Peptides In Model Membranes Research Articles

NMR techniques for investigating antimicrobial peptides in model membranes and bacterial cells

AMPs are short, mainly cationic membrane-active peptides found in all living organism. They perform diverse roles including signaling and acting as a line of defense against bacterial infections. AMPs have been extensively investigated as templates to facilitate the development of novel antimicrobial therapeutics. Understanding the interplay between these membrane-active peptides and the lipid membranes is considered to be a significant step in elucidating the specific mechanism of action of AMPs against prokaryotic and eukaryotic cells to aid the development of new therapeutics. In this review, we have provided a brief overview of various NMR techniques commonly used for studying AMP structure and AMP-membrane interactions in model membranes and whole cells.

Integrin Facilitates the Internalization of TAT Peptide Conjugated to RGD Motif in Model Lipid Membranes.

In recent years, targeted drug delivery has attracted a great interest for enhanced therapeutic efficiency, with diminished side effects, especially in cancer therapy. Cell penetrating peptides (CPPs) like HIV1-TAT peptides, appear to be the perfect vectors for translocating drugs or other cargoes across the plasma membrane, but their application is limited mostly due to insufficient specificity for intended targets. Although these molecules were successfully used, the mechanism by which the peptides enter the cell interior still needs to be clarified. The tripeptide motif RGD (arginine-glycine-aspartate), found in extracellular matrix proteins has high affinity for integrin receptors overexpressed in cancer and it is involved in different phases of disease progression, including proliferation, invasion and migration. Discovery of new peptides with high binding affinity for disease receptors and permeability of plasma membranes is desirable for both, development of targeted drug delivery systems and early detection and diagnosis. To complement the TAT peptide with specific targeting ability, we conjugated it with an integrin-binding RGD motif. Although the idea of RGD-CPPs conjugates is not entirely new,[1] here we describe the permeability abilities and specificity of integrin receptors of RGD-TAT peptides in model membranes. Our findings reveal that this novel RGD sequence based on TAT peptide maintains its ability to permeate lipid membranes and exhibits specificity for integrin receptors embedded in giant unilamellar vesicles. This promising outcome suggests that the RGD-TAT peptide has significant potential for applications in the field of targeted drug delivery systems.

Membrane permeabilization can be crucially biased by a fusogenic lipid composition - leaky fusion caused by antimicrobial peptides in model membranes.

Induced membrane permeabilization or leakage is often taken as an indication for activity of membrane-active molecules, such as antimicrobial peptides (AMPs). The exact leakage mechanism is often unknown, but important, because certain mechanisms might actually contribute to microbial killing, while others are unselective, or potentially irrelevant in an in vivo situation. Using an antimicrobial example peptide (cR3W3), we illustrate one of the potentially misleading leakage mechanisms: leaky fusion, where leakage is coupled to membrane fusion. Like many others, we examine peptide-induced leakage in model vesicles consisting of binary mixtures of anionic and zwitterionic phospholipids. In fact, phosphatidylglycerol and phosphatidylethanolamine (PG/PE) are supposed to reflect bacterial membranes, but exhibit a high propensity for vesicle aggregation and fusion. We describe the implications of this vesicle fusion and aggregation for the reliability of model studies. The ambiguous role of the relatively fusogenic PE-lipids becomes clear as leakage decreases significantly when aggregation and fusion are prevented by sterical shielding. Furthermore, the mechanism of leakage changes if PE is exchanged for phosphatidylcholine (PC). We thus point out that the lipid composition of model membranes can be biased towards leaky fusion. This can lead to discrepancies between model studies and activity in true microbes, because leaky fusion is likely prevented by bacterial peptidoglycan layers. In conclusion, choosing the model membrane might implicate the type of effect (here leakage mechanism) that is observed. In the worst case, as with leaky fusion of PG/PE vesicles, this is not directly relevant for the intended antimicrobial application.

