Peptide-membrane Interactions Research Articles

Conjugation of Cholesterol to HIV-1 Fusion Inhibitor C34 Increases Peptide-Membrane Interactions Potentiating Its Action

Recently, the covalent binding of a cholesterol moiety to a classical HIV-1 fusion inhibitor peptide, C34, was shown to potentiate its antiviral activity. Our purpose was to evaluate the interaction of cholesterol-conjugated and native C34 with membrane model systems and human blood cells to understand the effects of this derivatization. Lipid vesicles and monolayers with defined compositions were used as model membranes. C34-cholesterol partitions more to fluid phase membranes that mimic biological membranes. Importantly, there is a preference of the conjugate for liquid ordered membranes, rich in cholesterol and/or sphingomyelin, as observed both from partition and surface pressure studies. In human erythrocytes and peripheral blood mononuclear cells (PBMC), C34-cholesterol significantly decreases the membrane dipole potential. In PBMC, the conjugate was 14- and 115-fold more membranotropic than T-1249 and enfuvirtide, respectively. C34 or cholesterol alone did not show significant membrane activity. The enhanced interaction of C34-cholesterol with biological membranes correlates with its higher antiviral potency. Higher partitions for lipid-raft like compositions direct the drug to the receptor-rich domains where membrane fusion is likely to occur. This intermediary membrane binding step may facilitate the drug delivery to gp41 in its pre-fusion state.

The Addressing Fragment of Mitogaligin: First Insights into Functional and Structural Properties

Mitogaligin is a mitochondrion-targeting protein involved in cell death. The sequence of the protein is unrelated to that of any known pro- or antiapoptotic protein. Mitochondrial targeting is controlled by an internal sequence from residues 31 to 53, and although this sequence is essential and sufficient to provoke cell death, the precise mechanism of action at the mitochondrial membrane remains to be elucidated. Here, by focusing on the [31-53] fragment, we first assessed and confirmed its cell cytotoxicity by microinjection. Subsequently, with the aid of membrane models, we evaluated the impact of the membrane environment on the 3D structure of the peptide and on how the peptide is embedded and oriented within membranes. The fragment is well organized, even though it does not contain a canonical secondary structure, and adopts an interfacial location. Structural comparison with other membrane-interacting Trp-rich peptides demonstrated similarities with the antimicrobial peptide tritrpcidin.

Biomolecular engineering of a human beta defensin model for increased salt resistance

Human beta defensins (hBDs) are natural antimicrobial peptides (AMPs) with broad spectrum antimicrobial activity. However, hBDs, like many AMPs, are easily inactivated by salt, which limits their extracellular applications as antimicrobial coating agents. In this study, a salt-resistant hBD28 peptide was designed by increasing C-terminus cationicity of the wild type peptide via rational amino acid substitution. The mutant hBD28 exhibited salt-tolerance behaviour and improved antimicrobial potency compared to wild type hBD28. Zeta potential analysis confirmed that increased cationicity was crucial to overcome salt-induced charge-shielding effects, which enhanced peptide–membrane interaction compared to the wild type peptide. The mutant hBD28 did not exhibit obvious differences with respect to hydrophobicity, oligomerization ability, and secondary structure compared to the wild type peptide. A simple design strategy to overcome salt-inactivation in hBD28 is demonstrated through this study, which will guide the design of other salt-resistant AMPs to accelerate their development as anti-infective agents in ionic environments.

Perfluoro‐tert‐butyl‐homoserine as a sensitive 19F NMR reporter for peptide–membrane interactions in solution

Fluorine ((19)F) NMR is a valuable tool for studying dynamic biological processes. However, increasing the sensitivity of fluorinated reporter molecules is a key to reducing acquisition times and accessing transient biological interactions. Here, we evaluate the utility a novel amino acid, L-O-(perfluoro-t-butyl)-homoserine (pFtBSer), that can easily be synthesized and incorporated into peptides and provides greatly enhanced sensitivity over currently used (19)F biomolecular NMR probes. Incorporation of pFtBSer into the potent antimicrobial peptide MSI-78 results in a sharp (19)F NMR singlet that can be readily detected at concentrations of 5 µm and lower. We demonstrate that pFtBSer incorporation into MSI-78 provides a sensitive tool to study binding through (19)F NMR chemical shift and nuclear relaxation changes. These results establish future potential for pFtBSer to be incorporated into various proteins where NMR signal sensitivity is paramount, such as in-cell investigations.

