Dimeric peptides with three different linkers self-assemble with phospholipids to form peptide nanodiscs that stabilize membrane proteins (original) (raw)
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Self-assembling peptides form nanodiscs that stabilize membrane proteins
Soft Matter, 2014
New methods to handle membrane bound proteins, e.g. G-protein coupled receptors (GPCRs), are highly desirable. Recently, apoliprotein A1 (ApoA1) based lipoprotein particles have emerged as a new platform for studying membrane proteins, and it has been shown that they can self-assemble in combination with phospholipids to form discoidal shaped particles that can stabilize membrane proteins. In the present study, we have investigated an ApoA1 mimetic peptide with respect to its solution structure when in complex with phospholipids. This was achieved using a powerful combination of small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) supported by coarse-grained molecular dynamics simulations. The detailed structure of the discs was determined in unprecedented detail and it was found that they adopt a discoidal structure very similar to the ApoA1 based nanodiscs. We furthermore show that, like the ApoA1 and derived nanodiscs, these peptide discs can accommodate and stabilize a membrane protein. Finally, we exploit their dynamic properties and show that the 18A discs may be used for transferring membrane proteins and associated phospholipids directly and gently into phospholipid nanodiscs.
Protein Engineering Design and Selection, 1998
To dissect the determinants of protein insertion into membranes, we designed a model peptide which partitions between water and phospholipid bilayers as an α-helical monomer. We used a simplex method to optimize the 'a, d hydrophobicity' and 'e, g charge' of a series of five peptides, where 'abcdefg' correspond to the positions in two turns of an α-helix. Circular dichroism and analytical ultracentrifugation experiments showed that the final peptide (helix5) is monomeric and has an α-helix content of approximately 89% at 0°C in aqueous solution. In the presence of large unilamellar vesicles (LUVs), helix5 partitions between the aqueous and membranous phases with a partition constant well suited for measurements by electron paramagnetic resonance (EPR) spectroscopy. EPR power saturation experiments with a cysteine-scanning strategy showed that the α-helicity of helix5 is conserved upon binding to LUVs and that the α-helix binds parallel to the membrane surface with the central axis approximately 5 Å below the lipid phosphate groups. Helix5 should be a useful model peptide for studies aimed at dissecting the determinants of the membrane binding of α-helices. The simplex-based strategy may be useful in the rational design of proteins when desired structural or partitioning properties cannot be selected or screened from libraries.
On the design of supramolecular assemblies made of peptides and lipid bilayers
Journal of Peptide Science, 2014
Peptides confer interesting properties to materials, supramolecular assemblies and to lipid membranes and are used in analytical devices or within delivery vehicles. Their relative ease of production combined with a high degree of versatility make them attractive candidates to design new such products. Here, we review and demonstrate how CD-and solid-state NMR spectroscopic approaches can be used to follow the reconstitution of peptides into membranes and to describe some of their fundamental characteristics. Whereas CD spectroscopy is used to monitor secondary structure in different solvent systems and thereby aggregation properties of the highly hydrophobic domain of p24, a protein involved in vesicle trafficking, solid-state NMR spectroscopy was used to deduce structural information and the membrane topology of a variety of peptide sequences found in nature or designed. 15 N chemical shift solid-state NMR spectroscopy indicates that the hydrophobic domain of p24 as well as a designed sequence of 19 hydrophobic amino acid residues adopt transmembrane alignments in phosphatidylcholine membranes. In contrast, the amphipathic antimicrobial peptide magainin 2 and the designed sequence LK15 align parallel to the bilayer surface. Additional angular information is obtained from deuterium solid-state NMR spectra of peptide sites labelled with 2 H 3 -alanine, whereas 31 P and 2 H solid-state NMR spectra of the lipids furnish valuable information on the macroscopic order and phase properties of the lipid matrix. Using these approaches, peptides and reconstitution protocols can be elaborated in a rational manner, and the analysis of a great number of peptide sequences is reviewed. Finally, a number of polypeptides with membrane topologies that are sensitive to a variety of environmental conditions such as pH, lipid composition and peptide-to-lipid ratio will be presented. /journal/jpepsci 15 N-Leu23]-p24TMD. (F) Deconvolution of the spectra shown in E provides positiondependent quadrupolar splittings and the corresponding deuterium order parameters S CD. The p24TMD peptide causes a small increase in several of the POPC-d 31 order parameters. The estimated error bar is <0.004 for S CD , which fits within the outline of the symbols used in panel E.
