Phospholipid Headgroups Research Articles

Defect Formation and Peptide Permeation across Phospholipid Membranes.

Cell penetrating peptides (CPPs) are natural agents that efficiently permeate biological membranes. They are frequently positively charged, which is surprising since membranes pose hydrophobic barriers. In this Perspective, I discuss computations and experiments of a permeation model that couples permeant displacement with a membrane defect. We call the proposed mechanism Defect Assisted by Charge (DAC) and illustrate that it reduces the free energy barrier for translocation. A metastable state at the center of the membrane may be observed due to the charge interactions with the phospholipid head groups at the two leaflets. The combination of experiments and simulations sheds light on the mechanisms of a charged peptide translocation across phospholipid membranes.

Phase behaviour and structural properties of SOPC model lipid system in a sucrose solution

Lipid membranes are an important component of the biological cell. The profound understanding of their structure and functionality, as well as, the influence of various biologically relevant admixtures on their main characteristics is of great importance for research and development in medicine and pharmacology. The effect of sugars on the behaviour of the membrane cell enjoys an ever-increasing interest as they are biologically significant substances. We have studied the influence of the disaccharide sucrose on the physicochemical properties of SOPC (1-stearoyl-2-oleoyl-sn- glycero-3-phosphocholine) lipid system aiming to gain better understanding of the mechanisms of the interaction between both substances. For that purpose, we have used differential scanning calorimetry and Fourier-transform infrared spectroscopy. Our results show that adding sugar up to 300 mM concentration substantially alters the thermodynamic and structural properties of SOPC. The DSC thermograms at heating reveal a general lowering of the SOPC transition temperature Tm from gel to liquid crystalline phase (main phase transition, ordered-disordered phase transition) in the presence of sugar. The corresponding peaks are smeared and harder to trace. In agreement with this, a gradual decrease of the enthalpy values up to 300 mM was measured. The IR spectroscopy study provided spectral evidence for two states of hydration of the phosphate groups in the sugar-SOPC model systems suggesting a mechanism of interaction where only part of the phospholipid headgroups are hydrogen bonded to the sugar molecules. The obtained results are in good agreement with various earlier data including results about the bending elasticity moduli, as well as, some theoretical simulations on the sugar-lipid interactions. The current results also reinforce the potential of sucrose to be used as a cell protector against drought at, both, high and low temperatures.

Quasi-elastic neutron scattering reveals the relationship between the dynamical behavior of phospholipid headgroups and hydration water.

The dynamics of hydration water (HW) in 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) was investigated by means of quasi-elastic neutron scattering (QENS) and compared with those observed in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The headgroup dynamics of DMPE was investigated using a mixture of tail-deuterated DMPE and D2O, and the QENS profiles were interpreted as consisting of three modes. The fast mode comprised the rotation of hydrogen atoms in -NH3+ and -CH2- groups in the headgroup of DMPE, the medium-speed mode comprised fluctuations in the entire DMPE molecule, and the slow mode comprised fluctuations in the membrane. These interpretations were confirmed using molecular dynamics (MD) simulations. The HW dynamics analysis was performed on a tail-deuterated DMPE and H2O mixture. The QENS profiles were analyzed in terms of three modes: (1) a slow mode, identified as loosely bound HW in the DMPC membrane; (2) a medium-speed mode similar to free HW in the DMPC membrane; and (3) a fast mode, identified as rotational motion. The relaxation time for the fast mode was approximately six times shorter than that of rotational water in DMPC, consistent with the results of terahertz time-domain spectroscopy. The activation energy of medium-speed HW in DMPE differed from that of free HW in DMPC, suggesting the presence of different hydration states or hydrogen-bonded networks around the phosphocholine and phosphoethanolamine headgroups.

