Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides (original) (raw)
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Most linear peptides directly interact with membranes, but the mechanisms of interaction are far from being completely understood. Here, we present an investigation of the membrane interactions of a designed peptide containing a non-natural, synthetic amino acid. We selected a nonapeptide that is reported to interact with phospholipid membranes, ALYLAIRKR, abbreviated as ALY. We designed a modified peptide (azoALY) by substituting the tyrosine residue of ALY with an antimicrobial azobenzene-bearing amino acid. Both of the peptides were examined for their ability to interact with model membranes, assessing the penetration of phospholipid monolayers, and leakage across the bilayer of large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs). The latter was performed in a microfluidic device in order to study the kinetics of leakage of entrapped calcein from the vesicles at the single vesicle level. Both types of vesicles were prepared from a 9:1 (mol/mol) mixture of POPC...
The impact of peptides on lipid membranes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2008
We review the fundamental strategies used by small peptides to associate with lipid membranes and how the different strategies impact on the structure and dynamics of the lipids. In particular we focus on the binding of amphiphilic peptides by electrostatic and hydrophobic forces, on the anchoring of peptides to the bilayer by acylation and prenylation, and on the incorporation of small peptides that form well-defined channels. The effect of lipid-peptide interactions on the lipids is characterized in terms of lipid acyl-chain order, membrane thickness, membrane elasticity, permeability, lipid-domain and annulus formation, as well as acyl-chain dynamics. The different situations are illustrated by specific cases for which experimental observations can be interpreted and supplemented by theoretical modeling and simulations. A comparison is made with the effect on lipids of trans-membrane proteins. The various cases are discussed in the context of the possible roles played by lipid-peptide interactions for the biological, physiological, and pharmacological function of peptides.
Biochemical and Biophysical Research Communications, 2008
In this study, we employed electrophysiology experiments carried out at the single-molecule level to study the mechanism of action of the HPA3 peptide, an analogue of the linear antimicrobial peptide, HP(2-20), isolated from the N-terminal region of the Helicobacter pylori ribosomal protein. Amplitude analysis of currents fluctuations induced by HPA3 peptide at various potentials in zwitterionic lipid membranes reveal the existence of reproducible conductive states in the stochastic behavior of such events, which directly supports the existence of transmembrane pores induced the peptide. From our data recorded both at the single-pore and macroscopic levels, we propose that the HPA3 pore formation is electrophoretically facilitated by trans-negative transmembrane potentials, and HPA3 peptides translocate into the trans monolayers after forming the pores. We present evidence according to which the decrease in the membrane dipole potential of a reconstituted lipid membranes leads to an augmentation of the membrane activity of HPA3 peptides, and propose that a lower electric dipole field of the interfacial region of the membrane caused by phloretin facilitates the surface-bound HPA3 peptides to break free from one leaflet of the membrane, insert into the membrane and contribute to pore formation spanning the entire thickness of the membrane.
Langmuir : the ACS journal of surfaces and colloids, 2018
Considering the known different mode of action of antimicrobial peptides in zwitterionic and anionic cell membranes, the present work compares the action of the antimicrobial peptide K0-W6-Hya1 (KIFGAIWPLALGALKNLIK-NH2) with zwitterionic and negatively charged model membranes, namely, liposomes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, and a mixture of the two. Differential scanning calorimetry (DSC), steady state fluorescence of the Trp residue, dynamic light scattering (DLS), and measurement of the leakage of an entrapped fluorescent dye (carboxyfluorescein, CF) were performed with large unilamellar vesicles (LUVs). All techniques evidenced the different action of the peptide in zwitterionic and anionic vesicles. Trp fluorescence spectroscopy shows that the differences are related not only to the partition of the cationic peptide in zwitterionic and anionic membranes, but also to the different penetration depth of the peptide into the lipid bila...
