Peptide-lipid Interactions Research Articles

Calcium-dependent membrane interactions of tumor-targeting peptide pHLIP.

Cancerous tissues undergo extensive changes to their cellular environments that differentiate them from healthy tissues. These changes include changes in extracellular pH and Ca2+ concentrations, and the exposure of phosphatidylserine (PS) to the extracellular environment, which can modulate the interaction of peptides and proteins with the plasma membrane. Deciphering the molecular mechanisms of such interactions is critical for advancing the knowledge-based design of cancer-targeting molecular tools, such as pHLIP (pH-Low Insertion Peptide). Here, we explore the effects of PS, Ca2+, and peptide protonation state on the interactions of pHLIP with lipid membranes. Cellular studies demonstrate that exposed PS on the plasma membrane promotes pHLIP targeting. The magnitude of this effect is dependent on extracellular Ca2+ concentration, indicating that divalent cations play an important role in pHLIP targeting in vivo. The targeting mechanism is further explored with a combination of fluorescence and circular dichroism experiments in model membranes and microsecond-timescale all-atom molecular dynamics simulations. Our results demonstrate that Ca2+ is engaged in coupling peptide-lipid interactions in the unprotonated transmembrane conformation of pHLIP. The simulations reveal that while the pH-induced insertion leads to a strong depletion of PS around pHLIP, the Ca2+-induced insertion has the opposite effect. Thus, extracellular levels of Ca2+ are crucial to link cellular changes in membrane lipid composition with the selective targeting and insertion of pHLIP. The characterized Ca2+-dependent coupling between pHLIP sidechains and PS provides atomistic insights into the general mechanism for lipid-coupled regulation of protein-membrane insertion by divalent cations.

Membrane cholesterol regulates the oligomerization and fusogenicity of SARS-CoV fusion peptide: implications in viral entry.

N-terminal residues (770-788) of the S2 glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) have been recognized as a potential fusion peptide that can be involved in the entry of the virus into the host cell. Membrane composition plays an important role in lipid-peptide interaction and the oligomeric status of the peptide. SARS-CoV fusion peptide (S2 fusion peptide) is known to undergo cholesterol-dependent oligomerization in the membrane; however, its significance in membrane fusion is still speculative. This study aimed to investigate the oligomerization of SARS-CoV fusion peptide in a membrane containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol, with varying concentrations of cholesterol, and to evaluate peptide-induced membrane fusion to correlate the importance of peptide oligomerization with membrane fusion. Peptide-induced modulation of membrane organization and dynamics was explored by steady-state and time-resolved fluorescence spectroscopic measurements using depth-dependent probes. The results clearly demonstrated the induction of S2 fusion peptide oligomerization by membrane cholesterol and the higher efficiency of the oligomer in promoting membrane fusion compared to its monomeric counterpart. Cholesterol-dependent peptide oligomerization and membrane fusion are important aspects of viral infection since the cholesterol level can change with age as well as with the onset of various pathophysiological conditions.

SARS-CoV-2 Envelope Protein Forms Clustered Pentamers in Lipid Bilayers.

The SARS-CoV-2 envelope (E) protein is a viroporin associated with the acute respiratory symptoms of COVID-19. E forms cation-selective ion channels that assemble in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment. The channel activity of E is linked to the inflammatory response of the host cell to the virus. Like many viroporins, E is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the E stoichiometry have led to inconclusive results and suggested mixtures of oligomers whose exact nature might vary with the detergent used. Here, we employ 19F solid-state nuclear magnetic resonance and the centerband-only detection of exchange (CODEX) technique to determine the oligomeric number of E’s transmembrane domain (ETM) in lipid bilayers. The CODEX equilibrium value, which corresponds to the inverse of the oligomeric number, indicates that ETM assembles into pentamers in lipid bilayers, without any detectable fraction of low-molecular-weight oligomers. Unexpectedly, at high peptide concentrations and in the presence of the lipid phosphatidylinositol, the CODEX data indicate that more than five 19F spins are within a detectable distance of about 2 nm, suggesting that the ETM pentamers cluster in the lipid bilayer. Monte Carlo simulations that take into account peptide–peptide and peptide–lipid interactions yielded pentamer clusters that reproduced the CODEX data. This supramolecular organization is likely important for E-mediated virus assembly and budding and for the channel function of the protein.

