Neutral Membranes Research Articles

Effects of cholesterol on the anionic magnetite nanoparticle-induced deformation and poration of giant lipid vesicles.

We have investigated the effects of cholesterol on the deformation and poration of giant unilamellar vesicles (GUVs) induced by anionic magnetite nanoparticles (NPs). Negatively charged lipid, neutral lipid, and cholesterol were used to prepare the charged GUVs (surface charge density of membranes - 0.16 C m-2), while only neutral lipid and cholesterol were used to prepare the neutral GUVs. Cholesterol content varied from 0 to 40 mole% for preparing the biologically relevant membranes. The degree of deformation has been characterized by compactness, the value of which remains at 1.0 for spherical GUVs. The value of compactness increases with time for both membranes, but this increase depends on cholesterol content. The average compactness decreases with cholesterol content, and at 60 min, the values are 1.280 ± 0.002 and 1.131 ± 0.010 for 0 and 40 mole% cholesterol containing charged GUVs. The average compactness is relatively lower for neutral GUVs for the corresponding cholesterol. Membrane poration has been investigated by the leakage of calcein, which indicates a two-state transition model. The fraction of deformation is higher for charged GUVs than for neutral ones, while the fraction of poration shows the opposite result. Both the fractions decrease with cholesterol content.

Keratinocyte cellular damage induced by pesticide doses below the cytotoxic level evidenced by electrical impedance and broadband dielectric spectroscopy

The cellular response of a normal human keratinocyte cell line exposed to non-cytotoxic doses of a deltamethrin-based pesticide was investigated by means of two different electrical impedance data spectroscopy approaches: Nyquist plot and broadband dielectric spectroscopy. The measurements have shown that the membrane capacity increases with pesticide concentration and this facilitates the electric current through cell membranes. Furthermore, the impedance of the extracellular matrix also increases with pesticide concentration, thus reducing the electric current outside the cell. Dielectric permittivity changes in the cellular samples at frequencies larger than 100 Hz. Fluorescence (FL) measurements emphasized an increase in neutral membrane lipids as a consequence of the pesticide exposure. Comparison of FL response of pesticide exposed cells with the control ones showed a time increase in the emission intensity, suggesting the existence of a membrane lipid response aimed at repairing the cell damage due to pesticide exposure. Therefore, both spectroscopic techniques have been demonstrated as potential means to investigate the response to cell stress and damage. This opens up new possibilities in the early diagnosis of cellular modifications related to the pesticide exposure of cells.

Lipids and Phosphorylation Conjointly Modulate Complex Formation of β2-Adrenergic Receptor and β-arrestin2.

G protein-coupled receptors (GPCRs) are the largest class of human membrane proteins that bind extracellular ligands at their orthosteric binding pocket to transmit signals to the cell interior. Ligand binding evokes conformational changes in GPCRs that trigger the binding of intracellular interaction partners (G proteins, G protein kinases, and arrestins), which initiate diverse cellular responses. It has become increasingly evident that the preference of a GPCR for a certain intracellular interaction partner is modulated by a diverse range of factors, e.g., ligands or lipids embedding the transmembrane receptor. Here, by means of molecular dynamics simulations of the β2-adrenergic receptor and β-arrestin2, we study how membrane lipids and receptor phosphorylation regulate GPCR-arrestin complex conformation and dynamics. We find that phosphorylation drives the receptor’s intracellular loop 3 (ICL3) away from a native negatively charged membrane surface to interact with arrestin. If the receptor is embedded in a neutral membrane, the phosphorylated ICL3 attaches to the membrane surface, which widely opens the receptor core. This opening, which is similar to the opening in the G protein-bound state, weakens the binding of arrestin. The loss of binding specificity is manifested by shallower arrestin insertion into the receptor core and higher dynamics of the receptor-arrestin complex. Our results show that receptor phosphorylation and the local membrane composition cooperatively fine-tune GPCR-mediated signal transduction. Moreover, the results suggest that deeper understanding of complex GPCR regulation mechanisms is necessary to discover novel pathways of pharmacological intervention.