Acta Naturae, 2014
Lipid-protein nanodiscs (LPNs) are nanoscaled fragments of a lipid bilayer stabilized in solution by the apolipoprotein or a special membrane scaffold protein (MSP). In this work, the applicability of LPN-based membrane mimetics in the investigation of water-soluble membrane-active peptides was studied. It was shown that a pore-forming antimicrobial peptide arenicin-2 from marine lugworm (charge of +6) disintegrates LPNs containing both zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids. In contrast, the spider toxin VSTx1 (charge of +3), a modifier of Kv channel gating, effectively binds to the LPNs containing anionic lipids (POPC/DOPG, 3 : 1) and does not cause their disruption. VSTx1 has a lower affinity to LPNs containing zwitterionic lipids (POPC), and it weakly interacts with the protein component of nanodiscs, MSP (charge of -6). The neurotoxin II (NTII, charge of +4) from cobra venom, an inhibitor of the nicotinic acetylcholine receptor, shows...
Biomacromolecules, 2016
Self-assembling amphiphilic designer peptides have been successfully applied as nanomaterials in biomedical applications. Understanding molecular interactions at the peptide-membrane interface is crucial, since interactions at this site often determine (in)compatibility. The present study aims to elucidate how model membrane systems of different complexity (in particular single-component phospholipid bilayers and lipoproteins) respond to the presence of amphiphilic designer peptides. We focused on two short anionic peptides, V4WD2 and A6YD, which are structurally similar but showed a different self-assembly behavior. A6YD self-assembled into high aspect ratio nanofibers at low peptide concentrations, as evidenced by synchrotron small-angle X-ray scattering and electron microscopy. These supramolecular assemblies coexisted with membranes without remarkable interference. In contrast, V4WD2 formed only loosely associated assemblies over a large concentration regime, and the peptide pro...
Biochemistry, 2007
The effects of the hydrophobicity and the distribution of hydrophobic residues on the surfaces of some designed α-helical transmembrane peptides (acetyl-K 2 -L m -A n -K 2 -amide, where m + n = 24) on their solution behavior and interactions with phospholipids were examined. We find that although these peptides exhibit strong α-helix forming propensities in water, membrane-mimetic media, and lipid model membranes, the stability of the helices decreases as the Leu content decreases. Also, their binding to reversed phase high-performance liquid chromatography columns is largely determined by their hydrophobicity and generally decreases with decreases in the Leu/ Ala ratio. However, the retention of these peptides by such columns is also affected by the distribution of hydrophobic residues on their helical surfaces, being further enhanced when peptide helical hydrophobic moments are increased by clustering hydrophobic residues on one side of the helix. This clustering of hydrophobic residues also increases peptide propensity for self-aggregation in aqueous media and enhances partitioning of the peptide into lipid bilayer membranes. We also find that the peptides LA 3 LA 2 [acetyl-K 2 -(LAAALAA) 3 LAA-K 2 -amide] and particularly LA 6 [acetyl-K 2 -(LAAAAAA) 3 LAA-K 2 -amide] associate less strongly with and perturb the thermotropic phase behavior of phosphatidylcholine bilayers much less than peptides with higher L/A ratios. These results are consistent with free energies calculated for the partitioning of these peptides between water and phospholipid bilayers, which suggest that LA 3 LA 2 has an equal tendency to partition into water and into the hydrophobic core of phospholipid model membranes, whereas LA 6 should strongly prefer the aqueous phase. We conclude that for α-helical peptides of this type, Leu/Ala ratios of greater than 7/17 are required for stable transmembrane associations with phospholipid bilayers.
Biochemistry, 2001
The extent of matching of membrane hydrophobic thickness with the hydrophobic length of transmembrane protein segments potentially constitutes a major director of membrane organization. Therefore, the extent of mismatch that can be compensated, and the types of membrane rearrangements that result, can provide valuable insight into membrane functionality. In the present study, a large family of synthetic peptides and lipids is used to investigate a range of mismatch situations. Peptide conformation, orientation, and extent of incorporation are assessed by infrared spectroscopy, tryptophan fluorescence, circular dichroism, and sucrose gradient centrifugation. It is shown that peptide backbone structure is not significantly affected by mismatch, even when the extent of mismatch is large. Instead, this study demonstrates that for tryptophan-flanked peptides the dominant response of a membrane to large mismatch is that the extent of incorporation is reduced, when the peptide is both too short and too long. With increasing mismatch, a smaller fraction of peptide is incorporated into the lipid bilayer, and a larger fraction is present in extramembranous aggregates. Relatively long peptides that remain incorporated in the bilayer have a small tilt angle with respect to the membrane normal. The observed effects depend on the nature of the flanking residues: long tryptophan-flanked peptides do not associate well with thin bilayers, while equisized lysine-flanked peptides associate completely, thus supporting the notion that tryptophan and lysine interact differently with membrane-water interfaces. The different properties that aromatic and charged flanking residues impart on transmembrane protein segments are discussed in relation to protein incorporation in biological systems.