Ca2+ -dependent interactions between lipids and the tumor-targeting peptide pHLIP.

Cancerous tissues undergo extensive changes to their cellular environments that differentiate them from healthy tissues. These changes include changes in extracellular pH and Ca2+ concentrations, and the exposure of phosphatidylserine (PS) to the extracellular environment, which can modulate the interaction of peptides and proteins with the plasma membrane. Deciphering the molecular mechanisms of such interactions is critical for advancing the knowledge-based design of cancer-targeting molecular tools, such as pH-low insertion peptide (pHLIP). Here, we explore the effects of PS, Ca2+ , and peptide protonation state on the interactions of pHLIP with lipid membranes. Cellular studies demonstrate that exposed PS on the plasma membrane promotes pHLIP targeting. The magnitude of this effect is dependent on extracellular Ca2+ concentration, indicating that divalent cations play an important role in pHLIP targeting in vivo. The targeting mechanism is further explored with a combination of fluorescence and circular dichroism experiments in model membranes and microsecond-timescale all-atom molecular dynamics simulations. Our results demonstrate that Ca2+ is engaged in coupling peptide-lipid interactions in the unprotonated transmembrane conformation of pHLIP. The simulations reveal that while the pH-induced insertion leads to a strong depletion of PS around pHLIP, the Ca2+ -induced insertion has the opposite effect. Thus, extracellular levels of Ca2+ are crucial to linking cellular changes in membrane lipid composition with the selective targeting and insertion of pHLIP. The characterized Ca2+ -dependent coupling between pHLIP sidechains and PS provides atomistic insights into the general mechanism for lipid-coupled regulation of protein-membrane insertion by divalent cations.

Flip-Flop Promotion Mechanisms by Model Transmembrane Peptides.

Lipid transbilayer movement (flip-flop) is regulated by membrane proteins that are involved in homeostasis and signaling in eukaryotic cells. In the plasma membrane, an asymmetric lipid composition is maintained by energy-dependent unidirectional transport. Energy-independent flip-flop promotion by phospholipid scramblases disrupts the asymmetry in several physiological processes, such as apoptosis and blood coagulation. In the endoplasmic reticulum, rapid flip-flop is essential for bilayer integrity because phospholipids are synthesized only in the cytoplasmic leaflet. Phospholipid scramblases are also involved in lipoprotein biogenesis, autophagosome formation, and viral infection. Although several scramblases have been identified and investigated, the precise flip-flop promotion mechanisms are not fully understood. Model transmembrane peptides are valuable tools for investigating the general effects of lipid-peptide interactions. We focus on the development of model transmembrane peptides with flip-flop promotion abilities and their mechanisms.

Amphiphilic Gold Nanoparticles: A Biomimetic Tool to Gain Mechanistic Insights into Peptide-Lipid Interactions.

Functional peptides are now widely used in a myriad of biomedical and clinical contexts, from cancer therapy and tumor targeting to the treatment of bacterial and viral infections. Underlying this diverse range of applications are the non-specific interactions that can occur between peptides and cell membranes, which, in many contexts, result in spontaneous internalization of the peptide within cells by avoiding energy-driven endocytosis. For this to occur, the amphipathicity and surface structural flexibility of the peptides play a crucial role and can be regulated by the presence of specific molecular residues that give rise to precise molecular events. Nevertheless, most of the mechanistic details regulating the encounter between peptides and the membranes of bacterial or animal cells are still poorly understood, thus greatly limiting the biomimetic potential of these therapeutic molecules. In this arena, finely engineered nanomaterials—such as small amphiphilic gold nanoparticles (AuNPs) protected by a mixed thiol monolayer—can provide a powerful tool for mimicking and investigating the physicochemical processes underlying peptide-lipid interactions. Within this perspective, we present here a critical review of membrane effects induced by both amphiphilic AuNPs and well-known amphiphilic peptide families, such as cell-penetrating peptides and antimicrobial peptides. Our discussion is focused particularly on the effects provoked on widely studied model cell membranes, such as supported lipid bilayers and lipid vesicles. Remarkable similarities in the peptide or nanoparticle membrane behavior are critically analyzed. Overall, our work provides an overview of the use of amphiphilic AuNPs as a highly promising tailor-made model to decipher the molecular events behind non-specific peptide-lipid interactions and highlights the main affinities observed both theoretically and experimentally. The knowledge resulting from this biomimetic approach could pave the way for the design of synthetic peptides with tailored functionalities for next-generation biomedical applications, such as highly efficient intracellular delivery systems.

