Anionic Bilayers Research Articles

Bilayer lipids modulate ligand binding to atypical chemokine receptor 3

Chemokine receptors belong to the large class of G protein-coupled receptors (GPCRs) and are involved in a number of (patho)physiological processes. Previous studies highlighted the importance of membrane lipids for modulating GPCR structure and function. However, the underlying mechanisms of how lipids regulate GPCRs are often poorly understood. Here, we report that anionic lipid bilayers increase the binding affinity of the chemokine CXCL12 for the atypical chemokine receptor 3 (ACKR3) by modulating the CXCL12 binding kinetics. Notably, the anionic bilayer favors CXCL12 over the more positively charged chemokine CXCL11, which we explained by bilayer interactions orienting CXCL12 but not CXCL11 for productive ACKR3 binding. Furthermore, our data suggest a stabilization of active ACKR3 conformations in anionic bilayers. Taken together, the described regulation of chemokine selectivity of ACKR3 by the lipid bilayer proposes an extended version of the classical model of chemokine binding including the lipid environment of the receptor.

Charge-Reversed Exosomes for Targeted Gene Delivery to Cartilage for Osteoarthritis Treatment.

Gene therapy has the potential to facilitate targeted expression of therapeutic proteins to promote cartilage regeneration in osteoarthritis (OA). The dense, avascular, aggrecan-glycosaminoglycan (GAG) rich negatively charged cartilage, however, hinders their transport to reach chondrocytes in effective doses. While viral vector mediated gene delivery has shown promise, concerns over immunogenicity and tumorigenic side-effects persist. To address these issues, this study develops surface-modified cartilage-targeting exosomes as non-viral carriers for gene therapy. Charge-reversed cationic exosomes are engineered for mRNA delivery by anchoring cartilage targeting optimally charged arginine-rich cationic motifs into the anionic exosome bilayer by using buffer pH as a charge-reversal switch. Cationic exosomes penetrated through the full-thickness of early-stage arthritic human cartilage owing to weak-reversible ionic binding with GAGs and efficiently delivered the encapsulated eGFP mRNA to chondrocytes residing in tissue deep layers, while unmodified anionic exosomes do not. When intra-articularly injected into destabilized medial meniscus mice knees with early-stage OA, mRNA loaded charge-reversed exosomes overcame joint clearance and rapidly penetrated into cartilage, creating an intra-tissue depot and efficiently expressing eGFP; native exosomes remained unsuccessful. Cationic exosomes thus hold strong translational potential as a platform technology for cartilage-targeted non-viral delivery of any relevant mRNA targets for OA treatment.

Revisiting the reaction pathways for phospholipid hydrolysis catalyzed by phospholipase A2 with QM/MM methods

This study unveils hGIIA sPLA2's affinity for anionic bilayers, pinpointing crucial interfacial residues. It also emphasizes the dominance of the single-water pathway in its reaction mechanism, crucial for drug design targeting transition states.

Enhanced Uptake of Anticancer C6-Pep Dimer in a Model Membrane Caused by Differential pKa in Acidic Microenvironment.

Acidic tumor microenvironment (TME) presents a challenge for the action of antitumor drugs by acting as an additional barrier for the passive crossing of the cell membrane by chemotherapic agents playing a critical role in the proliferation of tumor cells. Anticancer lipopeptide C6-Pep dimer containing the leucine zipper motif shows an increased uptake into the model tumor membrane in TME, and application of external heat might lead to the uncoiling of the zipper, which could result in cell lysis. This work investigated the cause of this increased uptake of C6-Pep dimer into the bilayer model in TME. Accurate protonation states of all the titratable residues of the C6-Pep dimer in TME were determined using constant pH molecular dynamics. In TME, except for two terminal Glu5 residues, all other Glu residues in the C6-Pep dimer were permanently protonated. The remaining Glu5 residues had differential pKa values, leading to the construction of four possible dimers with different fixed protonation states, and molecular dynamics was used to study their interaction with the anionic bilayer. Except for the dimer at a physiological pH, the other dimers were positively charged and could readily adsorb on the membrane surface. The free energy of insertion of these dimers in the bilayer was lower for single and double protonated Glu5-containing dimers than for the others. After the insertion of the lipopeptides into the membrane, thinning of the bilayer in the vicinity of dimers and an increase in area per lipid of the bilayer were observed for all systems, indicating destabilization of the bilayer due to this intercalation. This study shows that the anticancer lipopeptide C6-Pep utilizes the TME around a tumor cell for insertion into the membrane.