Conformational exchange of fatty acid binding protein induced by protein-nanodisc interactions

Fatty acid binding proteins (FABPs) can facilitate the transfer of long-chain fatty acids between intracellular membranes across considerable distances. The transfer process involves fatty acids, their donor membrane and acceptor membrane, and FABPs, implying that potential protein-membrane interactions exist. Despite intensive studies on FABP-membrane interactions, the interaction mode remains elusive, and the protein-membrane association and dissociation rates are inconsistent. In this study, we used nanodiscs (NDs) as mimetic membranes to investigate FABP-membrane interactions. Our NMR experiments showed that human intestinal FABP interacts weakly with both negatively charged and neutral membranes, but it prefers the negatively charged one. Through simultaneous analysis of NMR relaxation in the rotating-frame (R1ρ), relaxation dispersion, chemical exchange saturation transfer, and dark-state exchange saturation transfer data, we estimated the affinity of the protein to negatively charged NDs, the dissociation rate, and apparent association rate. We further showed that the protein in the ND-bound state adopts a conformation different from the native structure and the second helix is very likely involved in interactions with NDs. We also found a membrane-induced FABP conformational state that exists only in the presence of NDs. This state is native-like, different from other conformational states in structure, unbound to NDs, and in dynamic equilibrium with the ND-bound state.

PH-Dependent Conformations of an Antimicrobial Spider Venom Peptide, Cupiennin 1a, from Unbiased HREMD Simulations.

Cupiennin 1a is an antimicrobial peptide found in the venom of the spider Cupiennius salei. A highly cationic peptide, its cell lysis activity has been found to vary between neutral and charged membranes. In this study, Hamiltonian replica-exchange molecular dynamics (HREMD) was used to determine the conformational ensemble of the peptide in both charged (pH 3) and neutral (pH 11) states. The obtained free energy landscapes demonstrated the conformational diversity of the neutral peptide. At high pH, the peptide was found to adopt helix–hinge–helix and disordered structures. At pH 3, the peptide is structured with a high propensity toward α-helices. The presence of these α-helices seems to assist the peptide in recognizing membrane surfaces. These results highlight the importance of the charged residues in the stabilization of the peptide structure and the subsequent effects of pH on the peptide’s conformational diversity and membrane activity. These findings may provide insights into the antimicrobial activity of Cupiennin 1a and other amphipathic linear peptides toward different cell membranes.

Inverse Conformational Selection in Lipid-Protein Binding.

Interest in lipid interactions with proteins and other biomolecules is emerging not only in fundamental biochemistry but also in the field of nanobiotechnology where lipids are commonly used, for example, in carriers of mRNA vaccines. The outward-facing components of cellular membranes and lipid nanoparticles, the lipid headgroups, regulate membrane interactions with approaching substances, such as proteins, drugs, RNA, or viruses. Because lipid headgroup conformational ensembles have not been experimentally determined in physiologically relevant conditions, an essential question about their interactions with other biomolecules remains unanswered: Do headgroups exchange between a few rigid structures, or fluctuate freely across a practically continuous spectrum of conformations? Here, we combine solid-state NMR experiments and molecular dynamics simulations from the NMRlipids Project to resolve the conformational ensembles of headgroups of four key lipid types in various biologically relevant conditions. We find that lipid headgroups sample a wide range of overlapping conformations in both neutral and charged cellular membranes, and that differences in the headgroup chemistry manifest only in probability distributions of conformations. Furthermore, the analysis of 894 protein-bound lipid structures from the Protein Data Bank suggests that lipids can bind to proteins in a wide range of conformations, which are not limited by the headgroup chemistry. We propose that lipids can select a suitable headgroup conformation from the wide range available to them to fit the various binding sites in proteins. The proposed inverse conformational selection model will extend also to lipid binding to targets other than proteins, such as drugs, RNA, and viruses.

Specific and nondisruptive interaction of guanidium-functionalized gold nanoparticles with neutral phospholipid bilayers

Understanding and controlling the interaction between nanoparticles and biological entities is fundamental to the development of nanomedicine applications. In particular, the possibility to realize nanoparticles capable of directly targeting neutral lipid membranes would be advantageous to numerous applications aiming at delivering nanoparticles and their cargos into cells and biological vesicles. Here, we use experimental and computational methodologies to analyze the interaction between liposomes and gold nanoparticles (AuNPs) featuring cationic headgroups in their protecting monolayer. We find that in contrast to nanoparticles decorated with other positively charged headgroups, guanidinium-coated AuNPs can bind to neutral phosphatidylcholine liposomes, inducing nondisruptive membrane permeabilization. Atomistic molecular simulations reveal that this ability is due to the multivalent H-bonding interaction between the phosphate residues of the liposome’s phospholipids and the guanidinium groups. Our results demonstrate that the peculiar properties of arginine magic, an effect responsible for the membranotropic properties of some naturally occurring peptides, are also displayed by guanidinium-bearing functionalized AuNPs.

