In situ characterization of lipid A interaction with antimicrobial peptides using surface X-ray scattering (original) (raw)
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European Biophysics Journal, 2002
We present a first study using synchrotron grazing incidence diffraction and X-ray reflectivity measurements on mixed phospholipid/peptide monolayers at the air/water interface. The thermodynamic properties of the pure and mixed monolayers were characterized using the classical film balance technique. Surface pressure/potential-area isotherms showed that the antimicrobial frog skin peptide PGLa formed a very stable monolayer with two PGLa molecules per kinetic unit and a collapse pressure of $22 mN/m. X-ray grazing incidence diffraction indicated that the peptidedimer formation did not lead to self-aggregation with subsequent crystallite formation. However, the scattering length density profiles derived from X-ray reflectivity measurements yield information on the PGLa monolayer that protrudes into the air phase by about 0.8 nm, suggesting that the peptide is aligned parallel to the air/ water interface. The monolayers, composed of disaturated phosphatidylcholines or phosphatidylglycerols, were stable up to 60 mN/m and exhibited a first-order transition from a liquid-expanded to a liquid-condensed state around 10 mN/m. Structural details of the phospholipid monolayers in the presence and absence of PGLa were obtained from synchrotron experiments. Thereby, the X-ray data of distearoylphosphatidylcholine/PGLa can be analyzed by being composed of the individual components, while the peptide strongly perturbed the lipid acyl chain order of distearoylphosphatidylglycerol. These results are in agreement that PGLa mixes at a molecular level with negatively charged lipids, but forms separate islands in zwitterionic phosphatidylcholine monolayers and demonstrates that antimicrobial peptides can discriminate between the major phospholipid components of bacterial and mammalian cytoplasmic membranes. Keywords X-ray diffraction AE Monolayer AE Antimicrobial peptides AE Lipid-peptide interaction AE Lipid specificity
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1999
Interest in biophysical studies on the interaction of antimicrobial peptides and lipids has strongly increased because of the rapid emergence of antibiotic-resistant bacterial strains. An understanding of the molecular mechanism(s) of membrane perturbation by these peptides will allow a design of novel peptide antibiotics as an alternative to conventional antibiotics. Differential scanning calorimetry and X-ray diffraction studies have yielded a wealth of quantitative information on the effects of antimicrobial peptides on membrane structure as well as on peptide location. These studies clearly demonstrated that antimicrobial peptides show preferential interaction with specific phospholipid classes. Furthermore, they revealed that in addition to charge-charge interactions, membrane curvature strain and hydrophobic mismatch between peptides and lipids are important parameters in determining the mechanism of membrane perturbation. Hence, depending on the molecular properties of both lipid and peptide, creation of bilayer defects such as phase separation or membrane thinning, pore formation, promotion of nonlamellar lipid structures or bilayer disruption by the carpet model or detergent-like action, may occur. Moreover, these studies suggest that these different processes may represent gradual steps of membrane perturbation. A better understanding of the mutual dependence of these parameters will help to elucidate the molecular mechanism of membrane damage by antimicrobial peptides and their target membrane specificity, keys for the rationale design of novel types of peptide antibiotics.
Investigation of interaction of antimicrobial peptides with lipid monolayers
2018
With the increasing occurrence of multi-drug resistant bacteria, which has put the health of modern society under serious threat, novel anti-infective agents are needed in order to tackle this crisis. Antimicrobial peptides are a class of molecules which, due to their non-selective interactions via disrupting or permeabilizing bacterial membranes, have big potential in being developed as such novel therapeutics. An important step in understanding the antimicrobial mechanism of action of antimicrobial peptides is to understand the binding and interaction behaviour of the peptides with the membranes and how the membrane composition and properties affect their action mechanisms. Thus, motivated by this fact, the scope of this thesis is to study how a de novo designed antimicrobial peptide with the sequence G(IIKK)4-I-NH2 (denoted G4) interacts with model lipid monolayers mimicking the cell membranes of hosts (mammals) and those of pathogens (Gram-positive and Gram-negative bacteria). T...
Colloids and surfaces. B, Biointerfaces, 2017
Many antimicrobial peptides (AMPs) target bacterial membranes and they kill bacteria by causing structural disruptions. One of the fundamental issues however lies in the selective responses of AMPs to different cell membranes as a lack of selectivity can elicit toxic side effects to mammalian host cells. A key difference between the outer surfaces of bacterial and mammalian cells is the charge characteristics. We report a careful study of the binding of one of the representative AMPs, with the general sequence G(IIKK)4I-NH2 (G4), to the spread lipid monolayers of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DPPG (1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)) mimicking the charge difference between them, using the combined measurements from Langmuir trough, Brewster angle microscopy (BAM) and neutron reflection (NR). The difference in pressure rise upon peptide addition into the subphase clearly demonstrated the different interactions arising fro...
Soft Matter, 2008
Interactions of the antimicrobial peptide protegrin-1 (PG-1) with phospholipid monolayers have been investigated by using grazing incidence X-ray diffraction (GIXD) and specular X-ray reflectivity (XR). The structure of a PG-1 film at the air-aqueous interface was also investigated by XR for the first time. Lipid A, dipalmitoyl-phosphatidylglycerol (DPPG) and dipalmitoyl-phosphatidylcholine (DPPC) monolayers were formed at the air-aqueous interface to mimic the surface of the bacterial cell wall and the outer leaflet of the erythrocyte cell membrane, respectively. Experiments were carried out under constant area conditions where the pressure changes upon insertion of peptide into the monolayer. GIXD data suggest that the greatest monolayer disruption produced by PG-1 is seen with the DPPG system at 20 mN/m since the Bragg peaks completely disappear after introduction of PG-1 to the system. PG-1 shows greater insertion into the lipid A system compared to the DPPC system when both films are held at the same initial surface pressure of 20 mN/m. The degree of insertion lessens at 30 mN/m with both DPPC and DPPG monolayer systems. XR data further reveal that PG-1 inserts primarily in the head group region of lipid monolayers. However, only the XR data of the anionic lipids suggest the existence of an additional adsorbed peptide layer below the head group of the monolayer. Overall the data show that the extent of peptide/lipid interaction and lipid monolayer disruption depends not only on the lipid composition of the monolayer, but the packing density of the lipids in the monolayer prior to the introduction of peptide to the subphase.