Interaction of an artificial antimicrobial peptide with lipid membranes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2009
Antimicrobial peptides constitute an important part of the innate immune defense and are promising new candidates for antibiotics. Naturally occurring antimicrobial peptides often possess hemolytic activity and are not suitable as drugs. Therefore, a range of new synthetic antimicrobial peptides have been developed in recent years with promising properties. But their mechanism of action is in most cases not fully understood. One of these peptides, called V4, is a cyclized 19 amino acid peptide whose amino acid sequence has been modeled upon the hydrophobic/cationic binding pattern found in Factor C of the horseshoe crab (Carcinoscorpius rotundicauda). In this work we used a combination of biophysical techniques to elucidate the mechanism of action of V4. Langmuir-Blodgett trough, atomic force microscopy, Fluorescence Correlation Spectroscopy, Dual Polarization Interference, and confocal microscopy experiments show how the hydrophobic and cationic properties of V4 lead to a) selective binding of the peptide to anionic lipids (POPG) versus zwitterionic lipids (POPC), b) aggregation of vesicles, and above a certain concentration threshold to c) integration of the peptide into the bilayer and finally d) to the disruption of the bilayer structure. The understanding of the mechanism of action of this peptide in relation to the properties of its constituent amino acids is a first step in designing better peptides in the future.
How lipids influence the mode of action of membrane-active peptides
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2007
The human, multifunctional peptide LL-37 causes membrane disruption by distinctly different mechanisms strongly dependent on the nature of the membrane lipid composition, varying not only with lipid headgroup charge but also with hydrocarbon chain length. Specifically, LL-37 induces a peptide-associated quasi-interdigitated phase in negatively charged phosphatidylglycerol (PG) model membranes, where the hydrocarbon chains are shielded from water by the peptide. In turn, LL-37 leads to a disintegration of the lamellar organization of zwitterionic dipalmitoyl-phosphatidylcholine (DPPC) into disk-like micelles. Interestingly, interdigitation was also observed for the longer-chain C18 and C20 PCs. This dual behavior of LL-37 can be attributed to a balance between electrostatic interactions reflected in different penetration depths of the peptide and hydrocarbon chain length. Thus, our observations indicate that there is a tight coupling between the peptide properties and those of the lipid bilayer, which needs to be considered in studies of lipid/peptide interaction. Very similar effects were also observed for melittin and the frog skin peptide PGLa. Therefore, we propose a phase diagram showing different lipid/peptide arrangements as a function of hydrocarbon chain length and LL-37 concentration and suggest that this phase diagram is generally applicable to membrane-active peptides localized parallel to the membrane surface.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2002
Model compounds of modified hydrophobicity (H), hydrophobic moment (W) and angle subtended by charged residues (x) were synthesized to define the general roles of structural motifs of cationic helical peptides for membrane activity and selectivity. The peptide sets were based on a highly hydrophobic, non-selective KLA model peptide with high antimicrobial and hemolytic activity. Variation of the investigated parameters was found to be a suitable method for modifying peptide selectivity towards either neutral or highly negatively charged lipid bilayers. H and W influenced selectivity preferentially via modification of activity on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers, while the size of the polar/hydrophobic angle affected the activity against 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol (POPG). The influence of the parameters on the activity determining step was modest in both lipid systems and the activity profiles were the result of the parameters' influence on the second less pronounced permeabilization step. Thus, the activity towards POPC vesicles was determined by the high permeabilizing efficiency, however, changes in the structural parameters preferentially influenced the relatively moderate affinity. In contrast, intensive peptide accumulation via electrostatic interactions was sufficient for the destabilization of highly negatively charged POPG lipid membranes, but changes in the activity profile, as revealed by the modification of x, seem to be preferentially caused by variation of the low permeabilizing efficiency. The parameters proved very effective also in modifying antimicrobial and hemolytic activity. However, their influence on cell selectivity was limited. A threshold value of hydrophobicity seems to exist which restricted the activity modifying potential of W and x on both lipid bilayers and cell membranes.
Antimicrobial Peptides Induce Growth of Phosphatidylglycerol Domains in a Model Bacterial Membrane
2010
We performed microsecond long coarse-grained molecular dynamics simulations to elucidate the lateral structure and domain dynamics of a phosphatidylethanolamine (PE)/phosphatidylglycerol (PG) mixed bilayer (7/3), mimicking the inner membrane of gram-negative bacteria. Specifically, we address the effect of surface bound antimicrobial peptides (AMPs) on the lateral organization of the membrane. We find that, in the absence of the peptides, the minor PG fraction only forms small clusters, but that these clusters grow in size upon binding of the cationic AMPs. The presence of AMPs systematically affects the dynamics and induces long-range order in the structure of PG domains, stabilizing the separation between the two lipid fractions. Our results help in understanding the initial stages of destabilization of cytoplasmic bacterial membranes below the critical peptide concentration necessary for disruption, and provide a possible explanation for the multimodal character of AMP activity.
