Diphytanoyl lipids as model systems for studying membrane-active peptides (original) (raw)
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The membrane interactions of antimicrobial peptides revealed by solid-state NMR spectroscopy
Chemistry and Physics of Lipids, 2012
Solid-state NMR spectroscopic techniques provide valuable information about the structure, dynamics and topology of membrane-inserted polypeptides. In particular antimicrobial peptides (or 'host defence peptides') have early on been investigated by solid-state NMR spectroscopy and many technical innovations in this domain have been developed with the help of these compounds when reconstituted into oriented phospholipid bilayers. Using solid-state NMR spectroscopy it could be shown for the first time that magainins or derivatives thereof exhibit potent antimicrobial activities when their cationic amphipathic helix is oriented parallel to the bilayer surface, a configuration found in later years for many other linear cationic amphipathic peptides. In contrast transmembrane alignments or lipid-dependent tilt angles have been found for more hydrophobic sequences such as alamethicin or β-hairpin antimicrobials. This review presents various solid-state NMR approaches and develops the basic underlying concept how angular information can be obtained from oriented samples. It is demonstrated how this information is used to calculate structures and topologies of peptides in their native liquid-disordered phospholipid bilayer environment. Special emphasis is given to discuss which NMR parameters provide the most complementary information, the minimal number of restraints needed and the effect of motions on the analysis of the NMR spectra. Furthermore, recent (31)P and (2)H solid-state NMR measurements of lipids are presented including some unpublished data which aim at investigating the morphological and structural changes of oriented or non-oriented phospholipids. Finally the structural models that have been proposed for the mechanisms of action of these peptides will be presented and discussed in view of the solid-state NMR and other biophysical experiments.
Biophysical Journal, 2006
We studied the interaction between synthetic amphipathic peptides and model membranes by solid-state NMR and infrared spectroscopies. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers were synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure. To shed light on their membrane interaction, 31 P and 2 H solid-state NMR experiments were performed on both peptides in interaction with dimyristoylphosphatidylcholine vesicles in the absence and presence of cholesterol, dimyristoylphosphatidylglycerol vesicles, and oriented bicelles. 31 P NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are weakly yet markedly affected by the presence of the peptides, whereas 31 P NMR experiments on bicelles indicate no significant changes in the morphology and orientation of the bicelles. On the other hand, 2 H NMR experiments on vesicles reveal that the acyl chain order is affected differently depending on the membrane lipidic composition and on the peptide hydrophobic length. Finally, infrared spectroscopy was used to study the interfacial region of the bilayer. Based on these studies, mechanisms of membrane perturbation are proposed for the 14-mer and 21-mer peptides in interaction with model membranes depending on the bilayer composition and peptide length.
Bulletin of the Korean Chemical Society, 2012
The activity of an antimicrobial peptide, protegrin-1 (PG-1), on lipid membranes was investigated using solidstate NMR and a new sampling method that employed mechanically aligned bilayers between thin glass plates. At 95% hydration and full hydration, the peptide respectively disrupted 25% and 86% of the aligned 1palmitoyl-2-oleoyl-sn-glycero-3-phosphotidylcholine (POPC) bilayers at a P/L (peptide-to-lipid) ratio of 1/20 under the new experimental conditions. The kinetics of the POPC bilayers disruption appeared to be diffusioncontrolled. The presence of cholesterol at 95% hydration and full hydration reduced the peptide disruption of the aligned POPC bilayers to less than 10% and 35%, respectively. A comparison of the equilibrium states of heterogeneously and homogeneously mixed peptides and lipids demonstrated the importance of peptide binding to the biomembrane for whole membrane disruption.
Biochimica et biophysica acta, 2014
Many amphiphilic antimicrobial peptides permeabilize bacterial membranes via successive steps of binding, re-alignment and/or oligomerization. Here, we have systematically compared the lipid interactions of two structurally unrelated peptides: the cyclic β-pleated gramicidin S (GS), and the α-helical PGLa. (19)F NMR was used to screen their molecular alignment in various model membranes over a wide range of temperatures. Both peptides were found to respond to the phase state and composition of these different samples in a similar way. In phosphatidylcholines, both peptides first bind to the bilayer surface. Above a certain threshold concentration they can re-align and immerse more deeply into the hydrophobic core, which presumably involves oligomerization. Re-alignment is most favorable around the lipid chain melting temperature, and also promoted by decreasing bilayer thickness. The presence of anionic lipids has no influence in fluid membranes, but in the gel phase the alignment s...
