Solid-state NMR investigations of interaction contributions that determine the alignment of helical polypeptides in biological membranes

AbstractHelical peptides reconstituted into oriented phospholipid bilayers were studied by proton-decoupled 15N solid-state NMR spectroscopy. Whereas hydrophobic channel peptides, such as the N-terminal region of Vpu of HIV-1, adopt transmembrane orientations, amphipathic peptide antibiotics are oriented parallel to the bilayer surface. The interaction contributions that determine the alignment of helical peptides in lipid membranes were analysed using model sequences, and peptides that change their topology in a pH-dependent manner have been designed. The energy contributions of histidines, lysines, leucines and alanines as well as the alignment of peptides and phospholipids under conditions of hydrophobic mismatch have been investigated in considerable detail

Detergent-like actions of linear amphipathic cationic antimicrobial peptides

AbstractAntimicrobial peptides have raised much interest as pathogens become resistant against conventional antibiotics. We review biophysical studies that have been performed to better understand the interactions of linear amphipathic cationic peptides such as magainins, cecropins, dermaseptin, δ-lysin or melittin. The amphipathic character of these peptides and their interactions with membranes resemble the properties of detergent molecules and analogies between membrane-active peptide and detergents are presented. Several models have been suggested to explain the pore-forming, membrane-lytic and antibiotic activities of these peptides. Here we suggest that these might be ‘special cases’ within complicated phase diagrams describing the morphological plasticity of peptide/lipid supramolecular assemblies

The SMART model: Soft Membranes Adapt and Respond, also Transiently, in the presence of antimicrobial peptides:

Biophysical and structural studies of peptide-lipid interactions, peptide topology and dynamics have changed our view on how antimicrobial peptides insert and interact with membranes. Clearly, both the peptides and the lipids are highly dynamic, change and mutually adapt their conformation, membrane penetration and detailed morphology on a local and a global level. As a consequence, the peptides and lipids can form a wide variety of supramolecular assemblies in which the more hydrophobic sequences preferentially, but not exclusively, adopt transmembrane alignments and have the potential to form oligomeric structures similar to those suggested by the transmembrane helical bundle model. In contrast, charged amphipathic sequences tend to stay intercalated at the membrane interface where they cause pronounced disruptions of the phospholipid fatty acyl packing. At increasing local or global concentrations, the peptides result in transient membrane openings, rupture and ultimately lysis. Depending on peptide-to-lipid ratio, lipid composition and environmental factors (temperature, buffer composition, ionic strength, etc.), the same peptide sequence can result in a variety of those responses. Therefore, the SMART model has been introduced to cover the full range of possibilities. With such a view in mind, novel antimicrobial compounds have been designed from amphipathic polymers, peptide mimetics, combinations of ultra-short polypeptides with hydrophobic anchors or small designer molecules

Membrane interactions and alignment of structures within the HIV-1 Vpu cytoplasmic domain: effect of phosphorylation of serines 52 and 56

AbstractThe cytoplasmic domain of the HIV-1 accessory protein Vpu is involved in the binding and degradation of the viral receptor CD4. In order to analyze previous structural models in the context of membrane environments, regions of VpuCYTO incorporating particular conformational features have been synthesized and labelled with 15N at selected backbone amides. Well-oriented proton-decoupled 15N solid-state NMR spectra with 15N chemical shifts at the most upfield position indicate that the amphipathic helix within [15N-Leu 45]-Vpu27–57 strongly interacts with mechanically aligned POPC bilayers and adopts an orientation parallel to the membrane surface. No major changes in the topology of this membrane-associated amphipathic helix were observed upon phosphorylation of serine residues 52 and 56, although this modification regulates biological function of Vpu. In contrast, [15N-Ala 62]-Vpu51–81 exhibits a pronounced 15N chemical shift anisotropy

Molecular packing of amphipathic peptides on the surface of lipid membranes

When polypeptides bind to the membrane surface, they become confined to a restricted quasi-two-dimensional space where peptide-peptide interactions become highly relevant, and the concept of a crowded medium is appropriate. Within this crowded environment interesting effects like clustering, separation of phases, cooperative alignment, and common movements occur. Here we investigated such effects by measuring distances between fluorophore-labeled peptides in the range </=1 nm by fluorescence self-quenching. For helical peptides with dimensions of approximately 1 x 3 nm such a small "ruler" is sensitive to the packing of the labeled peptides and thereby to their molecular arrangement. A novel approach to characterize peptide-peptide interactions within membranes is presented using the designer peptide LAH4. This sequence changes membrane topology in a controlled manner being transmembrane at neutral conditions but oriented parallel to the surface at low pH. Experimental measurements of the fluorescence self-quenching of close-by chromophores and the changes that occur upon dilution with unlabeled peptides are used to analyze the peptide distribution within the membrane surface. The data show a strong effect of electrostatic interactions and under some experimental conditions clustering of the peptides. Furthermore, the results suggest that at pH 4 the peptides arrange along the membrane surface in an ordered mesophase-like arrangement

