fragment, with the C-terminal cationic sequence being extended. The dimerization of the peptides by the oxidation of the terminal cysteine promoted the formation of nonselective pores. It was shown that nonselective pores are not formed in membranes of erythrocytes, which may indicate a dependence of the channel-forming ability on the membrane type. The results may be of interest for the directed synthesis of peptides with antibacterial activity.     

Interaction of delta-sleep-inducing peptide with cell membranes in vitro]

The effect of delta-sleep-inducing peptide (DSIP) on erythrocytic membranes of human donor blood was studied by the spin label and spin probe methods. The spin-labeled derivative of DSIP containing the N-terminal residue of 1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxylic acid was synthesized. An analysis of the ESR spectra of the spin-labeled DSIP derivative recorded after its incubation with a human erythrocyte suspension at 37 degrees C revealed a decrease in the rotational correlation time (tau c) and molecular order parameter (S) in comparison with the control solutions of the peptide in phosphate buffer (pH 7.4). The application of paramagnetic probes, 5-, 12-, and 16-doxylstearic acids and 3-doxylandrostanol, demonstrated that the introduction of DSIP in an erythrocytic suspension significantly increased the mobility of the hydrophobic area of the membrane bilayer both at a depth of 20-22 A and in the subsurface area (4-6 A). The dependence of these effects on the DSIP concentration was shown to have the form of a curve with well-defined extremes. The maximal disordering of membrane lipids was observed at peptide concentrations of 10(-9) and 10(-6) M. These results suggested that DSIP significantly affected the structure of plasmatic membranes in vitro by changing the physical state of their lipid components.     

Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae

In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127-6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1? deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1? mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.     

The membrane insertion of helical antimicrobial peptides from the N-terminus of Helicobacter pylori ribosomal protein L1

The interaction of two helical antimicrobial peptides, HPA3 and HPA3P with planar supported lipid membranes was quantitatively analysed using two complementary optical biosensors. The peptides are analogues of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1). The binding of these two peptide analogues to zwitterionic dimyristoyl-phosphatidylcholine (DMPC) and negatively charged membranes composed of DMPC/dimyristoylphosphatidylglycerol (DMPG) (4:1) was determined using surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). Using SPR analysis, it was shown that the proline substitution in HPA3P resulted in much lower binding for both zwitterionic and anionic membranes than HPA3. Structural changes in the planar DMPC and DMPC/DMPG (4:1) bilayers induced by the binding of both Hp(2-20) analogues were then resolved in real-time with DPI. The overall process of peptide-induced changes in membrane structure was analysed by the real-time changes in bound peptide mass as a function of bilayer birefringence. The insertion of both HPA3 and HPA3P into the supported lipid bilayers resulted in a decrease in birefringence with increasing amounts of bound peptide which reflects a decrease in the order of the bilayer. The binding of HPA3 to each membrane was associated with a higher level of bound peptide and greater membrane lipid disordering and a faster and higher degree of insertion into the membrane than HPA3P. Furthermore, the binding of both HPA3 and HPA3P to negatively charged DMPC/DMPG bilayers also leads to a greater disruption of the lipid ordering. These results demonstrate the geometrical changes in the membrane upon peptide insertion and the extent of membrane structural changes can be obtained quantitatively. Moreover, monitoring the effect of peptides on a structurally characterised bilayer has provided further insight into the role of membrane structure changes in the molecular basis of peptide selectivity and activity and may assist in defining the mode of antimicrobial action.     

Action of the multifunctional peptide BP100 on native biomembranes examined by solid-state NMR

Membrane composition is a key factor that regulates the destructive activity of antimicrobial peptides and the non-leaky permeation of cell penetrating peptides in vivo. Hence, the choice of model membrane is a crucial aspect in NMR studies and should reflect the biological situation as closely as possible. Here, we explore the structure and dynamics of the short multifunctional peptide BP100 using a multinuclear solid-state NMR approach. The membrane alignment and mobility of this 11 amino acid peptide was studied in various synthetic lipid bilayers with different net charge, fluidity, and thickness, as well as in native biomembranes harvested from prokaryotic and eukaryotic cells. ¹⁹F-NMR provided the high sensitivity and lack of natural abundance background that are necessary to observe a labelled peptide even in protoplast membranes from Micrococcus luteus and in erythrocyte ghosts. Six selectively ¹⁹F-labeled BP100 analogues gave remarkably similar spectra in all of the macroscopically oriented membrane systems, which were studied under quasi-native conditions of ambient temperature and full hydration. This similarity suggests that BP100 has the same surface-bound helical structure and high mobility in the different biomembranes and model membranes alike, independent of charge, thickness or cholesterol content of the system. ³¹P-NMR spectra of the phospholipid components did not indicate any bilayer perturbation, so the formation of toroidal wormholes or micellarization can be excluded as a mechanism of its antimicrobial or cell penetrating action. However, ²H-NMR analysis of the acyl chain order parameter profiles showed that BP100 leads to considerable membrane thinning and thereby local destabilization.     