Biomimetic Model Membrane Systems Serve as Increasingly Valuable in Vitro Tools
Advances in Biomimetics, 2011
Biological membranes contain a multitude of lipids, proteins, and carbohydrates unique for any given cell or organism, and are a critical component of many biological processes. Animal and cell cultures have been used to understand these biological processes at the membrane level and more traditionally, to assess toxicity. However, the complex composition does not allow understanding of the detailed role of each membrane component, such as individual lipid species. This insight can be obtained from using simplified model systems, which include various kinds of vesicles (unilamellar or multilamellar), micelles, monolayers at an air-water interface, planar lipid bilayers/black lipid membranes, bicelles (bilayered micelles) and supported bilayers. All systems allow detailed control of composition and experimental conditions, and have been used to mimic various different membrane types, such as mammalian and bacterial. Using various physicochemical techniques including nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), isothermal calorimetry (ITC), electron spin resonance, fluorescence spectroscopy, and X-ray diffraction, it is possible to investigate the mechanisms of membrane toxicity through differential changes in acyl chain melting temperature, membrane fluidity, and permeability of these different membrane models upon ligand binding. Moreover, the effects of ions (Na + , K + , Li + , Ca 2+ , Mg 2+ , Ba 2+), toxic heavy metals (Hg 2+ , Cd 2+) and a variety of drugs (e.g. Ellipticine for tumors and H1N1 virus or cyclosporine A to prevent graft rejection) have been evaluated on mammalian systems. For bacterial model membranes, the effects of antimicrobial peptides, antibiotics, the interaction of proteins with model membranes, and the insertion or reconstitution of membrane proteins into such systems have also been investigated. When interpreting the results, it is important to note that some models may be better representatives of the natural membrane than others, and consequently, some results more relevant than others. Factors to consider include-but are not limited to-lipid composition, membrane curvature, or ionic strength of the solution, which all impart certain characteristics on the membrane model, influencing the results. Thus, while a singlecomponent lipid model can be informative, it is important to consider its applications and limitations. Overall, this chapter will provide insight as to the different lipid models used to mimic mammalian and bacterial membranes and how they have been found to be effective and useful research tools. Future development of these membrane models to more closely mimic www.intechopen.com Advances in Biomimetics 252 the composition and complexity of the natural membrane will provide further insight into the mechanisms of membrane processes in biological systems. 1.1 Membranes As lipids are small amphiphilic molecules, there are three aspects that define the physical characteristics of a lipid: the polar headgroup, the hydrophobic acyl chains and the interface between them. There are several different lipid headgroup classes, each with unique chemical properties. Some biological headgroups are negatively charged and exhibit charge-charge repulsions, which result in larger effective cross-sectional areas (Cullis et al., 1986). However, the charge, and thus the area, is subject to the experimental conditions. Changes in the pH of the solution can impart or eliminate charges from the lipid based on the specific pKa values of the headgroup. The presence of mono-or divalent cations can serve to shield or neutralize the charge-charge repulsions, thus decreasing their effective cross-sectional area and consequently altering the properties of the lipid (Tate et al., 1991). Unlike the polar headgroups, which can be altered by the environment, the behavior of the hydrophobic acyl chains is mainly based on their chemical structure. Acyl chains are typically 14 to 22 carbons long and can be fully saturated, mono-unsaturated, or polyunsaturated. Length and degree of saturation play a major role in lipid packing and the behaviour of the membrane. Fully saturated lipids pack more tightly than lipids with unsaturated acyl chains, changing the fluidity, transition temperature, and the lateral membrane pressure profile. Longer chains also have greater van der Waals interactions that stabilize membranes (Birdi, 1988). In contrast, the increased cross-sectional area of unsaturated lipids enhances membrane fluidity (de Kruijff, 1997). Membranes are known to play an important role in many crucial biological functions, be it as the cellular membrane or as barrier of