The conformational dynamics of the flanking polyQ regions in the membrane-bound state of huntingtin exon 1

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an abnormal expansion of the polyglutamine (polyQ) repeat within the first exon of huntingtin protein (httex1). The membrane interaction of httex1 is critical for its self-assembly and toxicity. While initial studies were essentially focused on polyQ peptide-lipid interaction, recent evidence supports that both flanking polyQ regions [the N17 segment and proline-rich domain (PRD)] strongly modulate the httex1 aggregation at membrane surfaces.

Electromechanical Properties of Lipid Bilayers in Peptide-Lipid Interactions: Clinical Applications

Despite the amazing diversity and complexity of living systems, all life shares the presence of a semi-permeable lipid membrane. Throughout evolution, this permeability has been finely regulated by the function of proteins that form ion channels. Similarly, some peptides can stabilize by forming aqueous pores in a dose-dependent manner. At high concentrations, however, these peptides can compromise the integrity of the membrane, destroying it. Given this behavior, a huge diversity of antimicrobial peptides and lipo-peptides has been identified. Understanding the physical-chemical principles of their interactions with lipids can contribute to the design of synthetic peptides with antimicrobial activity as an alternative to the use of conventional antibiotics.

Design of a specific peptide against phenolic glycolipid-1 from Mycobacterium leprae and its implications in leprosy bacilli entry.

BACKGROUND Mycobacterium leprae, the causative agent of Hansen’s disease, causes neural damage through the specific interaction between the external phenolic glycolipid-1 (PGL-1) and laminin subunit alpha-2 (LAMA2) from Schwann cells.OBJECTIVE To design a LAMA2-based peptide that targets PGL-1 from M. leprae.METHODS We retrieved the protein sequence of human LAMA2 and designed a specific peptide using the Antimicrobial Peptide Database and physicochemical parameters for antimycobacterial peptide-lipid interactions. We used the AlphaFold2 server to predict its three-dimensional structure, AUTODOCK-VINA for docking, and GROMACS programs for molecular dynamics simulations.FINDINGS We analysed 52 candidate peptides from LAMA2, and subsequent screening resulted in a single 60-mer peptide. The mapped peptide comprises four β-sheets and a random coiled region. This peptide exhibits a 45% hydrophobic ratio, in which one-third covers the same surface. Molecular dynamics simulations show that our predicted peptide is stable in aqueous solution and remains stable upon interaction with PGL-1 binding. In addition, we found that PGL-1 has a preference for one of the two faces of the predicted peptide, which could act as the preferential binding site of PGL-1.MAIN CONCLUSIONS Our LAMA2-based peptide targeting PGL-1 might have the potential to specifically block this key molecule, suggesting that the preferential region of the peptide is involved in the initial contact during the attachment of leprosy bacilli to Schwann cells.

Improving Membrane Activity and Cargo Delivery Efficacy of a Cell-Penetrating Peptide by Loading with Carboranes.

Cell-penetrating peptides (CPPs) have emerged as versatile tools to increase the intracellular accumulation of different kinds of cargoes. For an efficient cellular uptake and drug delivery, their organization into a distinct and stable secondary structure at the outer surface of the plasma membrane is a hallmark and supports optimal lipid–peptide interactions. Incorporation of hydrophobic moieties, such as carboranes (CBs), has the potential to increase the lipophilicity of peptides, and thus, to facilitate the formation of secondary structures. Herein, we present synthesis and biophysical as well as biological characterization of carborane-CPP conjugates having incorporated one or more CB clusters. Our results highlight the possibility to modulate the secondary structure of CPPs by the addition of CB’s leading to constructs with altered membrane activity and promising use in terms of nucleic acid delivery.