Linear and Cyclic α-Melanotropin [4–10]-Fragment Analogues That Exhibit Superpotency and Residual Activity

Two analogues of α-MSH (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂), Ac-[Nle⁴, Asp⁵, D-Phe⁷, Lys¹⁰]α-MSH_4–10NH₂ and Ac-[Nle⁴, Asp⁵, D-Phe⁷, Lys¹⁰] α-MSH_4–10-NH₂, were synthesized, and the melanotropic activities of the peptides were compared in several bioassays. Potencies were determined in the in vitro frog and lizard skin bioassays and in the S91 melanoma cell tyrosinase assay. Both analogues were equipotent or more potent than α-MSH in all bioassays, and the activities of the analogues were prolonged compared to α-MSH. The two analogues were very resistant to inactivation by purified proteolytic enzymes (α-chymotrypsin, trypsin, and pepsin). The two peptides could be topically applied and transdermally delivered across the skin of mice in vivo, resulting in a shift from pheomelanogenesis to eumelanogenesis within follicular melanocytes. The cyclic analogue exhibited greater potency, prolonged activity, and stability against enzyme inactivation than did the linear peptide. The significance of the findings for the further design of melanotropin analogues is discussed, as in the possible relevance of these melanotropin analogues for use in biomedical studies.     

The effect of binding of spider-derived antimicrobial peptides, oxyopinins, on lipid membranes

Oxyopinins (Oxki1 and Oxki2) are antimicrobial peptides isolated from the crude venom of the wolf spider Oxyopes kitabensis. The effect of oxyopinins on lipid bilayers was investigated using high-sensitivity titration calorimetry and ³¹P solid-state NMR spectroscopy. High-sensitivity titration calorimetry experiments showed that the binding of oxyopinins was exothermic, and the binding enthalpies (ΔH) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) small unilamellar vesicles (SUVs) were − 18.1 kcal/mol and − 15.0 kcal/mol for Oxki1 and Oxki2, respectively, and peptide partition coefficient (K_p) was found to be 3.9 × 10³ M^− 1. ³¹P NMR spectra of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes in the presence of oxyopinins indicated that they induced a positive curvature in lipid bilayers. The induced positive curvature was stronger in the presence of Oxki2 than in the presence of Oxki1. ³¹P NMR spectra of phosphaditylcholine (PC) membranes in the presence of Oxki2 showed that Oxki2 produced micellization of membranes at low peptide concentrations, but unsaturated PC membranes or acidic phospholipids prevented micellization from occurring. Furthermore, ³¹P NMR spectra using membrane lipids from E. coli suggested that Oxki1 was more disruptive to bacterial membranes than Oxki2. These results strongly correlate to the known biological activity of the oxyopinins.     

Action mechanism of tachyplesin I and effects of PEGylation

PEGylation of protein and peptide drugs is frequently used to improve in vivo efficacy. We investigated the action mechanism of tachyplesin I, a membrane-acting cyclic antimicrobial peptide from Tachypleus tridentatus and the effects of PEGylation on the mechanism. The PEGylated peptide induced the leakage of calcein from egg yolk l-α-phosphatidylglycerol/egg yolk l-α-phosphatidylcholine large unilamellar vesicles similarly to the parent peptide. Both peptides induced lipid flip-flop coupled to leakage and was translocated into the inner leaflet of the bilayer, indicating that tachyplesin I forms a toroidal pore and that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. Despite their similar activities against model membranes, the peptides showed very different biological activities. The cytotoxicity of tachyplesin I was greatly reduced by PEGylation, although the antimicrobial activity was significantly weakened. We investigated the enhancement of the permeability of inner membranes induced by the peptides. Our results suggested that outer membranes and peptidoglycan layers play an inhibitory role in the permeation of the PEG moiety. Furthermore, a reduction in DNA binding by PEGylation may also contribute to the weak activity of the PEGylated peptide.     