Membrane defects enhance the interaction of antimicrobial peptides, aurein 1.2 versus caerin 1.1

The membrane interactions of the antimicrobial peptides aurein 1.2 and caerin 1.1 were observed by 31P and 2H solid-state NMR and circular dichroism spectroscopy. Both peptides were relatively unstructured in water. In the presence of dimyristoylphosphatidylcholine (DMPC) and mixed DMPC and dimyristoylphosphatidylglycerol (DMPG) vesicles, both peptides displayed a considerable increase in helical content with the shorter aurein peptide having a higher α-helix content in both lipid systems. In fluid phase DMPC vesicles, the peptides displayed differential interactions: aurein 1.2 interacted primarily with the bilayer surface, while the longer caerin 1.1 was able to penetrate into the bilayer interior. Both peptides displayed a preferential interaction with the DMPG component in DMPC/DMPG bilayers, with aurein 1.2 limited to interaction with the surface and caerin 1.1 able to penetrate into the bilayer and promote formation of a mixture of lipid phases or domains. In gel phase DMPC vesicles, aurein 1.2 disrupted the bilayer apparently through a carpet mechanism, while no additional interaction was seen with caerin 1.1. Although a lamellar bilayer was retained with the mixed DMPC/DMPG vesicles below the phase transition, both caerin 1.1 and aurein 1.2 promoted disruption of the bilayer and formation of an isotropic phase. The peptide interaction was enhanced relative to the fluid phase and was likely driven by co-existence of membrane defects. This study thus demonstrates that the effects of the lipid phase and domains need to be considered when studying membrane interactions of antimicrobial peptides.

Rational design of cationic antimicrobial peptides by the tandem of leucine-rich repeat

Antimicrobial peptides represent ancient host defense effector molecules present in organisms across the evolutionary spectrum. Lots of antimicrobial peptides were synthesized based on well-known structural motif widely existed in a variety of lives. Leucine-rich repeats (LRRs) are sequence motifs present in over 60,000 proteins identified from viruses, bacteria, and eukaryotes. To elucidate if LRR motif possesses antimicrobial potency, two peptides containing one or two LRRs were designed. The biological activity and membrane-peptide interactions of the peptides were analyzed. The results showed that the tandem of two LRRs exhibited similar antibacterial activity and significantly weaker hemolytic activity against hRBCs than the well-known membrane active peptide melittin. The peptide with one LRR was defective at antimicrobial and hemolytic activity. The peptide containing two LRRs formed α-helical structure, respectively, in the presence of membrane-mimicking environment. LRR-2 retained strong resistance to cations, heat, and some proteolytic enzymes. The blue shifts of the peptides in two lipid systems correlated positively with their biological activities. Other membrane-peptide experiments further provide the evidence that the peptide with two LRRs kills bacteria via membrane-involving mechanism. The present study increases our new understanding of well-known LRR motif in antimicrobial potency and presents a potential strategy to develop novel antibacterial agents.

Impact of pH on ultrafiltration membrane selectivity during electrodialysis with ultrafiltration membrane (EDUF) purification of soy peptides from a complex matrix

The aim of the present study was to investigate the influence of pH on the separation of a pepsin-pancreatin soy proteins isolate (SPI) hydrolysate, by electrodialysis with ultrafiltration membranes (EDUF) and particularly the influence of pH on ultrafiltration (UF) membrane selectivity. In this context, changes in conductivity, UF and ion-exchange membranes (IEM) electric resistance and peptide migration rates were monitored during a 4h-EDUF treatment, under three controlled pH conditions (3, 6 and 9).According to these results, the pH value was demonstrated to influence the composition of recovery compartments, but not the migration rate of peptides. However, in contrary to the general accepted idea, even if pH was increased, migration rate in anionic peptides recovery compartment (KCl 1) was stable (no significant effect of pH was reported). As proposed in a tentative model, modification of the UF membrane global charge affected the peptide–membrane interactions which may limit peptide migration to the anionic peptides recovery compartment (KCl 1). Finally, pH modulation appeared to be an efficient way to concentrate the low molecular weight peptides (<400Da) in the cationic peptides recovery compartment (KCl 2), and to limit the diversity of peptides recovered in the anionic peptides recovery compartment.