Applying flow cytometry to identify the modes of action of membrane-active peptides in a label-free and high-throughput fashion

Membrane-active peptides (MAPs) have several potential therapeutic uses, including as antimicrobial drugs. Many traditional methods used to evaluate the membrane interactions of MAPs have limited applicability. Low-throughput methods, such as microscopy, provide detailed information but often rely on fluorophore-labeled MAPs, and high-throughput assays, such as the calcein release assay, cannot assess the mechanism behind the disruption of vesicular-based lipid membranes. Here we present a flow cytometric assay that provides detailed information about the peptide-lipid membrane interactions on single artificial lipid vesicles while being high-throughput (1000–2000 vesicles/s) and based on label-free MAPs. We synthesized and investigated six MAPs with different modes of action to evaluate the versatility of the assay. The assay is based on the flow cytometric readouts from artificial lipid vesicles, including the fluorescence from membrane-anchored and core-encapsulated fluorophores, and the vesicle concentration. From these parameters, we were able to distinguish between MAPs that induce vesicle solubilization, permeation (pores/membrane distortion), and aggregation or fusion. Our flow cytometry findings have been verified by traditional methods, including the calcein release assay, dynamic light scattering, and fluorescence microscopy on giant unilamellar vesicles. We envision that the presented flow cytometric assay can be used for various types of peptide-lipid membrane studies, e.g. to identify new antibiotics. Moreover, the assay can easily be expanded to derive additional valuable information.

Laser Modes: Topological Encoded Vector Beams for Monitoring Amyloid‐Lipid Interactions in Microcavity (Adv. Sci. 12/2021)

In article number 2100096, Pin Chieh Wu, Yu-Cheng Chen, and co-workers report a novel concept to study tiny structural molecular interactions through the dynamic changes of vector laser beams. The cover shows the rapid changes of the topological laser modes which resembles chinese face changing. The complex topology of laser modes is discovered and developed into laser barcode to monitor peptide-lipid membrane interactions for a wide range of biomedical applications.

FRET detects lateral interaction between transmembrane domain of EGF receptor and ganglioside GM3 in lipid bilayers

Ganglioside GM3 in the plasma membranes suppresses cell growth by preventing the autophosphorylation of the epidermal growth factor receptor (EGFR). Biological studies have suggested that GM3 interacts with the transmembrane segment of EGFR. Further biophysical experiments are particularly important for quantitative evaluation of the peptide-glycolipid interplay in bilayer membranes using a simple reconstituted system. To examine these interactions in this way, we synthesized the transmembrane segment of EGFR bearing a nitrobenzoxadiazole fluorophore (NBD-TM) at the N-terminus. The affinity between EGFR and GM3 was evaluated based on Förster resonance energy transfer (FRET) between NBD-TM and ATTO594-labeled GM3 in bilayers where their non-specific interaction due to lateral proximity was subtracted by using NBD-labeled phospholipid. This method for selectively detecting the specific lipid-peptide interactions in model lipid bilayers disclosed that the lateral interaction between GM3 and the transmembrane segment of EGFR plays a certain role in disturbing the formation of active EGFR dimers.

High-resolution structural profile of hylaseptin-4: Aggregation, membrane topology and pH dependence of overall membrane binding process

Hylaseptin-4 (HSP-4, GIGDILKNLAKAAGKAALHAVGESL-NH2) is an antimicrobial peptide originally isolated from Hypsiboas punctatus tree frog. The peptide has been chemically synthetized for structural investigations by CD and NMR spectroscopies. CD experiments reveal the high helical content of HSP-4 in biomimetic media. Interestingly, the aggregation process seems to occur at high peptide concentrations either in aqueous solution or in presence of biomimetic membranes, indicating an increase in the propensity of the peptide for adopting a helical conformation. High-resolution NMR structures determined in presence of DPC-d38 micelles show a highly ordered α-helix from amino acid residues I2 to S24 and a smooth bend near G14. A large separation between hydrophobic and hydrophilic residues occurs up to the A16 residue, from which a shift in the amphipathicity is noticed. Oriented solid-state NMR spectroscopy show a roughly parallel orientation of the helical structure along the POPC lipid bilayer surface, with an insertion of the hydrophobic N-terminus into the bilayer core. Moreover, a noticeable pH dependence of the aggregation process in both aqueous and in biomimetic membrane environments is attributed to a single histidine residue (H19). The protonation degree of the imidazole side-chain might help in modulating the peptide-peptide or peptide-lipid interactions. Finally, molecular dynamics simulations confirm the orientation and preferential helical conformation and in addition, show that HSP-4 tends to self-aggregate in order to stabilize its active conformation in aqueous or phospholipid bilayer environments.