Scientific Reports, 2016
Dynamic Nuclear Polarization (DNP) has been introduced to overcome the sensitivity limitations of nuclear magnetic resonance (NMR) spectroscopy also of supported lipid bilayers. When investigated by solid-state NMR techniques the approach typically involves doping the samples with biradicals and their investigation at cryo-temperatures. Here we investigated the effects of temperature and membrane hydration on the topology of amphipathic and hydrophobic membrane polypeptides. Although the antimicrobial PGLa peptide in dimyristoyl phospholipids is particularly sensitive to topological alterations, the DNP conditions represent well its membrane alignment also found in bacterial lipids at ambient temperature. With a novel membrane-anchored biradical and purpose-built hardware a 17-fold enhancement in NMR signal intensity is obtained by DNP which is one of the best obtained for a truly static matrix-free system. Furthermore, a membrane anchor sequence encompassing 19 hydrophobic amino acid residues was investigated. Although at cryotemperatures the transmembrane domain adjusts it membrane tilt angle by about 10 degrees, the temperature dependence of two-dimensional separated field spectra show that freezing the motions can have beneficial effects for the structural analysis of this sequence.
Biochimie, 2009
The 2 H 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 2 H 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.
Biophysical Journal, 2005
The membrane-disruptive antimicrobial peptide PGLa is found to change its orientation in a dimyristoylphosphatidylcholine bilayer when its concentration is increased to biologically active levels. The alignment of the a-helix was determined by highly sensitive solid-state NMR measurements of 19 F dipolar couplings on CF 3 -labeled side chains, and supported by a nonperturbing 15 N label. At a low peptide/lipid ratio of 1:200 the amphiphilic peptide resides on the membrane surface in the so-called S-state, as expected. However, at high peptide concentration ($1:50 molar ratio) the helix axis changes its tilt angle from ;90°to ;120°, with the C-terminus pointing toward the bilayer interior. This tilted ''T-state'' represents a novel feature of antimicrobial peptides, which is distinct from a membrane-inserted I-state. At intermediate concentration, PGLa is in exchange between the S-and T-state in the timescale of the NMR experiment. In both states the peptide molecules undergo fast rotation around the membrane normal in liquid crystalline bilayers; hence, large peptide aggregates do not form. Very likely the obliquely tilted T-state represents an antiparallel dimer of PGLa that is formed in the membrane at increasing concentration.
Control and role of pH in peptide-lipid interactions in oriented membrane samples
Biochimica et biophysica acta, 2015
To understand the molecular mechanisms of amphiphilic membrane-active peptides, one needs to study their interactions with lipid bilayers under ambient conditions. However, it is difficult to control the pH of the sample in biophysical experiments that make use of mechanically aligned multilamellar membrane stacks on solid supports. HPLC-purified peptides tend to be acidic and can change the pH in the sample significantly. Here, we have systematically studied the influence of pH on the lipid interactions of the antimicrobial peptide PGLa embedded in oriented DMPC/DMPG bilayers. Using solid-state NMR ((31)P, (2)H, (19)F), both the lipid and peptide components were characterized independently, though in the same oriented samples under typical conditions of maximum hydration. The observed changes in lipid polymorphism were supported by DSC on multilamellar liposome suspensions. On this basis, we can present an optimized sample preparation protocol and discuss the challenges of performi...
Membrane Topology of a 14-mer Model Amphipathic Peptide: A Solid-State NMR Spectroscopy Study
Biochemistry, 2007
We have investigated the interaction between a synthetic amphipathic 14-mer peptide and model membranes by solid-state NMR. The 14-mer peptide is composed of leucines and phenylalanines modified by the addition of crown ethers and forms a helical amphipathic structure in solution and bound to lipid membranes. To shed light on its membrane topology, 31 P, 2 H, 15 N solid-state NMR experiments have been performed on the 14-mer peptide in interaction with mechanically oriented bilayers of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC). The 31 P, 2 H, and 15 N NMR results indicate that the 14-mer peptide remains at the surface of the DLPC, DMPC, and DPPC bilayers stacked between glass plates and perturbs the lipid orientation relative to the magnetic field direction. Its membrane topology is similar in DLPC and DMPC bilayers, whereas the peptide seems to be more deeply inserted in DPPC bilayers, as revealed by the greater orientational and motional disorder of the DPPC lipid headgroup and acyl chains. 15 N{ 31 P} rotational echo double resonance experiments have also been used to measure the intermolecular dipole-dipole interaction between the 14-mer peptide and the phospholipid headgroup of DMPC multilamellar vesicles, and the results indicate that the 14-mer peptide is in contact with the polar region of the DMPC lipids. On the basis of these studies, the mechanism of membrane perturbation of the 14-mer peptide is associated to the induction of a positive curvature strain induced by the peptide lying on the bilayer surface and seems to be independent of the bilayer hydrophobic thickness.
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