Effects of Lipid Membrane Curvature on Lipid Packing State Evaluated by Isothermal Titration Calorimetry

In this report, we present a novel approach for the elucidation of the physicochemical properties of lipid membranes by isothermal titration calorimetry (ITC) to quantify the heat absorbed during the solubilization of vesicles into TritonX-100 micelles. By using large and small unilamellar vesicles for comparison, this method provides calorimetric data on the gel-to-liquid-crystalline phase transition and its curvature effects and, in particular, the enthalpy change upon membrane deformation from a planar to a curved shape, which cannot be obtained by the conventional approach using differential scanning calorimetry. The results showed quantitatively that the increase in membrane curvature increases the enthalpy of 1,2-dimyristoyl-sn-glycero-3-phosphocholine membranes both below and above the phase-transition temperature, and that the effect is more significant for the former condition. The calorimetric data obtained are further discussed in relation to the elastic bending energy of the membranes and membrane-peptide interaction.

Effect of Curcumin on Peptide-Membrane Interaction: In Kinetics and in Equilibrium

We performed a three-chamber aspirated GUV experiment to study the competitive binding between curcumin and melittin. GUVs were produced and aspirated in chamber 1. Chamber 2 contained 10 microM curcumin. Chamber 3 contained 10 microM curcumin and various concentrations of peptides: penetratin, melittin or magainin. Aspirated GUVs were first equilibrated in chamber 2 and then transferred to chamber 3, where we observed the response of the GUV to the addition of peptides. Without the peptides in chamber 3, the GUV should remain unchanged. The results can be described as the peptides replacing curcumin bound on the bilayer interface and shifting curcumin into the hydrocarbon region, except for magainin. The bound curcumin essentially blocked magainin from binding to the membranes. This can be explained as a kinetic effect because the effect size of a bound magainin is twice the effective size of a bound curcumin, whereas the effective sizes of penetratin and melittin are comparable to curcumin (this is an usual property of curcumin for its small molecular size). Thus for penetratin and melittin, the behavior of the peptides is the same with or without curcumin. On the other hand, the curcumin/peptide/lipid mixtures produce bilayers totally unlike either curcumin with lipid or peptide with lipid. Instead of steady membrane thinning by either melittin or curcumin alone, the addition of melittin to curcumin/DOPC actually thickening the bilayers. This is the first example of incompatible results from equilibrium experiment and from kinetic experiment. It implies that competitive binding of two different membrane-active molecules can be complicated, a caution for drawing conclusion about synergy effects.

Dendrimers functionalized with membrane-interacting peptides for viral inhibition

This contribution reports the synthesis of a poly(amide)-based dendrimer functionalized at the termini with a membrane-interacting peptide derived from the herpes simplex virus (HSV) type 1 glycoprotein H, namely gH625–644. This peptide has been shown to interact with model membranes and to inhibit viral infectivity. The peptidodendrimer inhibits both HSV-1 and HSV-2 at a very early stage of the entry process, most likely through an interaction with the viral envelope glycoproteins; thus, preventing the virus from coming into close contact with cellular membranes, a prerequisite of viral internalization. The 50% inhibitory concentration was 100 and 300 nM against HSV-1 and HSV-2 respectively, with no evidence of cell toxicity at these concentrations. These results show that the functionalization of a dendrimer with the peptide sequence derived from an HSV glycoprotein shows promising inhibitory activity towards viruses of the Herpesviridae family.