intracellular compartments. The fluid mosaic model of biological membranes (Singer and Nicolson, 1972) was groundbreaking in the understanding of membrane dynamics and organization, and the main concept of free diffusion of lipid and protein molecules within a dynamic fluid bilayer is still relevant. Current research supports the fact that several proteins are sensitive to the presence of specific lipids, with some experiencing an increase in activity while others require the presence of certain lipids for proper membrane insertion or multimeric stability (van der Does et al., 2000; van Dalen et al., 2002; van den Brink-van der Laan et al., 2004). However, one of the main emphases of the fluid mosaic model was that proteins and lipids were free to diffuse within the membrane, distributed randomly throughout with no regions of distinct composition. Research now supports the existence of lipid domains, distinct regions of specific lipid composition within the fluid bilayer (Rietveld and Simons, 1998; Zerrouk et al., 2008). These domains possess unique physical properties and could be vital for many cell processes such as signal transduction, cell adhesion, and the function of several membrane proteins (Simons and Ikonen, 1997; Harder et al., 1998). 1.2 The mammalian membrane Mammalian membranes are primarily composed of phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS), phosphatidylethanolamine (PE), and cholesterol (Chol) lipid species in various ratios depending on cell type. The human erythrocyte membrane, one of the best characterized systems, is composed of 19.5% (w/w) of water, 39.5% of proteins, 35.1% of lipids, and 5.8% of carbohydrates (Yawata, 2003).
The Journal of Physical Chemistry B, 2008
Membrane pores that are induced in oriented membranes by an antimicrobial peptide (AMP), protegrin-1 (PG-1), are investigated by 31 P and 2 H solid state NMR spectroscopy. We incorporated a well-studied peptide, protegrin-1 (PG-1), a-sheet AMP, to investigate AMP-induced dynamic supramolecular lipid assemblies at different peptide concentrations and membrane compositions. Anisotropic NMR line shapes specifying toroidal pores and thinned membranes, which are formed in membrane bilayers by the binding of AMPs, have been analyzed for the first time. Theoretical NMR line shapes of lipids distributed on the surface of toroidal pores and thinned membranes reproduce reasonably well the line shape characteristics of our experimentally measured 31 P and 2 H solid-state NMR spectra of oriented lipids binding with PG-1. The lateral diffusions of lipids are also analyzed from the motionally averaged one-and two-dimensional 31 P and 2 H solid-state NMR spectra of oriented lipids that are binding with AMPs. 1. Introduction Membrane interactions of membrane-acting antimicrobial peptides (AMPs) 1-8 are still one of the more poorly understood areas in modern structural biology. As the components of immune systems of mammals, insects, amphibians, and plants, AMPs directly modify and/or destroy the structures of cell membranes of invaded microorganisms, such as bacteria, fungi, and enveloped viruses as well as malignant cells and parasites. 1-9 AMPs are categorized into five major classes: R-helical, defensin-like (cystein-rich),-sheet, peptides with an unusual composition of regular amino acids, and bacterial and fungal peptides containing modified amino acids. 10 Despite their diversely different structures, all AMPs display a similar motif: an amphiphilic structure with one surface highly positive (hence, hydrophilic) and the other hydrophobic. Classical uptake mechanisms relying on protein-based receptors and transporters appear not to be involved in the membrane interactions of these peptides because D-enantiomers of AMPs are equally active as the naturally occurring all-L peptides, indicating that chiral molecules are not involved. 11-14 While the antimicrobial action of some AMPs appears to involve attack on intracellular targets, in most cases direct attack on the microbial cell membrane itself results in depolarization, permeabilization, and lysis. 15-18 The most plausible mechanisms suggested for these membrane-acting peptides to interact with oriented membrane bilayers include formations of inverted micelles, 14 carpets, 19 or toroidal pores 20,21 in/on membranes via electrostatic adsorption. Yet, to our knowledge, how these peptides interact with lipid membranes on a molecular level, and what structural properties of these peptides endow their potent and selective membrane disruptive abilities are not fully understood. AMPs have two binding states 22-25 in lipid bilayers: a surfacebound S-state and a pore-forming I-state. According to the S-state (carpet) model, 19 AMPs initially bind on the surface of