Replica Exchange with Hybrid Tempering Efficiently Samples PGLa Peptide Binding to Anionic Bilayer.

We evaluated the utility of a variant of the replica exchange method, a replica exchange with hybrid tempering (REHT), for all-atom explicit water biomolecular simulations and compared it with a more traditional replica exchange with the solute tempering (REST) algorithm. As a test system, we selected a 21-mer antimicrobial peptide PGLa binding to an anionic DMPC/DMPG lipid bilayer. Application of REHT revealed the following binding mechanism. Due to the strong hydrophobic moment, the bound PGLa adopts an extensive helical structure. The binding free energy landscape identifies two major bound states, a metastable surface bound state and a dominant inserted state. In both states, positively charged PGLa amino acids maintain electrostatic interactions with anionic phosphate groups by rotating the PGLa helix around its axis. PGLa binding causes an influx of anionic DMPG and an efflux of zwitterionic DMPC lipids from the peptide proximity. PGLa thins the bilayer and disorders the adjacent fatty acid tails. Deep invasion of water wires into the bilayer hydrophobic core is detected in the inserted peptide state. The analysis of charge density distributions indicated that peptide positive charges are nearly compensated for by lipid negative charges and water dipole ordering, whereas ions play no role in peptide binding. Thus, electrostatic interactions are the key energetic factor in binding cationic PGLa to an anionic DMPC/DMPG bilayer. Comparison of REHT and REST shows that due to exclusion of lipids from tempered partition, REST lags behind REHT in peptide equilibration, particularly, with respect to peptide insertion and helix acquisition. As a result, REST struggles to provide accurate details of PGLa binding, although it still qualitatively maps the bimodal binding mechanism. Importantly, REHT not only equilibrates PGLa in the bilayer faster than REST, but also with less computational effort. We conclude that REHT is a preferable choice for studying interfacial biomolecular systems.

Curvature Matters: Modeling Calcium Binding to Neutral and Anionic Phospholipid Bilayers.

In this work, the influence of membrane curvature on the Ca2+ binding to phospholipid bilayers is investigated by means of molecular dynamics simulations. In particular, we compared Ca2+ binding to flat, elastically buckled, or uniformly bent zwitterionic and anionic phospholipid bilayers. We demonstrate that Ca2+ ions bind preferably to the concave membrane surfaces in both types of bilayers. We also show that the membrane curvature leads to pronounced changes in Ca2+ binding including differences in free ion concentrations, lipid coordination distributions, and the patterns of ion binding to different chemical groups of lipids. Moreover, these effects differ substantially for the concave and convex membrane monolayers. Comparison between force fields with either full or scaled charges indicates that charge scaling results in reduction of the Ca2+ binding to curved phosphatidylserine bilayers, while for phosphatidylcholine membranes, calcium binds only weakly for both force fields.

Design and synthesis of novel N-terminal peptides of integrin and aminopeptidase are new finding for anticancer activity

The short peptides, containing the amino acid sequence asparagine-glycine-arginine (NGR) and arginine-glycine-aspartic acid (RGD), possess the strong binding ability to N (APN/CD13) aminopeptidase receptor and integrin proteins involved in antitumor properties are overexpressed. A novel short N-terminal modified hexapeptides P1 and P2 was designed and synthesized using the Fmoc-chemistry solid phase peptide synthesis protocol. Notably, the cytotoxicity of the MTT assay demonstrated the viability of normal and cancer cells up to lower peptide concentrations. Interestingly, both peptides show good anticancer activities against the four cancer cells and normal cells namely, Hep-2, HepG2, MCF-7, A375, and Vero and compared with standard drugs, doxorubicin and paclitaxel. Additionally, in silico studies were applied to predict the binding sites and binding orientation of the peptides for potential anticancer targets. Steady-state fluorescence measurements showed that peptide P1 exhibits preferential interactions with POPC/POPG anionic bilayers rather than the zwitterionic POPC lipid bilayers and peptide P2, did not show any preferential interaction with lipids bilayers. But impressively, peptide P2 shows anticancer activity due to the NGR/RGD motif. Circular dichroism studies demonstrated that the peptide's secondary structure changes only minimally upon binding to the anionic lipid bilayers.