Comparative Life Cycle Assessment of CO2 Electrosynthesis to Fuels and Feedstocks

Development of electrochemical pathways to convert CO2 into fuels and feedstock is rapidly progressing over the past decade. Here we present a comparative cradle-to-gate life cycle assessment (LCA) of one and two-step electrochemical conversion of CO2 to eight major value-added products; wherein we consider CO2 capture, conversion and product separation in our process model. We measure the carbon intensity (i.e., global warming impact) of one and two-step electrochemical routes with its counterparts – thermochemical CO2 utilization and fossil-fuel based incumbent synthesis routes for those products. Due to inevitable carbonate formation or CO2 crossover in one-step CO2 electrolysis using neutral pH membrane electrode assembly, the two-step electrosynthesis pathways (CO2 to CO in solid oxide electrolysis cell followed by CO electroreduction in alkaline flow cell) would be distinctively compelling through the lens of climate benefits. This analysis further reveals that the carbon intensity of electrosynthesis products is due to significant energy requirement for conversion (75–85% of total energy consumption for gas products) and product separation (30–85% of total energy consumption for liquid products) phases. Electrochemical route is highly sensitive to the electricity emission intensity and is compelling only when coupled with low emission intensity (<0.25 kg CO2e per kWh). As the technology advances, we identify near-term compelling products that would provide climate benefits over incumbent routes, including syngas, ethylene and n-propanol. We further identify technological goals required for electrochemical route to be competitive, notably achieving liquid product concentration >20 wt%. It is our hope that this analysis will guide the CO2 electrosynthesis community to target achieving these technological goals, such that when coupled with low-carbon electricity, electrochemical route would bring climate benefits in near future.

Recent advances in catalyst materials for proton exchange membrane fuel cells

Research on fuel cell technology is constantly gaining importance, while global emission requirements are becoming more and more restrictive. For environmentally neutral proton exchange membrane fuel cells (PEMFCs) to become a competitive technology, sustainable infrastructures need to be established. One of the main showstoppers is the utilization of the rare and therefore costly precious metal Pt as the key element in the electrocatalysis of hydrogen and oxygen. A huge amount of research is done on immensely reducing or even replacing Pt for future PEMFC technology. In this research update, the progress on oxygen reduction reaction catalysts in acidic media over the past two years is reviewed, with special attention to their durability.

Tuning of a Membrane-Perforating Antimicrobial Peptide to Selectively Target Membranes of Different Lipid Composition.

The use of designed antimicrobial peptides as drugs has been impeded by the absence of simple sequence-structure-function relationships and design rules. The likely cause is that many of these peptides permeabilize membranes via highly disordered, heterogeneous mechanisms, forming aggregates without well-defined tertiary or secondary structure. We suggest that the combination of high-throughput library screening with atomistic computer simulations can successfully address this challenge by tuning a previously developed general pore-forming peptide into a selective pore-former for different lipid types. A library of 2916 peptides was designed based on the LDKA template. The library peptides were synthesized and screened using a high-throughput orthogonal vesicle leakage assay. Dyes of different sizes were entrapped inside vesicles with varying lipid composition to simultaneously screen for both pore size and affinity for negatively charged and neutral lipid membranes. From this screen, nine different LDKA variants that have unique activity were selected, sequenced, synthesized, and characterized. Despite the minor sequence changes, each of these peptides has unique functional properties, forming either small or large pores and being selective for either neutral or anionic lipid bilayers. Long-scale, unbiased atomistic molecular dynamics (MD) simulations directly reveal that rather than rigid, well-defined pores, these peptides can form a large repertoire of functional dynamic and heterogeneous aggregates, strongly affected by single mutations. Predicting the propensity to aggregate and assemble in a given environment from sequence alone holds the key to functional prediction of membrane permeabilization.