Mismatch Dependent Tilt of Amphipathic α-Helical Antimicrobial Peptides Inserted in Positive Spontaneous Curvature Lipid Membranes

We have used solid state NMR to study the orientation of membrane-active peptides in model membranes with different properties. We found that the peptide behavior can to a large extent be explained by the simple biophysical concepts of hydrophobic matching and lipid spontaneous curvature. Hydrophobic peptides insert in the membrane in a transmembrane orientation, and it is well established that peptides which have a longer hydrophobic stretch than the bilayer thickness start to tilt to compensate the hydrophobic mismatch. Here we show that the hydrophobic matching principle also applies to cationic amphipathic α-helical antimicrobial peptides. Using a series of peptides called KIAn, based on the repetitive amino acid sequence KIAGKIA, we found that only peptides long enough to span the hydrophobic thickness of the membrane induce leakage in lipid vesicles. Interestingly, a certain minimum length was also needed to induce hemolysis and to kill bacteria, and this minimum length was different for different bacterial strains, so that the series of peptides can be used as a “molecular ruler” to determine the thickness of erythrocyte or bacterial membranes in vivo. Using solid-state NMR we found that the orientation depends strongly on the lipid spontaneous curvature. The KIA peptides are always staying flat on the membrane surface in lipids with a negative spontaneous curvature. On the other hand, in lipids with a positive spontaneous curvature, like lyso-lipids, they can go deeper into the membrane and have a tilted or even inserted orientation. In the inserted state, the tilt of the peptides is found to be mismatch dependent. This is the first time a mismatch effect has been found for amphipathic peptides, and shows that the effect is more generally valid than has been appreciated until now.

The cooperative behaviour of antimicrobial peptides in model membranes

A systematic analysis of the hypothesis of the antimicrobial peptides' (AMPs) cooperative action is performed by means of full atomistic molecular dynamics simulations accompanied by circular dichroism experiments. Several AMPs from the aurein family (2.5,2.6, 3.1), have a similar sequence in the first ten amino acids, are investigated in different environments including aqueous solution, trifluoroethanol (TFE), palmitoyloleoylphosphatidylethanolamine (POPE), and palmitoyloleoylphosphatidylglycerol (POPG) lipid bilayers. It is found that the cooperative effect is stronger in aqueous solution and weaker in TFE. Moreover, in the presence of membranes, the cooperative effect plays an important role in the peptide/lipid bilayer interaction. The action of AMPs is a competition of the hydrophobic interactions between the side chains of the peptides and the hydrophobic region of lipid molecules, as well as the intra peptide interaction. The aureins 2.5-COOH and 2.6-COOH form a hydrophobic aggregate to minimize the interaction between the hydrophobic group and the water. Once that the peptides reach the water/lipid interface the hydrophobic aggregate becomes smaller and the peptides start to penetrate into the membrane. In contrast, aurein 3.1-COOH forms only a transient aggregate which disintegrates once the peptides reached the membrane, and it shows no cooperativity in membrane penetration.

Aggregation of Alzheimer’s Amyloid β-Peptide in Biological Membranes: A Molecular Dynamics Study

Numerous studies have concluded that the interaction of the amyloid β-peptide (Aβ) and cellular membranes contributes to the toxicity and cell death observed in the progression of Alzheimer's disease. Aggregated Aβ species disrupt membranes, leading to physical instability and ion leakage. Further, the presence of Aβ on the membrane surface increases the aggregation rate of the peptide, as diffusion occurs in two dimensions, increasing the probability of interpeptide interactions. Molecular dynamics (MD) simulations have been used to investigate Aβ in a number of environments, including aqueous solution and membranes. We previously showed that monomeric Aβ40 remains embedded in membranes composed of the most common lipids found in the cell membrane, but that the presence of ganglioside GM1 promotes release of the peptide into the extracellular medium. Here, we explore the interactions of two Aβ40 peptides in model membranes to understand whether aggregation can occur prior to the release of the peptide into the aqueous environment. We found that aggregation occurred, to different extents, in each of the model membranes and that the aggregates, once formed, did not exit the membrane environment. This information may have important implications for understanding the affinity of Aβ for membranes and the mechanism of Aβ toxicity in Alzheimer's disease.

What Determines the Activity of Antimicrobial and Cytolytic Peptides in Model Membranes

We previously proposed three hypotheses relating the mechanism of antimicrobial and cytolytic peptides in model membranes to the Gibbs free energies of binding and insertion into the membrane [Alme...

