Temperature-Dependent Transmembrane Insertion of the Amphiphilic Peptide PGLa in Lipid Bilayers Observed by Solid State 19F NMR Spectroscopy (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Biomembranes, 2006
The cationic antimicrobial peptide PGLa is electrostatically attracted to bacterial membranes, binds as an amphiphilic α-helix, and is thus able to permeabilize the lipid bilayer. Using solid state 2 H-NMR of non-perturbing Ala-d 3 labels on the peptide, we have characterized the helix alignment under a range of different conditions. Even at a very high peptide-to-lipid ratio (1:20) and in the presence of negatively charged lipids, there was no indication of a toroidal wormhole structure. Instead, PGLa realigns from a surface-bound S-state to an obliquely tilted T-state, which is presumably dimeric. An intermediate structure halfway between the Sand T-state was observed in fully hydrated multilamellar DMPC vesicles at 1:50, suggesting a fast exchange between the two states on the time scale of >50 kHz. We demonstrate that this equilibrium is shifted from the S-towards the T-state either upon (i) increasing the peptide concentration, (ii) adding negatively charged DMPG, or (iii) decreasing the level of hydration. The threshold concentration for realignment in DMPC is found to be between 1:200 and 1:100 in oriented samples at 96% humidity. In fully hydrated multilamellar DMPC vesicles, it shifts to an effective peptide-to-lipid ratio of 1:50 as some peptides are able to escape into the bulk water phase.
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.
Biophysical Journal, 2006
The structure and alignment of the amphipathic a-helical antimicrobial peptide PGLa in a lipid membrane is determined with high accuracy by solid-state 2 H-NMR. Orientational constraints are derived from a series of eight alanine-3,3,3d 3 -labeled peptides, in which either a native alanine is nonperturbingly labeled (4 3 ), or a glycine (2 3 ) or isoleucine (2 3 ) is selectively replaced. The concentration dependent realignment of the a-helix from the surface-bound ''S-state'' to a tilted ''T-state'' by 30°is precisely calculated using the quadrupole splittings of the four nonperturbing labels as constraints. The remaining, potentially perturbing alanine-3,3,3-d 3 labels show only minor deviations from the unperturbed peptide structure and help to single out the unique solution. Comparison with previous 19 F-NMR constraints from 4-CF 3 -phenylglycine labels shows that the structure and orientation of the PGLa peptide is not much disturbed even by these bulky nonnatural side chains, which contain CF 3 groups that offer a 20-fold better NMR sensitivity than CD 3 groups.
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.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2014
We have investigated in the present study the effect of both non-selective and selective cationic 14-mer peptides on the lipid orientation of DMPC bilayers by 31 P solid-state nuclear magnetic resonance (NMR) spectroscopy. Depending on the position of substitution, these peptides adopt mainly either an α-helical structure able to permeabilize DMPC and DMPG vesicles (non-selective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). Several systems have been investigated, namely bilayers mechanically oriented between glass plates as well as bicelles oriented with their normal perpendicular or parallel to the external magnetic field. The results have been compared with spectral simulations with the goal of elucidating the difference in the interaction of these two types of peptides with zwitterionic lipid bilayers. The results indicate that the perturbation induced by selective peptides is much greater than that induced by nonselective peptides in all the lipid systems investigated, and this perturbation has been associated to the aggregation of the selective β-sheet peptides in these systems. On the other hand, the oriented lipid spectra obtained in the presence of non-selective peptides suggest the presence of toroidal pores.
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
Journal of Magnetic Resonance, 2004
A highly sensitive solid state 19 F-NMR strategy is described to determine the orientation and dynamics of membrane-associated peptides from specific fluorine labels. Several analogues of the antimicrobial peptide PGLa were synthesized with the non-natural amino acid 4-trifluoromethyl-phenylglycine (CF 3-Phg) at different positions throughout the a-helical peptide chain. A simple 1-pulse 19 F experiment allows the simultaneous measurement of both the anisotropic chemical shift and the homonuclear dipolar coupling within the rotating CF 3-group in a macroscopically oriented membrane sample. The value and sign of the dipolar splitting determines the tilt of the CF 3-rotational axis, which is rigidly attached to the peptide backbone, with respect to the external magnetic field direction. Using four CF 3-labeled peptide analogues (with L L-CF 3-Phg at Ile9, Ala10, Ile13, and Ala14) we confirmed that PGLa is aligned at the surface of lipid membranes with its helix axis perpendicular to the bilayer normal at a peptide:lipid ratio of 1:200. We also determined the azimuthal rotation angle of the helix, which agrees well with the orientation expected from its amphiphilic character. Peptide analogues with a D D-CF 3-Phg label resulting from racemization of the amino acid during synthesis were separately collected by HPLC. Their spectra provide additional information about the PGLa structure and orientation but allow only to discriminate qualitatively between multiple solutions. The structural and functional characterization of the individual CF 3-labeled peptides by circular dichroism and antimicrobial assays showed only small effects for our four substitutions on the hydrophobic face of the helix, but a significant disturbance was observed in a fifth analogue where Ala8 on the hydrophilic face had been replaced. Even though the hydrophobic CF 3-Phg side chain cannot be utilized in all positions, it allows highly sensitive NMR measurements over a wide range of experimental conditions and dynamic regimes of the peptide.
Biochemistry, 2002
Protegrin-1 (PG-1) is a broad-spectrum-sheet antimicrobial peptide found in porcine leukocytes. The mechanism of action and the orientation of PG-1 in lipid bilayers are here investigated using 2 H, 31 P, 13 C, and 15 N solid-state NMR spectroscopy. 2 H spectra of mechanically aligned and chainperdeuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers indicate that PG-1 at high concentrations destroys the orientational order of the aligned lamellar bilayer. The conformation of the lipid headgroups in the unoriented region is significantly altered, as seen from the 31 P spectra of POPC and the 2 H spectra of headgroup-deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine. These observations indicate that PG-1 disrupts microbial membranes by breaking the extended bilayer into smaller disks, where a significant fraction of lipids is located in the edges of the disks with a distribution of orientations. These edges allow the lipid bilayer to bend back on itself as in toroidal pores. Interestingly, this loss of bilayer orientation occurs only in long-chain lipids such as POPC and not in shorter chain lipids such as 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC). To understand the mode of binding of PG-1 to the lipid bilayer, we determined the orientation of PG-1 in DLPC bilayers. The 13 CO and 15 N chemical shifts of Val-16 labeled PG-1 indicate that the-strand axis is tilted by 55°(5°from the bilayer normal while the normal of the-sheet plane is 48°(5°from the bilayer normal. This orientation favors interaction of the hydrophobic backbone of the peptide with the hydrophobic core of the bilayer and positions the cationic Arg side chains to interact with the anionic phosphate groups. This is the first time that the orientation of a disulfide-stabilized-sheet membrane peptide has been determined by solidstate NMR.
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.