Solid-State NMR Investigations of Peptide−Lipid Interaction and Orientation of a β-Sheet Antimicrobial Peptide, Protegrin † (original) (raw)
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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.
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.
Journal of Molecular Biology, 2008
The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The 13 C chemical shifts of 13 C, 15 N-labeled residues in the peptide indicate a reversible conformational change from β-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25-50% anionic lipids, and in both saturated DMPC/ DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C-H order parameters of 0.23-0.52 for C α and C β sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet-turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage.
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.
Conformational study of the protegrin-1 (PG-1) dimer interaction with lipid bilayers and its effect
BMC Structural Biology, 2007
Background: Protegrin-1 (PG-1) is known as a potent antibiotic peptide; it prevents infection via an attack on the membrane surface of invading microorganisms. In the membrane, the peptide forms a pore/channel through oligomerization of multiple subunits. Recent experimental and computational studies have increasingly unraveled the molecular-level mechanisms underlying the interactions of the PG-1 β-sheet motifs with the membrane. The PG-1 dimer is important for the formation of oligomers, ordered aggregates, and for membrane damaging effects. Yet, experimentally, different dimeric behavior has been observed depending on the environment: antiparallel in the micelle environment, and parallel in the POPC bilayer. The experimental structure of the PG-1 dimer is currently unavailable.
Orientations of helical peptides in membrane bilayers by solid state NMR spectroscopy
Solid State Nuclear Magnetic Resonance, 1996
The orientations of helical peptides in membrane bilayers provide important structural information that is directly relevant to their functional roles, both alone and within the context of larger membrane proteins. The orientations can be readily determined with solid state NMR experiments on samples of "N-labeled peptides in lipid bilayers aligned between glass plates. The observed "N chemical shift frequencies can be directly interpreted to indicate whether the peptide's helix axis has a trans-membrane or an in-plane orientation. In order to distinguish between these possibilities on the basis of a single spectral parameter, e.g. the easily measured 15N chemical shift frequency, it is necessary to demonstrate that the secondary structure of the peptide is helical, generally by solution NMR spectroscopy of the same peptide in micelle samples, and that it is immobile in bilayers, generally from solid state NMR spectra of unoriented samples, Six different 20-30 residue peptides are shown to have orientations that fall into the categories of trans-membrane or in-plane helices. A model hydrophobic peptide was found to be trans-membrane, several different amphipathic helical peptides were found to have either trans-membrane or in-plane orientations, and a leader or signal peptide, generally regarded as hydrophobic, was found to have a significant population with an in-plane orientation.
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
Biophysical Journal, 2004
Solid-state NMR methods employing 2 H NMR and geometric analysis of labeled alanines (GALA) were used to study the structure and orientation of the transmembrane a-helical peptide acetyl-GWW(LA) 8 LWWA-amide (WALP23) in phosphatidylcholine (PC) bilayers of varying thickness. In all lipids the peptide was found to adopt a transmembrane a-helical conformation. A small tilt angle of 4.5°was observed in di-18:1-PC, which has a hydrophobic bilayer thickness that approximately matches the hydrophobic length of the peptide. This tilt angle increased slightly but systematically with increasing positive mismatch to 8.2°in di-C12:0-PC, the shortest lipid used. This small increase in tilt angle is insufficient to significantly change the effective hydrophobic length of the peptide and thereby to compensate for the increasing hydrophobic mismatch, suggesting that tilt of these peptides in a lipid bilayer is energetically unfavorable. The tilt and also the orientation around the peptide axis were found to be very similar to the values previously reported for a shorter WALP19 peptide (GWW(LA) 6 LWWA). As also observed in this previous study, the peptide rotates rapidly around the bilayer normal, but not around its helix axis. Here we show that these properties allow application of the GALA method not only to macroscopically aligned samples but also to randomly oriented samples, which has important practical advantages. A minimum of four labeled alanine residues in the hydrophobic transmembrane sequence was found to be required to obtain accurate tilt values using the GALA method.
Biochemistry, 2004
RTD-1 is a cyclic-hairpin antimicrobial peptide isolated from rhesus macaque leukocytes. Using 31 P, 2 H, 13 C, and 15 N solid-state NMR, we investigated the interaction of RTD-1 with lipid bilayers of different compositions. 31 P and 2 H NMR of uniaxially oriented membranes provided valuable information about how RTD-1 affects the static and dynamic disorder of the bilayer. Toward phosphatidylcholine (PC) bilayers, RTD-1 causes moderate orientational disorder independent of the bilayer thickness, suggesting that RTD-1 binds to the surface of PC bilayers without perturbing its hydrophobic core. Addition of cholesterol to the POPC membrane does not affect the orientational disorder. In contrast, binding of RTD-1 to anionic bilayers containing PC and phosphatidylglycerol lipids induces much greater orientational disorder without affecting the dynamic disorder of the membrane. These correlate with the selectivity of RTD-1 for anionic bacterial membranes as opposed to cholesterol-rich zwitterionic mammalian membranes. Line shape simulations indicate that RTD-1 induces the formation of micrometer-diameter lipid cylinders in anionic membranes. The curvature stress induced by RTD-1 may underlie the antimicrobial activity of RTD-1. 13 C and 15 N anisotropic chemical shifts of RTD-1 in oriented PC bilayers indicate that the peptide adopts a distribution of orientations relative to the magnetic field. This is most likely due to a small fraction of lipid cylinders that change the RTD-1 orientation with respect to the magnetic field. Membranebound RTD-1 exhibits narrow line widths in magic-angle spinning spectra, but the sideband intensities indicate rigid-limit anisotropies. These suggest that RTD-1 has a well-defined secondary structure and is likely aggregated in the membrane. These structural and dynamical features of RTD-1 differ significantly from those of PG-1, a related-hairpin antimicrobial peptide.