Antimicrobial peptides alter lipid bilayer properties before disrupting membrane barrier property.

Antimicrobial peptides (AMPs) have been the subject of inquiry since the initial search for antibiotics in the ­first half of 20th century; the first antibiotic used in humans was the linear pentadecapeptide gramicidin. Much of this interest comes from the modest frequency of bacterial resistance against AMPs. Though AMPs differ in sequence and structure, the majority share three core physicochemical properties; they are short, cationic and amphipathic. Moreover, enantiomers of AMPs tend to have similar potencies. These common features have led to a focus on the bacterial membrane as a target for AMPs. High concentrations of AMPs cause gross bilayer defects, through pore formation or micellar breakdown. At lower concentrations, AMPs have effects on bacterial physiology that appear unrelated to membrane permeabilization, suggesting that they may act through bilayer-mediated regulation (BMR) of membrane protein function. If correct, this offers a mechanism of action that integrates the known membrane-modifying effects of AMPs with their ability to broadly alter bacterial physiology at sub-pore forming concentrations. We tested this hypothesis using melittin, gramicidin S, and indolicidin, three AMPs with distinct structures, that indeed alter bilayer properties at concentrations below those required for pore formation. Contrary to expectation, AMP induced BMR is attenuated in anionic bilayers despite increased AMP binding, suggesting an organizing effect of membrane-bound counterions. We further found that amphiphilic bilayer modifiers promote membrane permeabilization, suggesting that BMR may be responsible for the cooperativity of AMP-induced permeabilization. Two membrane-impermeant detergents, CHAPS and zwittergent, promote pore formation despite inhibiting AMP binding, suggesting a role for leaflet-specific lateral pressure differences. These results suggest a new mechanism by which AMPs exert their bactericidal effect, which may guide antibiotic design and provide a path for circumventing bacterial resistance.

Synaptotagmin rings as high-sensitivity regulators of synaptic vesicle docking and fusion

Synchronous release at neuronal synapses is accomplished by a machinery that senses calcium influx and fuses the synaptic vesicle and plasma membranes to release neurotransmitters. Previous studies suggested the calcium sensor synaptotagmin (Syt) is a facilitator of vesicle docking and both a facilitator and inhibitor of fusion. On phospholipid monolayers, the Syt C2AB domain spontaneously oligomerized into rings that are disassembled by Ca2+, suggesting Syt rings may clamp fusion as membrane-separating "washers" until Ca2+-mediated disassembly triggers fusion and release [J. Wang etal., Proc. Natl. Acad. Sci. U.S.A. 111, 13966-13971 (2014)].). Here, we combined mathematical modeling with experiment to measure the mechanical properties of Syt rings and to test this mechanism. Consistent with experimental results, the model quantitatively recapitulates observed Syt ring-induced dome and volcano shapes on phospholipid monolayers and predicts rings are stabilized by anionic phospholipid bilayers or bulk solution with ATP. The selected ring conformation is highly sensitive to membrane composition and bulk ATP levels, a property that may regulate vesicle docking and fusion in ATP-rich synaptic terminals. We find the Syt molecules hosted by a synaptic vesicle oligomerize into a halo, unbound from the vesicle, but in proximity to sufficiently phosphatidylinositol 4,5-bisphosphate (PIP2)-rich plasma membrane (PM) domains, the PM-bound trans Syt ring conformation is preferred. Thus, the Syt halo serves as landing gear for spatially directed docking at PIP2-rich sites that define the active zones of exocytotic release, positioning the Syt ring to clamp fusion and await calcium. Our results suggest the Syt ring is both a Ca2+-sensitive fusion clamp and a high-fidelity sensor for directed docking.

3,4,5-Trimethoxybenzoate of Catechin, an Anticarcinogenic Semisynthetic Catechin, Modulates the Physical Properties of Anionic Phospholipid Membranes