The impact of membrane lipid composition on antimicrobial function of an α-helical peptide
Chemistry and Physics of Lipids, 2008
VP1, a putative ␣-helical antimicrobial peptide (␣-AMP) inhibited growth of Bacillus subtilis and Escherichia coli at 500 M. The peptide induced stable surface pressure changes in monolayers formed from B. subtilis native lipid extract (circa 4.5 mN m −1 ) but transient pressure changes in corresponding E. coli monolayers (circa 1.0 mN m −1 ), which led to monolayer disintegration. Synthetic lipid monolayers mimetic of the extracts were used to generate compression isotherms. Thermodynamic analysis of B. subtilis isotherms indicated membrane stabilisation by VP1 ( G Mix < 0), via a mechanism dependent upon the phosphatidylglycerol to cardiolipin ratio. Corresponding analysis of E. coli isotherms indicated membrane destabilisation by the peptide ( G Mix > 0). Destabilisation correlated with PE levels present and appeared to involve a mechanism resembling those used by tilted peptides. These data emphasise that structure/function analysis of ␣-AMPs must consider not only their structural characteristics but also the lipid make-up of the target microbial membrane.
Physical Review E, 2013
As a defined model of outer membranes of gram negative bacteria, we investigated the interaction of monolayers of lipopolysacchrides from Salmonella enterica rough strains R90 (LPS Ra) with natural and synthetic peptides. The fine structures perpendicular to the membrane plane and the ion distribution near the interface were determined by specular x-ray reflectivity (XRR) and grazing-incidence x-ray fluorescence (GIXF) in the presence and absence of divalent cations. The unique combination of XRR and GIXF allows for the quantitative identification of different modes of interactions in a high spatial resolution, which cannot be assessed by other experimental methods. Natural fish protamine disrupts the stratified membrane structures in the absence of Ca 2+ ions, while staying away from the membrane surface in the presence of Ca 2+ ions. In contrast, synthetic antisepsis peptide Pep 19-2.5 weakly adsorbs to the membrane and stays near the uncharged sugar units even in the absence of Ca 2+ . In the presence of Ca 2+ , Pep 19-2.5 can reach the negatively charged inner core without destroying the barrier capability against ions.
Single-Molecule Resolution of Antimicrobial Peptide Interactions with Supported Lipid A Bilayers
Biophysical journal, 2018
The molecular interactions between antimicrobial peptides (AMPs) and lipid A-containing supported lipid bilayers were probed using single-molecule total internal reflection fluorescence microscopy. Hybrid supported lipid bilayers with lipid A outer leaflets and phospholipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)) inner leaflets were prepared and characterized, and the spatiotemporal trajectories of individual fluorescently labeled LL37 and Melittin AMPs were determined as they interacted with the bilayer surfaces comprising either monophosphoryl or diphosphoryl lipid A (from Escherichia coli) to determine the impact of electrostatic interactions. Large numbers of trajectories were obtained and analyzed to obtain the distributions of surface residence times and the statistics of the spatial trajectories. Interestingly, the AMP species were sensitive to subtle differences in the charge of the lipid, with both peptides diffusing more slowly and residing longer on the dip...
Biochemistry, 2010
In a minimalist design approach, a synthetic peptide MSI-367 [(KFAKKFA) 3-NH 2)] was designed and synthesized with an objective towards generating cell-selective non-lytic peptides, which have a significant bearing in cell-targeting. The peptide showed potent activity against both bacteria and fungi, but no toxicity to human cells at micromolar concentrations. Bacterial versus human cell membrane selectivity of the peptide was determined from membrane permeabilization assays. Circular dichroism investigations revealed the intrinsic helix propensity of the peptide, β-turn structure in aqueous buffer and, extended and turn conformations upon binding to lipid vesicles. DSC experiments on DiPOPE bilayers indicated the induction of positive curvature strain and repression of fluid lamellar to inverted hexagonal phase transition by MSI-367. Results from ITC experiments suggested the possibility of formation of specific lipid-peptide complexes leading to aggregation. 2 H NMR of deuterated-POPC multilamellar vesicles confirmed the limited effect of membrane embedded peptide at the lipid-water interface. 31 P NMR data indicated changes in the lipid head group orientation of POPC, POPG, and POPE lipid bilayers upon peptide binding. Membrane-embedded and membrane-inserted states of the peptide were observed in sum frequency generation vibrational spectroscopy. CD, ITC and 31 P NMR data on E. coli lipids agree with the hypothesis that strong electrostatic lipid-peptide interactions embrace the peptide at the lipid-water interface and provide the basis for bacterial cell selectivity.
Physica B: Condensed Matter, 2005
Peptide secretion by living organisms constitutes an integral response process exploited by natural immune systems. In this work we present a model study and insight into this process reporting the thermodynamic and structural effects induced in phospholipid monolayers due to peptide insertion into the layer. Synchrotron X-ray radiation is combined with the Langmuir technique and exploited to form 'lipid-peptide' monolayers and probe the physical characteristics of the fundamental biological process of 'peptide secretion'. Our experiments show that the insertion of peptides in the phospholipid layer has adverse effects on the elastic properties of the layer manifested through the bending rigidity.