Non-disruptive uptake of anionic and cationic gold nanoparticles in neutral zwitterionic membranes

The potential toxicity of ligand-protected nanoparticles (NPs) on biological targets is crucial for their clinical translation. A number of studies are aimed at investigating the molecular mechanisms shaping the interactions between synthetic NPs and neutral plasma membranes. The role played by the NP surface charge is still widely debated. We compare, via liposome leakage assays, the perturbation induced by the penetration of sub-6 nm anionic and cationic Au NPs into model neutral lipid membranes composed of the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Our charged Au NPs are functionalized by a mixture of the apolar 1-octanethiol and a ω-charged thiol which is either the anionic 11-mercapto-1-undecanesulfonate or the cationic (11-mercaptoundecyl)-N,N,N-trimethylammonium. In both cases, the NP uptake in the bilayer is confirmed by quartz crystal microbalance investigations. Our leakage assays show that both negatively and positively charged Au NPs do not induce significant membrane damage on POPC liposomes when penetrating into the bilayer. By means of molecular dynamics simulations, we show that the energy barrier for membrane penetration is the same for both NPs. These results suggest that the sign of the NP surface charge, per se, does not imply different physicochemical mechanisms of interaction with zwitterionic lipid membranes.

A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids.

Infections caused by Gram-negative bacteria are difficult to fight because these pathogens exclude or expel many clinical antibiotics and host defense molecules. However, mammals have evolved a substantial immune arsenal that weakens pathogen defenses, suggesting the feasibility of developing therapies that work in concert with innate immunity to kill Gram-negative bacteria. Using chemical genetics, we recently identified a small molecule, JD1, that kills Salmonella enterica serovar Typhimurium (S. Typhimurium) residing within macrophages. JD1 is not antibacterial in standard microbiological media, but rapidly inhibits growth and curtails bacterial survival under broth conditions that compromise the outer membrane or reduce efflux pump activity. Using a combination of cellular indicators and super resolution microscopy, we found that JD1 damaged bacterial cytoplasmic membranes by increasing fluidity, disrupting barrier function, and causing the formation of membrane distortions. We quantified macrophage cell membrane integrity and mitochondrial membrane potential and found that disruption of eukaryotic cell membranes required approximately 30-fold more JD1 than was needed to kill bacteria in macrophages. Moreover, JD1 preferentially damaged liposomes with compositions similar to E. coli inner membranes versus mammalian cell membranes. Cholesterol, a component of mammalian cell membranes, was protective in the presence of neutral lipids. In mice, intraperitoneal administration of JD1 reduced tissue colonization by S. Typhimurium. These observations indicate that during infection, JD1 gains access to and disrupts the cytoplasmic membrane of Gram-negative bacteria, and that neutral lipids and cholesterol protect mammalian membranes from JD1-mediated damage. Thus, it may be possible to develop therapeutics that exploit host innate immunity to gain access to Gram-negative bacteria and then preferentially damage the bacterial cell membrane over host membranes.

Non-Receptor-Mediated Lipid Membrane Permeabilization by the SARS-CoV-2 Spike Protein S1 Subunit.

Due to the pressing need to generate specific drugs or vaccines for COVID-19 and management of its outbreak, detailed knowledge regarding the SARS-CoV-2 entry into host cells and timely, cheap, and easy-to-use detection methods are of critical importance for containing the SARS-CoV-2 epidemic. Through electrophysiology and fluorescence spectroscopy experiments, we show that even in the absence of the angiotensin-converting enzyme 2 receptor, the S1 subunit from SARS-CoV-2 spike protein binding to neutral phospholipid membranes leads to their mechanical destabilization and permeabilization. A similar cytotoxic effect of the protein was seen in human lung epithelial cells. A monoclonal antibody generated toward the S1 subunit alleviates to a considerable extent the destabilizing potential of the protein in such model membranes. Finally, we demonstrate the proof-of-concept capability of an α-hemolysin (α-HL) protein nanopore to detect in aqueous buffer and real time the region-binding domain of the S1 subunit from SARS-CoV-2 spike protein by monitoring its immunological interaction with a target antibody. Our results may offer new perspectives in understanding the pathogenesis of the SARS-CoV-2 infection, its treatment, and real-time detection.

Interactions of an Anionic Antimicrobial Peptide with Zinc(II): Application to Bacterial Mimetic Membranes.