3,4,5-Trimethoxybenzoate of catechin (TMBC) is a semisynthetic catechin which shows strong antiproliferative activity against malignant melanoma cells. The amphiphilic nature of the molecule suggests that the membrane could be a potential site of action, hence the study of its interaction with lipid bilayers is mandatory in order to gain information on the effect of the catechin on the membrane properties and dynamics. Anionic phospholipids, though being minor components of the membrane, possess singular physical and biochemical properties that make them physiologically essential. Utilizing phosphatidylserine biomimetic membranes, we study the interaction between the catechin and anionic bilayers, bringing together a variety of experimental techniques and molecular dynamics simulation. The experimental data suggest that the molecule is embedded into the phosphatidylserine bilayers, where it perturbs the thermotropic gel to liquid crystalline phase transition. In the gel phase, the catechin promotes the formation of interdigitation, and in the liquid crystalline phase, it decreases the bilayer thickness and increases the hydrogen bonding pattern of the interfacial region of the bilayer. The simulation data agree with the experimental ones and indicate that the molecule is located in the interior of the anionic bilayer as monomer and small clusters reaching the carbonyl region of the phospholipid, where it also disturbs the intermolecular hydrogen bonding between neighboring lipids. Our observations suggest that the catechin incorporates well into phosphatidylserine bilayers, where it produces structural changes that could affect the functioning of the membrane.

Small molecules enhance the potency of natural antimicrobial peptides

The skin-associated microbiome plays an important role in general well-being and in a variety of treatable skin conditions. In this regard, endogenous antimicrobial peptides have both a direct and indirect role in determining the composition of the microbiota. We demonstrate here that certain small molecular species can amplify the antimicrobial potency of naturally occurring antimicrobial peptides. In this study, we have used niacinamide, a form of vitamin B3 naturally found in foods and widely used in cosmetic skincare products, and two of its structural analogs, to investigate their cooperativity with the human antimicrobial peptide LL37 on the bacterium Staphylococcus aureus. We observed a clear synergistic effect of niacinamide and, to some extent, N-methylnicotinamide, whereas isonicotinamide showed no significant cooperativity with LL37. Adaptively biased molecular dynamics simulations using simplified model membrane substrates and single peptides revealed that these molecules partition into the headgroup region of an anionic bilayer used to mimic the bacterial membrane. The simulated effects on the physical properties of the simulated model membrane are well correlated with experimental activity observed in real biological assays despite the simplicity of the model. In contrast, these molecules have little effect on zwitterionic bilayers that mimic a mammalian membrane. We conclude that niacinamide and N-methylnicotinamide can therefore potentiate the activity of host peptides by modulating the physical properties of the bacterial membrane, and to a lesser extent through direct interactions with the peptide. The level of cooperativity is strongly dependent on the detailed chemistry of the additive, suggesting an opportunity to fine-tune the behavior of host peptides.

Forces, Kinetics, and Fusion Efficiency Altered by the Full-Length Synaptotagmin-1 -PI(4,5)P2 Interaction in Constrained Geometries.

A mechanism for full-length synaptotagmin-1 (syt-1) to interact with anionic bilayers and to promote fusion in the presence of SNAREs is proposed. Colloidal probe force spectroscopy in conjunction with tethered particle motion monitoring showed that in the absence of Ca2+ the binding of syt-1 to membranes depends on the presence and content of PI(4,5)P2. Addition of Ca2+ switches the interaction forces from weak to strong, eventually exceeding the cohesion of the C2A domain of syt-1 leading to partial unfolding of the protein. Fusion of single unilamellar vesicles equipped with syt-1 and synaptobrevin 2 with planar pore-spanning target membranes containing PS and PI(4,5)P2 shows an almost complete suppression of stalled intermediate fusion states and an accelerated fusion kinetics in the presence of Ca2+, which is further enhanced upon addition of ATP.

Mechanical properties of anionic asymmetric bilayers from atomistic simulations.

Mechanotransduction, the biological response to mechanical stress, is often initiated by activation of mechanosensitive (MS) proteins upon mechanically induced deformations of the cell membrane. A current challenge in fully understanding this process is in predicting how lipid bilayers deform upon the application of mechanical stress. In this context, it is now well established that anionic lipids influence the function of many proteins. Here, we test the hypothesis that anionic lipids could indirectly modulate MS proteins by alteration of the lipid bilayer mechanical properties. Using all-atom molecular dynamics simulations, we computed the bilayer bending rigidity (KC), the area compressibility (KA), and the surface shear viscosity (ηm) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC) lipid bilayers with and without phosphatidylserine (PS) or phosphatidylinositol bisphosphate (PIP2) at physiological concentrations in the lower leaflet. Tensionless leaflets were first checked for each asymmetric bilayer model, and a formula for embedding an asymmetric channel in an asymmetric bilayer is proposed. Results from two different sized bilayers show consistently that the addition of 20% surface charge in the lower leaflet of the PC bilayer with PIP2 has minimal impact on its mechanical properties, while PS reduced the bilayer bending rigidity by 22%. As a comparison, supplementing the PIP2-enriched PC membrane with 30% cholesterol, a known rigidifying steroid lipid, produces a significant increase in all three mechanical constants. Analysis of pairwise splay moduli suggests that the effect of anionic lipids on bilayer bending rigidity largely depends on the number of anionic lipid pairs formed during simulations. The potential implication of bilayer bending rigidity is discussed in the framework of MS piezo channels.