Balance between Additional Van Der Waals and Electrostatic Components of 107-115 hLZ Synergistically Increase Peptide-Membrane Interactions Potentiating its Antimicrobial Activity

Substitution of Ala 108 and Ala 111 in the 107-115 human lysozyme peptide results in a 20 fold increase in the anti-staphylococcal activity and decrease its hemolytic activity which becomes significant only at 10 fold its MIC. This analog displays an additional positive charge near the N-terminus (108) and an extra Trp residue at the center of the molecule (111), suggesting that this constellation improves its interaction with the bacterial membrane. In order to understand the role of this arrangement in the membrane interaction, studies with model lipids membranes were carried out. The interaction of both peptides, 107-115 hLz and the novel analog ([K108 W111]107-115 hLz) were evaluated on liposomes by monitoring the fluorescence changes of its Trp residues and on lipid monolayers by monitoring the variation of the surface pressure. Results from both techniques revealed significant increase of lipid affinity of [K108 W111]107-115 hLz, specially in the presence of negatively charged lipid as POPG, but also a significant interaction with zwiterionics lipids that suggest that not only the electrostatic force are involved in the affinity increase. The peptides locations into membranes were also studied by fluorescence quenching techniques without any significant difference between both peptides, suggesting that the enhanced in the antimicrobial activity might be ascribed to an increased affinity. All together the data suggest that the enhanced activity of peptide [K108 W111]107-115 hLz might be explained by a synergy effect between the increased electrostatic forces (substitution of A by K at position 108) as well as the increased hydrophobic interactions (substitution of A by W at position 111). The substitutions might stabilize the peptide in the membrane.

Peptide-Induced Bilayer Thinning Structure of Unilamellar Vesicles and the Related Binding Behavior as Revealed by X-ray Scattering

To quantitatively correlate membrane thinning with peptide binding affinity, we have studied the bilayer thinning structure of unilamellar vesicles (ULV) of a phospholipid 1,2-dierucoyl-sn-glycero-3-phosphocholine (di22:1PC) upon binding of melittin, a water-soluble amphipathic peptide. Successive thinning of the ULV bilayers with increasing peptide concentration was monitored via small-angle X-ray scattering (SAXS). Results suggest that the two leaflets of the ULV of closed bilayers are perturbed and thinned asymmetrically upon free peptide binding, in contrast to the centro-symmetric bialyer thinning of the substrate-oriented multilamellar membranes (MLM) with premixed melittin. Moreover, thinning of the melittin-ULV bilayer saturates at ∼4 %, significantly lower than the critical thinning of ∼8% (determined via the correspondingly premixed peptide-MLM bilayers) for thermally equilibriated formation of membrane pores, revealing a critical influence of binding affinity for water soluble peptides. Scaling the peptide-ULV bilayer thinning to that of the corresponding peptide-MLM, of a calibrated peptide-to-lipid ratio, we have deduced the number of bound peptides on the ULV bilayers as a function of free peptide concentration in solution. The hence derived X-ray-based binding isotherm allows extraction of a low binding constant for melittin to the ULV bilayers, on the basis of surface partition equilibrium and the Gouy-Chapman theory. Moreover, we show that the ULV and MLM bilayers of di22:1PC may have a same thinning constant upon binding of a same peptide, hence providing a basis in establishing X-ray-based binding isotherms for thermodynamic binding parameters of late stage peptide-membrane interactions prior to pore formation.

Rapid Cytotoxicity Screening Platform for Amyloid Inhibitors Using a Membrane-Potential Sensitive Fluorescent Probe

The growing interest in membrane interactions of amyloidogenic proteins indicates that lipid binding and the regulation of membrane potential are critical to the onset and progression of neurodegenerative diseases such as Parkinson's (PD), Alzheimer's (AD), and prion diseases. Advancing the understanding of this field requires the application of varied biophysical and biological techniques designed to probe the characteristics and underlying mechanisms of membrane-peptide interactions. Therefore, the development of a rapid cytotoxicity evaluation system using a membrane potential-sensitive bis-oxonol fluorescent dye, DiBAC4(3) is reported here. The exposure of C-terminal truncated α-synuclein 119 (α-Syn119) and amyloid-β(1-42) (Aβ(1-42)) to U2-OS cell cultures resulted in an immediate, significant, and concentration-dependent increase in fluorescence response of DiBAC4(3). This response was strongly correlated with the cytotoxicity of α-Syn119 and Aβ(1-42) as determined by conventional CC8 and ATP assays. Furthermore, the capacity of well-defined polyphenolic antioxidants (i.e., pyrroloquinoline quinone (PQQ), baicalein, (-)-epigallocatechin-3-gallate (EGCG), and myricetin) to mitigate amyloid-induced cytotoxicity was evaluated using the developed biosensing system. We envisage that this work would accelerate the development of a rapid and cost-effective high-throughput screening platform in drug discovery for AD and PD.