Phospholipid spin probes measure the effects of ethanol on the molecular order of liver microsomes

Ethanol, in vitro, is known to perturb the molecular order of the phospholipids in biological membranes, while chronic ethanol exposure, in vivo, leads to resistance to disordering. Such changes have usually been measured by electron spin resonance, utilizing fatty acid spin probes. The use of such probes is controversial, since their orientation in the membrane may not accurately represent that of individual phospholipids. We, therefore, compared ethanol-induced structural perturbations in the membranes of rat hepatic microsomes measured with the spin probe 12-doxylstearic acid (SA 12) with those assayed with various phospholipid spin probes. With SA 12, the addition of increasing amounts of ethanol (50-250 mM) in vitro caused a progressive decrease in the membrane molecular order, as measured by electron spin resonance (ESR). By contrast, microsomes obtained from rats chronically fed ethanol were resistant to the disordering effect of ethanol. Microsomes labeled with the phospholipid spin probes 1-palmitoyl-2-(12-doxylstearoyl)phosphatidylcholine, -phosphatidylethanolamine, or -phosphatidic acid also exhibited increased disordering with the addition of increasing amounts of ethanol. However, the effect noted with phospholipid spin probes was less than that observed with the fatty acid probe. Microsomes obtained from the livers of chronically intoxicated animals labeled with the phospholipid probes were also resistant to the disordering effects of ethanol in vitro. These results suggest that fatty acid spin probes are qualitatively valid for measuring membrane perturbations in biological membranes, ethanol affects all microsomal phospholipids, regardless of chemical dissimilarities (e.g., head-group structure), in a qualitatively similar fashion, and the fluidization of fatty acyl chains in microsomal membranes is comparable in different membrane phospholipids.     

Antioxidant and detoxifying activities of analogues of the delta sleep inducing peptide

Sixteen peptides differing in structure from the delta sleep inducing peptide (DSIP) by one to two substitutions of amino acid residues have been synthesized to study the possibility of their application in oncology. The antioxidant properties of the peptides in vitro and their detoxifying activity in vivo have been examined on a model of toxicosis caused by cisplatin, a cytostatic drug widely used in the therapy of tumor diseases. It has been shown that almost all DSIP analogues examined exhibit a direct antioxidant activity, with the activity of the ID-6 analogue being higher than that of DSIP and comparable with that of vitamin C and β-carotene. This analogue shows the most pronounced detoxifying activity toward the action of cisplatin, which manifests itself as a decrease in the death of animals from acute toxicity to 17% compared to 50–67% in the control and the restoration of some biochemical parameters of blood, in particular, a decrease in the activity of the enzymes, aspartate and alanine aminotransferases, and the concentration of the end products of nitrogen exchange: creatinine and urea. Thus, the peptides of the DSIP family may appear promising agents for decreasing the toxic effects of cytostatics used in oncology.     

Coupling Molecular Dynamics Simulations with Experiments for the Rational Design of Indolicidin-Analogous Antimicrobial Peptides

Antimicrobial peptides (AMPs) have attracted much interest in recent years because of their potential use as new-generation antibiotics. Indolicidin (IL) is a 13-residue cationic AMP that is effective against a broad spectrum of bacteria, fungi, and even viruses. Unfortunately, its high hemolytic activity retards its clinical applications. In this study, we adopted molecular dynamics (MD) simulations as an aid toward the rational design of IL analogues exhibiting high antimicrobial activity but low hemolysis. We employed long-timescale, multi-trajectory all-atom MD simulations to investigate the interactions of the peptide IL with model membranes. The lipid bilayer formed by the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was chosen as the model erythrocyte membrane; lipid bilayers formed from a mixture of POPC and the negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol were chosen to model bacterial membranes. MD simulations with a total simulation time of up to 4 μs revealed the mechanisms of the processes of IL adsorption onto and insertion into the membranes. The packing order of these lipid bilayers presumably correlated to the membrane stability upon IL adsorption and insertion. We used the degree of local membrane thinning and the reduction in the order parameter of the acyl chains of the lipids to characterize the membrane stability. The order of the mixed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol/POPC lipid bilayer reduced significantly upon the adsorption of IL. On the other hand, although the order of the pure-POPC lipid bilayer was perturbed slightly during the adsorption stage, the value was reduced more dramatically upon the insertion of IL into the membrane's hydrophobic region. The results imply that enhancing IL adsorption on the microbial membrane may amplify its antimicrobial activity, while the degree of hemolysis may be reduced through inhibition of IL insertion into the hydrophobic region of the erythrocyte membrane. In addition, through simulations, we identified the amino acids that are most responsible for the adsorption onto or insertion into the two model membranes. Positive charges are critical to the peptide's adsorption, whereas the presence of hydrophobic Trp8 and Trp9 leads to its deeper insertion. Combining the hypothetical relationships between the membrane disordering and the antimicrobial and hemolytical activities with the simulated results, we designed three new IL-analogous peptides: IL-K7 (Pro7 → Lys), IL-F89 (Trp8 and Trp9 → Phe), and IL-K7F89 (Pro7 → Lys; Trp8 and Trp9 → Phe). The hemolytic activity of IL-F89 is considerably lower than that of IL, whereas the antimicrobial activity of IL-K7 is greatly enhanced. In particular, the de novo peptide IL-K7F89 exhibits higher antimicrobial activity against Escherichia coli; its hemolytic activity decreased to only 10% of that of IL. Our simulated and experimental results correlated well. This approach—coupling MD simulations with experimental design—is a useful strategy toward the rational design of AMPs for potential therapeutic use.     