Binding of Ca2+-independent C2 domains to lipid membranes: A multi-scale molecular dynamics study

SummaryC2 domains facilitate protein interactions with lipid bilayers in either a Ca2+-dependent or -independent manner. We used molecular dynamics (MD) simulations to explore six Ca2+-independent C2 domains, from KIBRA, PI3KC2α, RIM2, PTEN, SHIP2, and Smurf2. In coarse-grained MD simulations these C2 domains formed transient interactions with zwitterionic bilayers, compared with longer-lived interactions with anionic bilayers containing phosphatidylinositol bisphosphate (PIP2). Type I C2 domains bound non-canonically via the front, back, or side of the β sandwich, whereas type II C2 domains bound canonically, via the top loops. C2 domains interacted strongly with membranes containing PIP2, causing bound anionic lipids to cluster around the protein. Binding modes were refined via atomistic simulations. For PTEN and SHIP2, CG simulations of their phosphatase plus C2 domains with PIP2-containing bilayers were also performed, and the roles of the two domains in membrane localization compared. These studies establish a simulation protocol for membrane-recognition proteins.

Two silver chalcogenidoantimonates synthesized in piperazine and their high performances for visible-light driven Cr(VI) reduction

In the synthesis of organic hybrid metal chalcogenides, piperazine (pip) has been widely applied as a template or charge-balancing agent in the structure assembly, but application of piperazine as a solvent to synthesize organic hybrid metal chalcogenides has not been reported. In this work, two new two-dimensional layered organic hybrid silver chalcogenidoantimonates, namely [pipH2]0.5[Ag2SbS3] (1), [pipH2]0.5[Ag2SbSe3] (2), have been synthesized by using piperazine as reaction medium. Both compounds possess the similar honeycombed anionic bilayers, and the biprotonated piperazine cations are resided between the bilayers. Thermal stabilities and optical properties of compounds 1 and 2 have been studied. Moreover, compounds 1 and 2 exhibited excellent photocatalytic activities for reducing aqueous Cr(VI) under visible light irradiation.

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In the synthesis of organic hybrid metal chalcogenides, piperazine (pip) has been widely applied as a template or charge-balancing agent in the structure assembly, but application of piperazine as a solvent to synthesize organic hybrid metal chalcogenides has not been reported. In this work, two new two-dimensional layered organic hybrid silver chalcogenidoantimonates, namely [pipH2]0.5[Ag2SbS3] (1), [pipH2]0.5[Ag2SbSe3] (2), have been synthesized by using piperazine as reaction medium. Both compounds possess the similar honeycombed anionic bilayers, and the biprotonated piperazine cations are resided between the bilayers. Thermal stabilities and optical properties of compounds 1 and 2 have been studied. Moreover, compounds 1 and 2 exhibited excellent photocatalytic activities for reducing aqueous Cr(VI) under visible light irradiation.

Binding Mechanisms of Amyloid-like Peptides to Lipid Bilayers and Effects of Divalent Cations.

In several neurodegenerative diseases, cell toxicity can emerge from damage produced by amyloid aggregates to lipid membranes. The details accounting for this damage are poorly understood including how individual amyloid peptides interact with phospholipid membranes before aggregation. Here, we use all-atom molecular dynamics simulations to investigate the molecular mechanisms accounting for amyloid-membrane interactions and the role played by calcium ions in this interaction. Model peptides known to self-assemble into amyloid fibrils and bilayer made from zwitterionic and anionic lipids are used in this study. We find that both electrostatic and hydrophobic interactions contribute to peptide-bilayer binding. In particular, the attraction of peptides to lipid bilayers is dominated by electrostatic interactions between positive residues and negative phosphate moieties of lipid head groups. This attraction is stronger for anionic bilayers than for zwitterionic ones. Hydrophobicity drives the burial of nonpolar residues into the interior of the bilayer producing strong binding in our simulations. Moreover, we observe that the attraction of peptides to the bilayer is significantly reduced in the presence of calcium ions. This is due to the binding of calcium ions to negative phosphate moieties of lipid head groups, which leaves phospholipid bilayers with a net positive charge. Strong binding of the peptide to the membrane occurs less frequently in the presence of calcium ions and involves the formation of a "Ca2+ bridge".