Microscopic Rates of Peptide–Phospholipid Bilayer Interactions from Single-Molecule Residence Times

The binding of glucagon-like peptide-1 (GLP-1) to a planar phospholipid bilayer is measured using single-molecule total internal reflection fluorescence microscopy. From several reports in the literature, GLP-1 has been shown to be a random coil in free solution, adopting a folded, α-helix conformation when intercalated into membrane environments. Single-molecule fluorescence measurements of GLP-1 binding to supported lipid bilayers show evidence of two populations of membrane-associated molecules having different residence times, suggesting weakly adsorbed peptides and strongly bound peptides in the lipid bilayer. The path to and from a strongly bound (folded, intercalated) state would likely include an adsorbed state as an intermediate, so that the resulting kinetics would correspond to a consecutive first-order reversible three-state model. In this work, the relationships between measured single-molecule residence times and the microscopic rates in a three-state kinetic model are derived and used to interpret the binding of GLP-1 to a supported lipid bilayer. The system of differential equations associated with the proposed consecutive-three state kinetics scheme is solved, and the solution is applied to interpret histograms of single-molecule, GLP-1 residence times in terms of the microscopic rates in the sequential two-step model. These microscopic rates are used to estimate the free energy barrier to adsorption, the fraction of peptides adsorbing to the membrane surface that successfully intercalate in the bilayer, the lifetime of inserted peptides in the membrane, and the free energy change of insertion into the lipid bilayer from the adsorbed state. The transition from a random coil in solution to a folded state in a membrane has been recognized as a common motif for insertion of membrane active peptides. Therefore, the relationships developed here could have wide application to the kinetic analysis of peptide-membrane interactions.

The defensin–lipid interaction: Insights on the binding states of the human antimicrobial peptide HNP-1 to model bacterial membranes

Antimicrobial peptides are an important component of innate immunity and have generated considerable interest as a new potential class of natural antibiotics. The biological activity of antimicrobial peptides is strongly influenced by peptide–membrane interactions. Human Neutrophil Peptide 1 (HNP-1) is a 30 aminoacid peptide, belonging to the class of α-defensins. Many biophysical studies have been performed on this peptide to define its mechanism of action. Combining spectroscopic and thermodynamic analysis, insights on the interaction of the α-defensin with POPE:POPG:CL negative charged bilayers are given. The binding states of the peptide below and above the threshold concentration have been analyzed showing that the interaction with lipid bilayers is dependent by peptide concentration. These novel results that indicate how affinity and biological activities of natural antibiotics are depending by their concentration, might open new way of investigation of the antimicrobial mode of action.

Multi-Scale Simulation of the Simian Immunodeficiency Virus Fusion Peptide

Fusion peptides of type I fusion glycoproteins are structural elements of several enveloped viruses which enable the fusion between host and virus membranes. It is generally suggested that these peptides can promote the early fusion steps by inducing membrane curvature and that they adopt a tilted helical conformation in membranes. Although this property has been the subject of several experimental and in silico studies, an extensive sampling of the membrane peptide interaction has not yet been done. In this study, we performed coarse-grained molecular dynamic simulations in which the lipid bilayer self-assembles around the peptide. The simulations indicate that the SIV fusion peptide can adopt two different orientations in a DPPC bilayer, a major population which adopts a tilted interfacial orientation and a minor population which is perpendicular to the bilayer. The simulations also indicate that for the SIV mutant that does not induce fusion in vitro the tilt is abolished.

Membrane Interacting Peptides: From Killers to Helpers

Membrane interacting peptides are reviewed in terms of structure and mode of action on lipid membranes. Helical, β-stranded, peptides containing both helices and strands, cyclic, lipopeptides and short linear peptides are seen to considerably modulate membrane function. Among peptides that lead to membrane alteration or permeation, antimicrobial peptides play an important role and some of them may be foreseen as potential new antibiotics. Alternatively, peptides that do not destroy the membrane are also very important in modulating the structure and dynamics of the lipid bilayer and play important roles in membrane protein functions. Peptide lipid complexes are shown to be very variable in structure and dynamics: "carpet", "barrel stave", toroid and disordered pores, electrostatic wedge and molecular electroporation models are discussed. Their assembly is reviewed in terms of electric, amphipathic and dynamic properties of both lipids and peptides.