Biogenesis of erythrocyte membrane proteins in vitro studies with rabbit reticulocytes

The capability of rabbit reticulocytes to synthesize red cell membrane proteins has been tested in vitro. Reticulocyte-rich blood from phenylhydrazine-treated rabbits was incubated in vitro in a complete amino acid medium containing ferrous salts, glucose, rabbit plasma and [³H]leucine. Red cell ghost membranes were prepared by hypotonic lysis and leucine incorporation into hemoglobin and total membrane proteins determined. The pattern of incorporation into individual peptides was determined by polycrylamide gel electrophoresis of labeled membranes on large (19 mm) gel which were then sliced into 1 mm sections; radioactivity was compared with densitometric tracings of Coomassie blue stained analytical (6 mm) gels. Incorporation of [³H]leucine into both hemoglobin and membrane protein was linear over 1 h. Gel analysis of labeled membranes revealed that the amino acid was primarily incorporated into peptides with molecular weights of 90 000 or less; three peptides of molecular weights 90 000, 60 000 and 33 000 showed the highest specific activity. Synthesis of the four largest peptide species was negligible. Removal of ferrous salts inhibited synthesis of both globin and membrane protein equally (approx. 50%). However, puromycin and cycloheximide preferentially inhibited the synthesis of globin as compared to membrane proteins. Reticulocytes remain capable of synthesizing a number of membrane proteins; these results are consistent with studies of red cell membrane synthesis in anemic rabbits in vivo.     

Synthetic analogues of antimicrobial peptides from the venom of the Central Asian spider <Emphasis Type="Italic">Lachesana tarabaevi</Emphasis>

Analogues of latarcins Ltc1 and Ltc3b, antimicrobial peptides from the venom of the Central Asian spider Lachesana tarabaevi capable of formation of amphiphilic structures in membranes without involvement of disulfide bonds, were synthesized. The amino acid sequences of the analogues correspond to immature forms of these peptides, each of them containing an additional C-terminal amino acid residue. It is concluded from the study of the biological activity of the synthesized peptides that the posttranslational C-terminal amidation of Ltc3b is a functionally important modification that ensures a high activity of the mature peptide. The lipid composition was shown to affect the interaction of synthesized peptides with artificial membranes. The analogue of Ltc3b manifested the highest activity on cholesterol-containing membranes. The mechanism of action of the studied antimicrobial peptides on membranes is discussed.     

Conformational and functional studies of gomesin analogues by CD, EPR and fluorescence spectroscopies

The aim of this work was to examine the bioactivity and the conformational behavior of some gomesin (Gm) analogues in different environments that mimic the biological membrane/water interface. Thus, manual peptide synthesis was performed by the solid-phase method, antimicrobial activity was evaluated by a liquid growth inhibition assay, and conformational studies were performed making use of several spectroscopic techniques: CD, fluorescence and EPR. [TOAC¹]-Gm; [TOAC¹, Ser^2,6,11,15]-Gm; [Trp⁷]-Gm; [Ser^2,6,11,15, Trp⁷]-Gm; [Trp⁹]-Gm; and [Ser^2,6,11,15, Trp⁹]-Gm were synthesized and tested. The results indicated that incorporation of TOAC or Trp caused no significant reduction of antimicrobial activity; the cyclic analogues presented a β-hairpin conformation similar to that of Gm. All analogues interacted with negatively charged SDS both above and below the detergent's critical micellar concentration (cmc). In contrast, while Gm and [TOAC¹]-Gm required higher LPC concentrations to bind to micelles of this zwitterionic detergent, the cyclic Trp derivatives and the linear derivatives did not seem to interact with this membrane-mimetic system. These data corroborate previous results that suggest that electrostatic interactions with the lipid bilayer of microorganisms play an important role in the mechanism of action of gomesin. Moreover, the results show that hydrophobic interactions also contribute to membrane binding of this antimicrobial peptide.     