What Determines the Activity of Antimicrobial and Cytolytic Peptides in Model Membranes

We previously proposed three hypotheses relating the mechanism of antimicrobial and cytolytic peptides in model membranes to the Gibbs free energies of binding and insertion into the membrane [Almeida, P. F., and Pokorny, A. (2009) Biochemistry 48, 8083-8093]. Two sets of peptides were designed to test those hypotheses, by mutating of the sequences of δ-lysin, cecropin A, and magainin 2. Peptide binding and activity were measured on phosphatidylcholine membranes. In the first set, the peptide charge was changed by mutating basic to acidic residues or vice versa, but the amino acid sequence was not altered much otherwise. The type of dye release changed from graded to all-or-none according to prediction. However, location of charged residues in the sequence with the correct spacing to form salt bridges failed to improve binding. In the second set, the charged and other key residues were kept in the same positions, whereas most of the sequence was significantly but conservatively simplified, maintaining the same hydrophobicity and amphipathicity. This set behaved completely different from predicted. The type of release, which was expected to be maintained, changed dramatically from all-or-none to graded in the mutants of cecropin and magainin. Finally, contrary to the hypotheses, the results indicate that the Gibbs energy of binding to the membrane, not the Gibbs energy of insertion, is the primary determinant of peptide activity.

The Interfacial Behaviour of Peptides Over Long Time Scales

Peptides are frequently used as models for membrane proteins due to their accessibility and low molecular weight, both of which facilitate the interpretation of complex spectroscopic data. Membrane proteins are recycled in vivo with half-lives ranging from minutes to days. It is therefore of interest to examine the behaviour of peptides in model membranes over extended periods in order to determine whether these systems reach equilibrium and remain stable. This timescale is seldom examined in studies using model peptides.

A Thermodynamic Approach to the Mechanism of Cell-Penetrating Peptides in Model Membranes

We report a first test of the hypothesis that the mechanism of antimicrobial, cytolytic, and amphipathic cell-penetrating peptides in model membranes is determined by the thermodynamics of insertion of the peptide into the lipid bilayer from the surface-associated state. Three peptides were designed with minimal mutations relative to the sequence of TP10W, the Y3W variant of transportan 10, which is a helical, amphipathic cell-penetrating peptide previously studied. Binding to 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes and release of dye from those vesicles were assessed by stopped-flow fluorescence, and the secondary structure of the peptides on the membrane was determined by circular dichroism. The Gibbs energy of binding determined experimentally was in excellent agreement with that calculated using the Wimley-White interfacial hydrophobicity scale, taking into account the helical content of the membrane-associated peptide. Release of dye from POPC vesicles remained graded, as predicted by the hypothesis. More significantly, as the Gibbs energy of insertion into the bilayer became more unfavorable, which was estimated using the Wimley-White octanol hydrophobicity scale, dye release became slower, in quantitative agreement with the prediction.

Global Bilayer Properties can Modulate Membrane Protein Oligomerization

While sequence dependent oligomerization of individual transmembrane α-helices has been studied to some extent in the recent years, the influence of the lipid bilayer properties on defined helix-helix interactions remains largely uncharacterized. To study the potential impact of changing bilayer properties on a defined transmembrane helix-helix interaction we have followed association of fluorescently labeled glycophorin A transmembrane peptides in model membranes by fluorescence spectroscopy. Changes in Forster resonance energy transfer strongly suggest that the lipid bilayer thickness does significantly influence the monomer-dimer equilibrium of the transmembrane domain. Furthermore, the presence of cholesterol in model membranes promotes self-association of transmembrane helices by modulating the bilayer thickness and -more importantly- by affecting lipid acyl chain ordering. In addition, changes in the lipid composition, which modulate lipid bilayer curvature elasticity and the lateral pressure profile, affect GpA dimerization. In conclusion, the findings show that the physical state of a membrane can be critically involved in controlling specific and promiscuous interactions of α-helical transmembrane domains, as e. g. involved in membrane protein folding and assembly as well as in transmembrane signaling.

Oriented Synchrotron Radiation Circular Dichroism and Linear Dichroism Spectroscopy of Peptides in Model Membranes

The orientation of membrane-associated alpha-helical peptides was investigated using novel methodologies of oriented Synchrotron Radiation Circular Dichroism (SRCD) and linear dichroism (SRLD) spectroscopies. Because of its enhanced signal-to-noise and a detector geometry that minimised optical artefacts associated with conventional CD studies of membrane suspensions, SRCD enabled the measurement of oriented CD spectra. To accomplish this a specially-designed sample cell holder was produced which would maintain constant humidity in hydrated film samples.