Cell-Penetrating Peptides: Correlation between Peptide-Lipid Interaction and Penetration Efficiency.

Cell-penetrating peptides are used in the delivery of peptides and biologics, with some cell-penetrating peptides found to be more efficient than others. The exact mechanism of how they interact with the cell membrane and penetrate it, however, remains unclear. This study attempts to investigate the difference in free energy profiles of three cell-penetrating peptides (TAT, CPP1 and CPP9) with a model lipid bilayer (DOPC) using molecular dynamics pulling simulations with umbrella sampling. Potential mean force (PMF) and free energy barrier between the peptides and DOPC are determined using WHAM analysis and MM-PBSA analysis, respectively. CPP9 is found to have the smallest PMF value, followed by CPP1 and TAT, consistent with the experimental data. YDEGE peptide, however, does not give the highest PMF value, although it is a non-cell-permeable peptide. YDEGE is also found to form water pores, alongside with TAT and CPP9, suggesting that it is difficult to distinguish true water pore formation from artefacts arising from pulling simulations. On the contrary, free energy analysis of the peptide-DOPC complex at the lipid-water interface with MM-PBSA provides results consistent with experimental data with CPP9 having the least interaction with DOPC and lowest free energy barrier, followed by CPP1, TAT and YDEGE. These findings suggest that peptide-lipid interaction at the lipid-water interface has a direct correlation with the penetration efficiency of peptides across the lipid bilayer.

Energetics and Kinetics of Peptide-Lipid Membrane Interaction: A Molecular Dynamics Study

Quantitative characterization of the binding strength of peptides to lipid bilayers is crucial in understanding the molecular mechanism of cellular processes triggered by peptide-lipid interactions. In general, single molecule methods, such as high precision AFM based dynamic force spectroscopy, can be used to measure the dissociation force distribution, P(F), and the corresponding force dependent rupture rate, k(F), of individual peptides bound to a lipid bilayer. However, the interpretation of the results require extensive theoretical and computational modeling that implies the knowledge of the free energy profile (potential of mean force or PMF) of the system as a function of the separation between peptide and membrane surface. To this end, we have used all atom MD simulations, to calculate the PMF of three representative Wimley-White pentapeptides (Ac-WLXLL, with guest residues X=R,I and L) interacting with two different lipid bilayers (zwitterionic POPC and charged POPG). The PMF, as a function of the distance between the residue W and the plane of the membrane, was calculated by using (i) the equilibrium umbrella-sampling method, and (ii) the nonequilibrium FR method. Both methods yielded comparable PMFs. In addition, the FR method also permitted the calculation of the position dependent diffusion coefficient of the peptide. In each case, the values of the activation energy (free energy barrier) and activation length extracted from the PMFs appear to be comparable to previous experimental and computational studies. The obtained energetic and kinetic parameters have been successfully used to fit P(F) and k(F) measured in AFM experiments.

Amphipathic Peptide-Phospholipid Nanofibers: Phospholipid Specificity and Dependence on Concentration and Temperature.

The design of nanoassemblies is an important part of the development of new materials for applications in nanomedicine and biosensors. In our previous study, cysteine substitution of the apolipoprotein A-I-derived peptide 18A at residue 11, 18A[A11C], bound to 1-palmitoyl-2-oleoylphosphatidylcholine to form fibrous aggregates at a lipid-to-peptide molar ratio of ≤2 and a fiber diameter of 10-20 nm. However, the mechanisms underlying the lipid-peptide interactions that enable nanofiber formation remain unclear. Here, we evaluated the phospholipid specificity, concentration dependence, and temperature dependence of the formation of 18A[A11C]-lipid nanofibers. Nanofibers were found to form in the presence of specific phospholipids and have a constant lipid/peptide stoichiometry of 1.2 ± 0.2. Moreover, an increase in the length of the acyl chain in phosphatidylcholines was found to increase the structural stability of the nanofibers. These results indicate that specific molecular interactions between peptides and both the headgroups and acyl chains of phospholipids are involved in nanofiber formation. Furthermore, the formation and disassembly of the nanofibers were reversibly controlled by changes in temperature and concentration. The results of the present study provide an insight into the creation of nanoassembling structures.