A 2H Solid-State NMR Study of Lipid Clustering by Cationic Antimicrobial and Cell-Penetrating Peptides in Model Bacterial Membranes

Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use ²H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the β-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. ²H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct ²H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ∼40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ∼80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.     

A H Solid-State NMR Study of Lipid Clustering by Cationic Antimicrobial and Cell-Penetrating Peptides in Model Bacterial Membranes

Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use ²H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the β-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. ²H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct ²H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ∼40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ∼80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.     

Membrane interactions and biological activity of antimicrobial peptides from Australian scorpion

UyCT peptides are antimicrobial peptides isolated from the venom of the Australian scorpion. The activity of the UyCT peptides against Gram positive and Gram negative bacteria and red blood cells was determined. The membrane interactions of these peptides were evaluated by dye release (DR) of the fluorophore calcein from liposomes and isothermal titration calorimetry (ITC); and their secondary structure was determined by circular dichroism (CD). Three different lipid systems were used to mimic red blood cells, Escherichia coli and Staphylococcus aureus membranes. UyCT peptides exhibited broad spectrum antimicrobial activity with low MIC for S. aureus and multi-drug resistant Gram negative strains. Peptide combinations showed some synergy enhancing their potency but not hemolytic activity. The UyCT peptides adopted a helical structure in lipid environments and DR results confirmed that the mechanism of action is by disrupting the membrane. ITC data indicated that UyCT peptides preferred prokaryotic rather than eukaryotic membranes. The overall results suggest that UyCT peptides could be pharmaceutical leads for the treatment of Gram negative multiresistant bacterial infections, especially against Acinetobacter baumanni, and candidates for peptidomimetics to enhance their potency and minimize hemolysis. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Membrane order perturbation in the presence of antimicrobial peptides by H solid-state NMR spectroscopy

The ²H solid-state NMR spectra of deuterated fatty acyl chains provide direct access to the order of the hydrophobic membrane interior. From the deuterium order parameter profiles of perdeuterated fatty acyl chains the membrane hydrophobic thickness can be calculated. Here we show data obtained from POPC, POPE and mixed POPE/POPG bilayers, representative of bacterial membranes, in the presence of cholesterol or ergosterol and antimicrobial peptaibols. Whereas sterols have a strong ordering effect also on these membranes, the peptides exhibit neutral or disordering effects. By comparing with data from the literature it becomes obvious that cationic amphipathic peptides that probably reside within the interface of phospholipid membranes tend to strongly disorder the packing of the fatty acyl chains, an effect that has been correlated to antimicrobial and DNA transfection activities. In contrast transmembrane sequences or hydrophobic peptides that probably partition deeply into the membrane tend to have only modest disordering activities. The ²H solid-state NMR approach has also been used to monitor the lateral separation of domains rich in anionic phospholipids in the presence of cationic peptides and has thereby provided important insights into their mechanisms of action.     

[TRP11]-Neurotensin and xenopsin discriminate between rat and guinea-pig neurotensin receptors

The binding and biological activities of neurotensin and two analogues, [Trp¹¹]-neurotensin and xenopsin, in which a tryptophan replaces the neurotensin residue Tyr¹¹, were compared in rat and guinea-pig. The binding activity of the three peptides was measured as their ability to inhibit the binding of [³H]neurotensin to rat and guinea-pig brain synaptic membranes. Their biological activities were measured as their effects on the contractility of rat and guinea-pig ileal smooth muscle preparations. In binding as well as biological assays, it was found that [Trp¹¹]-neurotensin and xenopsin were as potent as neurotensin in the rat. In contrast, the two analogues were about 10 times less potent than neurotensin in the guinea-pig. These findings reveal differences between rat and guinea-pig neurotensin receptors. Such species-related differences in neurotensin receptors should be considered when comparing the activity of neurotensin analogues in assays using tissue preparations from various animal species.