Biophysical Investigations Elucidating the Mechanisms of Action of Antimicrobial Peptides and Their Synergism

Biophysical and structural investigations are presented with a focus on the membrane lipid interactions of cationic linear antibiotic peptides such as magainin, PGLa, LL37, and melittin. Observations made with these peptides are distinct as seen from data obtained with the hydrophobic peptide alamethicin. The cationic amphipathic peptides predominantly adopt membrane alignments parallel to the bilayer surface; thus the distribution of polar and non-polar side chains of the amphipathic helices mirror the environmental changes at the membrane interface. Such a membrane partitioning of an amphipathic helix has been shown to cause considerable disruptions in the lipid packing arrangements, transient openings at low peptide concentration, and membrane disintegration at higher peptide-to-lipid ratios. The manifold supramolecular arrangements adopted by lipids and peptides are represented by the 'soft membranes adapt and respond, also transiently' (SMART) model. Whereas molecular dynamics simulations provide atomistic views on lipid membranes in the presence of antimicrobial peptides, the biophysical investigations reveal interesting details on a molecular and supramolecular level, and recent microscopic imaging experiments delineate interesting sequences of events when bacterial cells are exposed to such peptides. Finally, biophysical studies that aim to reveal the mechanisms of synergistic interactions of magainin 2 and PGLa are presented, including unpublished isothermal titration calorimetry (ITC), circular dichroism (CD) and dynamic light scattering (DLS) measurements that suggest that the peptides are involved in liposome agglutination by mediating intermembrane interactions. A number of structural events are presented in schematic models that relate to the antimicrobial and synergistic mechanism of amphipathic peptides when they are aligned parallel to the membrane surface.PMC602300

The histidine-rich peptide LAH4-L1 strongly promotes PAMAM-mediated transfection at low nitrogen to phosphorus ratios in the presence of serum

Non-viral vectors are widely used and investigated for the delivery of genetic material into cells. However, gene delivery barriers like lysosomal degradation, serum inhibition and transient gene expression so far still limit their clinical applications. Aiming to overcome these limitations, a pH-sensitive hybrid gene vector (PSL complex) was designed by self-assembly of poly(amidoamine) (PAMAM) dendrimers, the histidine-rich peptide LAH4-L1 and the sleeping beauty transposon system (SB transposon system, a plasmid system capable of efficient and precise genomic insertion). Transfection studies revealed that PSL complexes achieved excellent efficiency in all investigated cell lines (higher than 90% in HeLa cells and over 30% in MDCK cells, a difficult-to-transfect cell line). Additionally, the PSL complexes showed high serum tolerance and exhibited outstanding transfection efficiency even in medium containing 50% serum (higher than 90% in HeLa cells). Moreover, a high level of long-term gene expression (over 30% in HeLa cells) was observed. Furthermore, PSL complexes not only resulted in high endocytosis, but also showed enhanced ability of endosomal escape compared to PAMAM/DNA complexes. These results demonstrate that simple association of PAMAM dendrimers, LAH4-L1 peptides and the SB transposon system by self-assembly is a general and promising strategy for efficient and safe gene delivery.PMC557505

Membrane topologies of the PGLa antimicrobial peptide and a transmembrane anchor sequence by Dynamic Nuclear Polarization/ solid-state NMR spectroscopy OPEN

International audienceDynamic 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

Simultaneous Analysis of Secondary Structure and Light Scattering from Circular Dichroism Titrations: Application to Vectofusin-1:

Circular Dichroism data are often decomposed into their constituent spectra to quantify the secondary structure of peptides or proteins but the estimation of the secondary structure content fails when light scattering leads to spectral distortion. If peptide-induced liposome self-association occurs, subtracting control curves cannot correct for this. We show that if the cause of the light scattering is independent from the peptide structural changes, the CD spectra can be corrected using principal component analysis (PCA). The light scattering itself is analysed and found to be in good agreement with backscattering experiments. This method therefore allows to simultaneously follow structural changes related to peptide-liposome binding as well as peptide induced liposome self-association. We apply this method to study the structural changes and liposome binding of vectofusin-1, a transduction enhancing peptide used in lentivirus based gene therapy. Vectofusin-1 binds to POPC/POPS liposomes, causing a reversal of the negative liposome charge at high peptide concentrations. When the peptide charges exactly neutralise the lipid charges on both leaflets reversible liposome self-association occurs. These results are in good agreement with biological observations and provide further insight into the conditions required for efficent transduction enhancement.PMC517791