Comparing the interaction of the antibiotic levofloxacin with zwitterionic and anionic membranes: Calorimetry, fluorescence, and spin label studies

The present work compares the interaction of the antibiotic levofloxacin (LVX) with zwitterionic and anionic liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG), respectively. By using differential scanning calorimetry (DSC), and with spin labels incorporated into liposomes at two different depths of the bilayers, we investigated the changes induced on the membrane by increasing concentrations of LVX. Further information was obtained using intrinsic LVX fluorescence. Under the conditions used here, all techniques evinced that LVX has little affinity for DPPC zwitterionic membrane. Opposite to that, LVX exhibits a considerable affinity for anionic bilayers, with membrane partition constants Kp = (3.3 ± 0.5) × 102 and (4.5 ± 0.3) × 102, for gel and fluid DPPG membranes, respectively. On binding to DPPG, LVX seems to give rise to the coexistence of LVX -rich and -poor domains on DPPG membranes, as detected by DSC. At the highest LVX concentration used (20 mol%), DSC trace shows an increase in the cooperativity of DPPG gel-fluid transition, also detected by spin labels as an increase in the bilayer packing. Moreover, LVX does not induce pore formation in either DPPG or POPG vesicles. Considering the possible relevance of LVX-membrane interaction for the biological and toxicological action of the antibiotic, the findings discussed here certainly contribute to a better understanding of its action, and the planning of new drugs.

The RIT1 C-terminus associates with lipid bilayers via charge complementarity

RIT1 is a member of the Ras superfamily of small GTPases involved in regulation of cellular signaling. Mutations to RIT1 are involved in cancer and developmental disorders. Like many Ras subfamily members, RIT1 is localized to the plasma membrane. However, RIT1 lacks the C-terminal prenylation that helps many other subfamily members adhere to cellular membranes. We used molecular dynamics simulations to examine the mechanisms by which the C-terminal peptide (CTP) of RIT1 associates with lipid bilayers. We show that the CTP is unstructured and that its membrane interactions depend on lipid composition. While a 12-residue region of the CTP binds strongly to anionic bilayers containing phosphatidylserine lipids, the CTP termini fray from the membrane allowing for accommodation of the RIT1 globular domain at the membrane-water interface.

PH-triggered pore-forming peptides with strong composition-dependent membrane selectivity

Peptides that self-assemble into nanometer-sized pores in lipid bilayers could have utility in a variety of biotechnological and clinical applications if we can understand their physical chemical properties and learn to control their membrane selectivity. To empower such control, we have used synthetic molecular evolution to identify the pH-dependent delivery peptides, a family of peptides that assemble into macromolecule-sized pores in membranes at low peptide concentration but only at pH < ∼6. Further advancements will also require better selectivity for specific membranes. Here, we determine the effect of anionic headgroups and bilayer thickness on the mechanism of action of the pH-dependent delivery peptides by measuring binding, secondary structure, and macromolecular poration. The peptide pHD15 partitions and folds equally well into zwitterionic and anionic membranes but is less potent at pore formation in phosphatidylserine-containing membranes. The peptide also binds and folds similarly in membranes of various thicknesses, but its ability to release macromolecules changes dramatically. It causes potent macromolecular poration in vesicles made from phosphatidylcholine with 14 carbon acyl chains, but macromolecular poration decreases sharply with increasing bilayer thickness and does not occur at any peptide concentration in fluid bilayers made from phosphatidylcholine lipids with 20-carbon acyl chains. The effects of headgroup and bilayer thickness on macromolecular poration cannot be accounted for by the amount of peptide bound but instead reflect an inherent selectivity of the peptide for inserting into the membrane-spanning pore state. Molecular dynamics simulations suggest that the effect of thickness is due to hydrophobic match/mismatch between the membrane-spanning peptide and the bilayer hydrocarbon. This remarkable degree of selectivity based on headgroup and especially bilayer thickness is unusual and suggests ways that pore-forming peptides with exquisite selectivity for specific membranes can be designed or evolved.