While the majority of known antimicrobial peptides are cationic, a small number consist of short Asp-rich sequences that are anionic. These require metal ions to become biologically active. Here, we report the study of the zinc complexes of the peptide GADDDDD (GAD5), an antimicrobial peptide. Using a combination of dynamic light scattering (DLS), ζ-potential, infrared, Raman, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM), we find that adding zinc ions to GAD5 forces it into a compact structure. Higher amounts of zinc ions favor a larger structure, possibly a dimer. SEM images show that zinc ions reduce the size of the fibrillar structures of GAD5. TGA curves show that the addition of zinc ions increases the thermal stability of the structure of the peptide. TGA and DSC indicate that the association of GAD5 with a zwitterionic phospholipid in the presence of zinc ions is the most stable. The stability of that complex is due to the presence of a sharp endothermic peak in the 200-300 °C range, suggesting the presence of interlamellar water that is essential to the stabilization of the structure. These results indicate that the Zn-GAD5 complex prefers the bacteria-mimicking neutral (zwitterionic) membranes. In the presence of negatively charged phospholipids, the complex remains unordered and unstable. In terms of mechanism of action, the Zn-GAD5 complex promotes a possible endocytic uptake with respect to neutral (zwitterionic) membranes while promoting membrane disruption by forming pores with respect to negatively charged phospholipids.

Electronic response of phagraphene membranes to excess charge carriers

We have investigated the technologically relevant, yet unmapped effect of excess charge carriers on electronic structure and electric conductivity of phagraphene (PG) membranes within the framework of density functional theory and the pseudopotential approach. The charge of the reference unit cell then changes from zero to n = ±e, ±2e, and ±3e, with e the magnitude of elementary charge. Electronic structure of the resulted (PG)n membranes have then been investigated at the PBE level via calculating the related electronic band structures and density of states. A non-zero electronegativity has accordingly been inferred for the neutral membrane showing its tendency to attract electron density when it is placed in contact with other materials. Results also broadly support the view that the electronic response of phagraphene to excess charge carriers manifests itself as augmentation in electric conductivity of the system.

Deformation and poration of giant unilamellar vesicles induced by anionic nanoparticles

The interaction of anionic magnetite nanoparticles (MNPs) of size 18 nm with negatively charged giant unilamellar vesicles (GUVs) formed from a mixture of neutral dioleoylphosphatidylcholine (DOPC) and negatively charged dioleoylphosphatidylglycerol (DOPG) lipids has been investigated. It has been obtained that NPs induces the deformation of spherical GUVs. The reaction of other GUVs on NPs consists in the appearance of pores in their membranes. We focused the effect of electrostatics on the interaction of charged membranes with MNPs. To study the influence of the surface charge of GUVs on the processes under consideration, we varied the fraction of DOPG in the vesicles from 0 to 100%. We examined the influence of salt concentration in the range of 50−300 mM NaCl concentration. To describe the degree of deformation, a special parameter compactness was introduced. The pore formation in the membranes of GUVs was investigated by the leakage of sucrose. The compactness increases with time and also NPs concentration. The fraction of deformed GUVs increases with the increase of surface charge density of membranes as well as the decrease of salt concentration in buffer. The value of compactness for neutral membrane is 1.25 times higher than that of charged ones. The fraction of deformed GUVs become constant after 20 min, however it increases with NPs concentration. The time taken for stochastic pore formation is less for charged membrane than neutral one. The physical mechanism explaining the experimental results obtained in these investigations.

Microfluidic-Based Holonomic Constraints of siRNA in the Kernel of Lipid/Polymer Hybrid Nanoassemblies for Improving Stable and Safe In Vivo Delivery.

A safe and efficient delivery system is critical for clinical application of siRNA. However, the conventional electrostatic interaction-based siRNA nanoplexes with bulk mixing preparation were always unsatisfactory for its stability and safety. In this study, the new core-shell lipid/PCL-PEI/siRNA nanoparticles (LPS NPs) endowing holonomic constraint of siRNA in the inner core were prepared by microfluidic technology. On the microfluidic chip, siRNAs were completely compressed into the inner hydrophilic core of reverse PCL-PEI micelles at a low N/P ratio of 5, followed by coating a neutral lipid membrane to form core-shell nanoparticles, which had a uniform size (120.2 ± 1.4 nm) and a negative charge (-8.8 ± 1.6 mV). Compared to bulk mixing-based LMS NPs, the lower usage of cationic PCL-PEI materials and stronger protection of siRNA in serum were found in the microfluidic-based LPS NPs. Furthermore, it was demonstrated that the LPS NPs exhibited significant downregulation of EGFR mRNA and protein expression level both in vitro and in vivo, and showed significant inhibition of tumor growth following systemic administration along with no obvious systemic toxicity. These findings demonstrated that the microfluidic-based lipid/polymer hybrid nanoassemblies would offer a promising siRNA delivery system for clinical application.