Translocon‐Assisted Folding of Membrane Proteins: New Insights into Lipid‐Protein Interactions

Recent studies of the translocon-assisted folding of membrane proteins have revealed two unexpected findings about the insertion of transmembrane helices across the endoplasmic reticulum membrane. First, the so-called S4 voltage-sensor helix of potassium channels, comprised of hydrophobic residues and four arginine residues, can be inserted. Second, polyleucine helices as short as 10 residues are readily inserted. Exploration of these observations using physical studies of synthetic peptides in model membranes and molecular dynamics simulations provide new insights into lipid-protein interactions. They reveal that the lipid bilayer is far more complex—and interesting—than its usual lollypop cartoon suggests. The biological, physical, and molecular dynamics data to be presented demonstrate the extreme adaptability of phospholipids that arises from the privileged relationship between their phosphate groups and lysine and arginine residues. This adaptability makes possible the transmembrane insertion of very short helices and the independent stability of potassium channel voltage-sensor domains in membranes. [Research supported by the National Institute of General Medical Sciences and the National Center for Research Resources.]

Palmitoylation and Intracellular Domain Interactions Both Contribute to Raft Targeting of Linker for Activation of T Cells

Some transmembrane proteins must associate with lipid rafts to function. However, even if acylated, transmembrane proteins should not pack well with ordered raft lipids, and raft targeting is puzzling. Acylation is necessary for raft targeting of linker for activation of T cells (LAT). To determine whether an acylated transmembrane domain is sufficient, we examined raft association of palmitoylated and nonpalmitoylated LAT transmembrane peptides in lipid vesicles by a fluorescence quenching assay, by microscopic examination, and by association with detergent-resistant membranes (DRMs). All three assays detected very low raft association of the nonacylated LAT peptide. DRM association was the same as a control random transmembrane peptide. Acylation did not measurably enhance raft association by the first two assays but slightly enhanced DRM association. The palmitoylated LAT peptide and a FLAG-tagged LAT transmembrane domain construct expressed in cells showed similar DRM association when both were reconstituted into mixed vesicles (containing cell-derived proteins and lipids and excess artificial raft-forming lipids) before detergent extraction. We conclude that the acylated LAT transmembrane domain has low inherent raft affinity. Full-length LAT in mixed vesicles associated better with DRMs than the peptide. However, cells appeared to contain two pools of LAT, with very different raft affinities. Since some LAT (but not the transmembrane domain construct) was isolated in a protein complex, and the Myc- and FLAG-tagged forms of LAT could be mutually co-immunoprecipitated, oligomerization or interactions with other proteins may enhance raft affinity of one pool of LAT. We conclude that both acylation and other factors, possibly protein-protein interactions, target LAT to rafts.

Farnesylated peptides in model membranes: a biophysical investigation

Commentary : Farnesylated peptides in model membranes: a biophysical investigation (vol 33, pg 300, 2003)

Farnesylated peptides in model membranes: a biophysical investigation.

Protein prenylation plays an important role in signal transduction, protein-protein interactions, and the localization and association of proteins with membranes. Using three different techniques, this study physically characterizes the interactions between model dimyristoylphosphatidylcholine membranes and a series of farnesylated peptides. Magic angle spinning nuclear Overhauser enhancement spectroscopy and differential scanning calorimetry reveal that both charged [Ac-Asn-Lys-Asn-Cys-(farnesyl)-OMe and Ac-Asn-Lys-Asn-Cys-(farnesyl)-NH(2)] and uncharged [Ac-Cys-(farnesyl)-OMe and farnesol] species partition into dimyristoylphosphatidylcholine bilayers. Calorimetry and vesicle fluctuation analysis of giant unilamellar vesicles show that the charged peptides modestly decrease the main gel-fluid phase transition and markedly increase the bending rigidity of large unilamellar vesicles. Uncharged species, on the other hand, dramatically decrease the main phase transition and modestly decrease the bending rigidity. No difference with carboxyl methylation is detected.

Phospholipid flop induced by transmembrane peptides in model membranes is modulated by lipid composition.