Antimicrobial peptides: mechanism of action and lipid-mediated synergistic interactions within membranes.

Biophysical and structural studies of peptide-lipid interactions, peptide topology and dynamics have changed our view of how antimicrobial peptides insert and interact with membranes. Clearly, both peptides and lipids are highly dynamic, and change and mutually adapt their conformation, membrane penetration and detailed morphology on a local and a global level. As a consequence, 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. Although the membranes are soft and can adapt, at increasing peptide density they cause pronounced disruptions of the phospholipid fatty acyl packing. At even higher local or global concentrations the peptides cause transient membrane openings, rupture and ultimately lysis. Interestingly, mixtures of peptides such as magainin 2 and PGLa, which are stored and secreted naturally as a cocktail, exhibit considerably enhanced antimicrobial activities when investigated together in antimicrobial assays and also in pore forming experiments applied to biophysical model systems. Our most recent investigations reveal that these peptides do not form stable complexes but act by specific lipid-mediated interactions and the nanoscale properties of phospholipid bilayers.

Revealing the Mechanisms of Synergistic Action of Two Magainin Antimicrobial Peptides.

The study of peptide-lipid and peptide-peptide interactions as well as their topology and dynamics using biophysical and structural approaches have changed our view how antimicrobial peptides work and function. It has become obvious that both the peptides and the lipids arrange in soft supramolecular arrangements which are highly dynamic and able to change and mutually adapt their conformation, membrane penetration, and detailed morphology. This can occur on a local and a global level. This review focuses on cationic amphipathic peptides of the magainin family which were studied extensively by biophysical approaches. They are found intercalated at the membrane interface where they cause membrane thinning and ultimately lysis. Interestingly, mixtures of two of those peptides namely magainin 2 and PGLa which occur naturally as a cocktail in the frog skin exhibit synergistic enhancement of antimicrobial activities when investigated together in antimicrobial assays but also in biophysical experiments with model membranes. Detailed dose-response curves, presented here for the first time, show a cooperative behavior for the individual peptides which is much increased when PGLa and magainin are added as equimolar mixture. This has important consequences for their bacterial killing activities and resistance development. In membranes that carry unsaturations both peptides align parallel to the membrane surface where they have been shown to arrange into mesophases involving the peptides and the lipids. This supramolecular structuration comes along with much-increased membrane affinities for the peptide mixture. Because this synergism is most pronounced in membranes representing the bacterial lipid composition it can potentially be used to increase the therapeutic window of pharmaceutical formulations.

Electrostatic effects in saturation of membrane binding of cationic cell-penetrating peptide.

Membrane-active peptides that demonstrate cell-penetrating, antimicrobial or cytotoxic functions are diverse in their amino acid sequences, but share common physicochemical features like short length, amphipathic conformation in membrane environment and high net charge. Nonspecific electrostatic interactions of basic peptide residues with anionic membrane lipids play a crucial role in the initial binding of such peptides to plasma membranes of bacterial and mammalian cells. At the same time, a number of membrane-active peptides functions when they are localized at high concentrations on the lipid membranes. Dissecting the role of electrostatics in this functional peptide conditions is important to understand why the majority of them bear high positive charge. We have studied interaction of EB1 cell-penetrating peptide (charge +8) with model anionic membranes. The saturation of peptide binding to liposomes that comprises 5%, 10% and 25% of negatively charged lipids (POPC/POPG mixture) was observed. We have found that peptide recharges liposomes and its surface saturating concentration increases with the amount of anionic lipids in a membrane so as a surface charge (bound peptide plus anionic lipids). This observation may be explained with the Gouy-Chapman theory based model with addition of independent effective peptide charges for peptide-peptide and peptide-lipid interactions, as well as steric saturation term. Additionally, in certain conditions, membrane bound peptide leads to liposome aggregation. In some lipid-to-peptide ratio regions disaggregation follows that may indicate an additional slow equilibration process after fast initial peptide binding.