A Synthetic Carrier of Nucleic Acids Structured as a Neutral Phospholipid Envelope Tightly Assembled on Polyplex Surface.

Synthetic carriers of nucleic acids remain inefficient for practical applications due to their insufficient functions as compared with viral vectors developed by evolution. Here, a synthetic carrier is designed to structurally mimic lentivirus, a widely-used viral vector in therapeutic developments, for its neutral phospholipid membrane tightly anchored on the surface of a packed nucleic acid core. Unlike the reported lipopolyplexes of which the surface membrane around the nucleic acid core is formed from charged lipids, the stable attachment of the neutral lipids to each polyplex core in the present system is achieved through preadsorbed micelles of multicarboxyl amphiphilic molecules as lipid bilayer anchors. The adsorbed micelles are under a tension of deformation due to the electrostatic attraction of the head groups to the cationic surface and their "thermodynamic responsibility" to cover the hydrophobic tails in water. When liposomes of neutral phospholipids approach, the hydrophobic tail groups of the adsorbed micelles may insert into the lipid bilayer matrix to induce them to fuse around polyplex and relieve the thermodynamic tension. The formed neutral phospholipid membrane may encapsulate the polyplex core stably, prevent siRNA from prephagocytic leaking and degrading, and immobilize functional agents with increased capacity.

Schwinger-Dyson equations for composite electrolytes governed by mixed electrostatic couplings strengths.

The electrostatic Schwinger-Dyson equations are derived and solved for an electrolyte mixture composed of monovalent and multivalent ions confined to a negatively charged nanoslit. The closure of these equations is based on an asymmetric treatment of the ionic species with respect to their electrostatic coupling strength: the weakly coupled monovalent ions are treated within a gaussian approximation, while the multivalent counterions of high coupling strength are incorporated with a strong-coupling approach. The resulting self-consistent formalism includes explicitly the interactions of the multivalent counterions with the monovalent salt. In highly charged membranes characterized by a pronounced multivalent counterion adsorption, these interactions take over the salt-membrane charge coupling. As a result, the increment of the negative membrane charge brings further salt anions into the slit pore and excludes salt cations from the pore into the reservoir. The corresponding like-charge attraction and opposite-charge repulsion effect is amplified by the pore confinement but suppressed by salt addition into the reservoir. The effect is particularly pronounced in high dielectric membranes where the attractive polarization forces lead to a dense multivalent cation layer at the membrane walls. These cation layers act as an effective positive surface charge, resulting in a total monovalent cation exclusion and a strong anion excess even in the case of neutral membrane walls.

Molecular dynamics study of membrane permeabilization by wild-type and mutant lytic peptides from the non-enveloped Flock House virus

Flock House virus (FHV) serves as a model system for understanding infection mechanisms utilized by non-enveloped viruses to transport across cellular membranes. During the infection cycle of FHV, a fundamental stage involves disruption of the endosomal membrane by membrane active peptides, following externalization of the peptides from the capsid interior. The FHV lytic agents are the 44 C-terminal amino acids residues of the capsid protein, which are auto-catalytically cleaved during the capsid maturation process. The cleaved peptides are termed γ peptides. In this study, we perform multi-scale molecular dynamics simulations including 40 μs all-atom molecular dynamics simulations to study the behavior of pre-inserted transmembrane lytic peptides at a high concentration in a neutral membrane. We study the dynamical organization among peptides to form oligomeric bundles in four systems including the wild-type γ peptide and three mutant forms; namely, a truncation mutant in which the 23 C-terminal residues are deleted (γ1), a construct where the 8 C-terminal residues of γ are fused to γ1 (Δ385–399 γ) and a single-point mutant (F402A γ), all of which have been experimentally shown to drastically affect infectivity and lytic activity compared to the wild-type γ. Our results shed light on the actions of varied forms of the FHV lytic peptide including membrane insertion, trans-membrane stability, peptide oligomerization, water permeation activity and dynamic pore formation. Findings from this study provide detailed structural information and rationale for the differences in lytic activity among variants of FHV γ.