Since phospholipid synthesis is generally confined to one leaflet of a membrane, membrane growth requires phospholipid translocation (flip-flop). It is generally assumed that this process is protein-mediated; however, the mechanism of flip-flop remains elusive. Previously, we have demonstrated flop of 2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl] (C6NBD) phospholipids, induced by the presence of membrane-spanning peptides in vesicles composed of an Escherichia coli phospholipid extract, supporting the hypothesis that the presence of transmembrane stretches of proteins in the bilayer is sufficient to allow phospholipid flip-flop in the inner membrane of E. coli [Kol et al. (2001) Biochemistry 40, 10500]. Here, we investigated whether the specific phospholipid composition of E. coli is a prerequisite for transmembrane helix-induced flop of phospholipids. This was tested by determining the amount of C6NBD-phospholipid that was translocated from the inner leaflet to the outer leaflet of a model membrane in time, using a dithionite reduction assay. The transmembrane peptides GWWL(AL)8WWA (WALP23) and GKKL(AL)8KKA (KALP23) induced phospholipid flop in model membranes composed of various lipid mixtures. The rate of peptide-induced flop was found to decrease with increasing dioleoylphosphatidylethanolamine (DOPE) content of vesicles composed of DOPE and dioleoylphosphatidylcholine (DOPC), and the rate of KALP23-induced flop was shown to be stimulated by higher dioleoylphosphatidylglycerol (DOPG) content in model membranes composed of DOPG and DOPC. Furthermore, the incorporation of cholesterol had an inhibitory effect on peptide-induced flop. Finally, flop efficiency was strongly dependent on the phospholipid headgroup of the NBD-phospholipid analogue. Possible implications for transmembrane helix-induced flop in biomembranes in general are discussed.

Hydrogen/deuterium exchange of hydrophobic peptides in model membranes by electrospray ionization mass spectrometry

We demonstrate here that the hydrogen/deuterium solvent exchange (HDX) properties of the transmembrane fragment of the M2 protein of Influenza A (M2-TM) incorporated into lipid vesicles or detergent micelles can be studied with straightforward electrospray (ESI) and nanospray mass spectrometry (MS) configurations provided that key factors, including sample preparation techniques, are optimized. Small unilamellar vesicle preparations were obtained by solubilizing dimyristoyl phosphatidylcholine (DMPC) and the M2-TM peptide in aqueous solution with n-octyl-β-D-glycopyranoside, followed by dialysis to remove the detergent. Electron microscopy experiments revealed that subsequent concentration by centrifugation introduced large multilamellar aggregates that were not compatible with ESI-MS. By contrast, a lyophilization-based concentration procedure, followed by thawing above the liquid crystal transition temperature of the lipid component, maintained the liposome size profile and yielded excellent ion fluxes in both ESI-MS and nano-ESI-MS. Using these methods the global HDX profile of M2-TM in aqueous DMPC vesicles was compared with that in methanol, demonstrating that several amide sites were protected from exchange by the lipid membrane. We also show that hydrophobic peptides can be detected by ESI-MS in the presence of a large molar excess of the detergent Triton X-100. The rate of HDX of M2-TM in Triton X-100 micelles was faster than that in DMPC vesicles but slower than when the peptide had been denatured in methanol. These results indicate that the accessibility of backbone amide sites to the solvent can be profoundly affected by membrane protein structure and dynamics, as well as the properties of model bilayer systems.

Influence of hydrophobic mismatch and palmitoylation on the association of transmembrane α-helical peptides with detergent-resistant membranes

The aim of this study was to gain insight into the mechanism through which transmembrane proteins are targeted to liquid ordered (L o) phase domains or rafts. This was investigated by analyzing the Triton X-100 resistance of designed transmembrane peptides in model membranes of 1,2-dioleoyl- sn-glycero-3-phosphocholine, sphingomyelin and cholesterol (1/1/1, molar ratio), which contain both L o phase domains and fluid bilayers. By using peptides with one or two palmitate chains covalently linked to their N-terminus or with variable hydrophobic lengths, the roles of protein palmitoylation and of mismatch between the transmembrane segment of the protein and the bilayer thickness, respectively, were investigated. The results show that neither hydrophobic matching nor palmitoylation is sufficient for partitioning of peptides into L o phase domains. It is concluded that the L o phase itself, due to the tight packing of the lipids, constitutes an unfavorable environment for accommodation of protein transmembrane segments.

Antimicrobial peptides in model membranes studied by in-plane neutron scattering

Antimicrobial peptides in model membranes studied by in-plane neutron scattering