European Journal of Biochemistry, 2002
The interaction of many lytic cationic antimicrobial peptides with their target cells involves electrostatic interactions, hydrophobic effects, and the formation of amphipathic secondary structures, such as a helices or b sheets. We have shown in previous studies that incorporating % 30% D-amino acids into a short a helical lytic peptide composed of leucine and lysine preserved the antimicrobial activity of the parent peptide, while the hemolytic activity was abolished. However, the mechanisms underlying the unique structural features induced by incorporating D-amino acids that enable short diastereomeric antimicrobial peptides to preserve membrane binding and lytic capabilities remain unknown. In this study, we analyze in detail the structures of a model amphipathic a helical cytolytic peptide KLLLKWLL KLLK-NH 2 and its diastereomeric analog and their interactions with zwitterionic and negatively charged membranes. Calculations based on high-resolution NMR experiments in dodecylphosphocholine (DPCho) and sodium dodecyl sulfate (SDS) micelles yield three-dimensional structures of both peptides. Structural analysis reveals that the peptides have an amphipathic organization within both membranes. Specifically, the a helical structure of the L-type peptide causes orientation of the hydrophobic and polar amino acids onto separate surfaces, allowing interactions with both the hydrophobic core of the membrane and the polar head group region. Significantly, despite the absence of helical structures, the diastereomer peptide analog exhibits similar segregation between the polar and hydrophobic surfaces. Further insight into the membranebinding properties of the peptides and their depth of penetration into the lipid bilayer has been obtained through tryptophan quenching experiments using brominated phospholipids and the recently developed lipid/polydiacetylene (PDA) colorimetric assay. The combined NMR, FTIR, fluorescence, and colorimetric studies shed light on the importance of segregation between the positive charges and the hydrophobic moieties on opposite surfaces within the peptides for facilitating membrane binding and disruption, compared to the formation of a helical or b sheet structures.
Interaction of a pH-Responsive Designed Nanostructured Peptide with a Model Lipid Membrane
Journal of Nanoscience and Nanotechnology, 2016
Studying molecular interactions of soft biomolecular materials is a key step to the development of novel functional nanostructures with potential application in nanobiotechnology and nanomedicine. In current work we aimed to study the interaction of a rationally designed peptide whose degree of molecular self-association depends on pH, with model membranes employing Fourier transform infrared spectroscopy (FTIR). Two types of model lipid membranes were used, one formed by the DPPC, a lipid with a zwitterionic head group, and consequently electrically neutral, and the other type formed by the DPPG, a lipid with an anionic head group, and therefore negatively charged. The results showed that in the range of pH 6.0-8.0 the peptide P11-4 shows spectral features which are evidence of the presence of peptides with anti-parallel beta-sheet conformation (bands in the range 1625-1615 cm −1 and 1680-1685 cm −1 , as also spectral features which indicate the existence of random coil conformation (bands around 1644 cm −1. According to the results obtained by FTIR, it was found that the peptide had its behavior modulated by the interaction with the model membranes. It was observed that DPPG model lipid membranes caused an effect on P11-4 molecular organization state under different pH values, pH 6.0, 7.0 and 8.0, showing the most prominent effect on the basic environment, while the DPPC membranes practically did not affect the peptide self-assembling behavior. Such results are important for the development of novel biomolecular nanostructured materials by the physico-chemical understanding of self-organizing molecular systems.
Biophysical Journal, 2005
The thermotropic phase behavior and lateral structure of dipalmitoylphosphatidylcholine (DPPC) lipid bilayers containing an acylated peptide has been characterized by differential scanning calorimetry (DSC) on vesicles and atomic force microscopy (AFM) on mica-supported bilayers. The acylated peptide, which is a synthetic decapeptide N-terminally linked to a C 14 acyl chain (C 14-peptide), is incorporated into DPPC bilayers in amounts ranging from 0-20 mol %. The calorimetric scans of the two-component system demonstrate a distinct influence of the C 14-peptide on the lipid bilayer thermodynamics. This is manifested as a concentration-dependent downshift of both the main phase transition and the pretransition. In addition, the main phase transition peak is significantly broadened, indicating phase coexistence. In the AFM imaging scans we found that the C 14-peptide, when added to supported gel phase DPPC bilayers, inserts preferentially into preexisting defect regions and has a noticeable influence on the organization of the surrounding lipids. The presence of the C 14-peptide gives rise to a laterally heterogeneous bilayer structure with coexisting lipid domains characterized by a 10 Å height difference. The AFM images also show that the appearance of the ripple phase of the DPPC lipid bilayers is unaffected by the C 14-peptide. The experimental results are supported by molecular dynamics simulations, which show that the C 14-peptide has a disordering effect on the lipid acyl chains and causes a lateral expansion of the lipid bilayer. These effects are most pronounced for gel-like bilayer structures and support the observed downshift in the phase-transition temperature. Moreover, the molecular dynamics data indicate a tendency of a tryptophan residue in the peptide sequence to position itself in the bilayer headgroup region.