Design of Peptide-Membrane Interactions to Modulate Single-File Water Transport through Modified Gramicidin Channels

Water permeability through single-file channels is affected by intrinsic factors such as their size and polarity and by external determinants like their lipid environment in the membrane. Previous computational studies revealed that the obstruction of the channel by lipid headgroups can be long-lived, in the range of nanoseconds, and that pore-length-matching membrane mimetics could speed up water permeability. To test the hypothesis of lipid-channel interactions modulating channel permeability, we designed different gramicidin A derivatives with attached acyl chains. By combining extensive molecular-dynamics simulations and single-channel water permeation measurements, we show that by tuning lipid-channel interactions, these modifications reduce the presence of lipid headgroups in the pore, which leads to a clear and selective increase in their water permeability.

Improving membrane binding as a design strategy for amphipathic peptide hormones: 2‐helix variants of PYY3‐36

It has been hypothesized that amphipathic peptides might bind to membranes prior to activating their cognate receptors, but this has proven difficult to test. The peptide hormone PYY3-36 is believed to perform its appetite-suppressing actions through binding to hypothalamic Y2 receptors. It has been proposed that PYY3-36 via its amphipathic α-helix binds to the plasma membrane prior to receptor docking. Here, our aim was to study the implication of this hypothesis using new analogs of PYY3-36. We first studied membrane binding of PYY3-36. Next, we designed a series of PYY3-36 analogs to increase membrane-binding affinity by substituting the N-terminal segment with a de novo designed α-helical, amphipathic sequence. These 2-helix variants of PYY3-36 were assembled by solid-phase peptide synthesis. Pharmacological studies demonstrated that even though the native peptide sequence was radically changed, highly active Y2 receptor agonists were generated. A potent analog, with a Kd of 4 nM for membranes, was structurally characterized by NMR in the membrane-bound state, which clearly showed that it formed the expected 2-helix. The topology of the peptide-micelle association was studied by paramagnetic relaxation enhancement using a spin label, which confirmed that the hydrophobic residues bound to the membrane. Our studies further support the hypothesis that PYY3-36 associates with the membrane and indicate that this can be used in the design of novel molecules with high receptor binding potency. These observations are likely to be generally important for peptide hormones and biopharmaceutical drugs derived from them. This new 2-helix variant of PYY3-36 will be useful as a tool compound for studying peptide-membrane interactions.

Spectroscopic and Computational Study of Melittin, Cecropin A, and the Hybrid Peptide CM15

Antimicrobial peptides (AMPs), such as cecropin A from silk moth, are key components of the innate immune system. They are effective defensive weapons against invading pathogens, yet they do not target host eukaryotic cells. In contrast, peptide toxins, such as honeybee melittin, are nondiscriminating and target both eukaryotic and prokaryotic cells. An AMP-toxin hybrid peptide that is composed of cecropin A and melittin (CM15) improves upon the antimicrobial activity of cecropin A without displaying the nonspecific, hemolytic properties of melittin. Here we report fluorescence and UV resonance Raman spectra of melittin, cecropin A, and CM15 with the goal of elucidating peptide-membrane interactions that help guide specificity. We have probed the potency for membrane disruption, local environment and structure of the single tryptophan residue, backbone conformation near the peptide hinge, and amide backbone structure of the peptides in lipid environments that mimic eukaryotic and prokaryotic membranes. These experimental results suggest that melittin inserts deeply into the bilayer, whereas cecropin A remains localized to the lipid headgroup region. A surprising finding is that CM15 is a potent membrane-disruptor despite its largely unfolded conformation. A molecular dynamics analysis complements these data and demonstrates the ability of CM15 to associate favorably with membranes as an unfolded peptide. This combined experimental–computational study suggests that new models for peptide–membrane interactions should be considered.