The structure of phosphatidylinositol remodeling MBOAT7 reveals its catalytic mechanism and enables inhibitor identification

Cells remodel glycerophospholipid acyl chains via the Lands cycle to adjust membrane properties. Membrane-bound O-acyltransferase (MBOAT) 7 acylates lyso-phosphatidylinositol (lyso-PI) with arachidonyl-CoA. MBOAT7 mutations cause brain developmental disorders, and reduced expression is linked to fatty liver disease. In contrast, increased MBOAT7 expression is linked to hepatocellular and renal cancers. The mechanistic basis of MBOAT7 catalysis and substrate selectivity are unknown. Here, we report the structure and a model for the catalytic mechanism of human MBOAT7. Arachidonyl-CoA and lyso-PI access the catalytic center through a twisted tunnel from the cytosol and lumenal sides, respectively. N-terminal residues on the ER lumenal side determine phospholipid headgroup selectivity: swapping them between MBOATs 1, 5, and 7 converts enzyme specificity for different lyso-phospholipids. Finally, the MBOAT7 structure and virtual screening enabled identification of small-molecule inhibitors that may serve as lead compounds for pharmacologic development.

Lysophosphatidylglycerol (LPG) phospholipase D maintains membrane homeostasis in Staphylococcus aureus by converting LPG to lysophosphatidic acid

Lysophospholipids are deacylated derivatives of their bilayer forming phospholipid counterparts that are present at low concentrations in cells. Phosphatidylglycerol (PG) is the principal membrane phospholipid in Staphylococcus aureus and lysophosphatidylglycerol (LPG) is detected in low abundance. Here, we used a mass spectrometry screen to identify locus SAUSA300_1020 as the gene responsible for maintaining low concentrations of 1-acyl-LPG in S.aureus. The SAUSA300_1020 gene encodes a protein with a predicted amino terminal transmembrane α-helix attached to a globular glycerophosphodiester phosphodiesterase (GDPD) domain. We determined that the purified protein lacking the hydrophobic helix (LpgDΔN) possesses cation-dependent lysophosphatidylglycerol phospholipase D activity that generates both lysophosphatidic acid (LPA) and cyclic-LPA products and hydrolyzes cyclic-LPA to LPA. Mn2+ was the highest affinity cation and stabilized LpgDΔN to thermal denaturation. LpgDΔN was not specific for the phospholipid headgroup and degraded 1-acyl-LPG, but not 2-acyl-LPG. Furthermore, a 2.1Å crystal structure shows that LpgDΔN adopts the GDPD variation of the TIM barrel architecture except for the length and positioning of helix α6 and sheet β7. These alterations create a hydrophobic diffusion path for LPG to access the active site. The LpgD active site has the canonical GDPD metal binding and catalytic residues, and our biochemical characterization of site-directed mutants support a two-step mechanism involving a cyclic-LPA intermediate. Thus, the physiological function of LpgD in S.aureus is to convert LPG to LPA, which is re-cycled into the PG biosynthetic pathway at the LPA acyltransferase step to maintain membrane PG molecular species homeostasis.

Gold-Supported Lipid Membranes Formed by Redox-Triggered Vesicle Fusion on Binary Self-Assembled Monolayers: Ion-Pairing Association and Surface Hydrophilicity.

The assembly of biomimetic, planar supported lipid bilayers (SLBs) by the popular vesicle fusion method, which relies on the spontaneous adsorption and rupture of small unilamellar vesicles from aqueous solution on a solid surface, typically works with a limited range of support materials and lipid systems. We previously reported a conceptual advance in the formation of SLBs from vesicles in the gel or fluid phase using the interfacial ion-pairing association of charged phospholipid headgroups with electrochemically generated cationic ferroceniums bound to a self-assembled monolayer (SAM) chemisorbed to gold. This redox-driven approach lays down a single bilayer membrane on the SAM-modified gold surface at room temperature within minutes and is compatible with both anionic and zwitterionic phospholipids. The present work explores the effects of the surface ferrocene concentration and hydrophobicity/hydrophilicity on the formation of continuous SLBs of dialkyl phosphatidylserine, dialkyl phosphatidylglycerol, and dialkyl phosphatidylcholine using binary SAMs of ferrocenylundecanethiolate (FcC11S) and dodecanethiolate (CH3C11S) or hydroxylundecanethiolate (HOC11S) comprising different surface mole fractions of ferrocene (χFcsurf). An increase in the surface hydrophilicity and surface free energy of the FcC11S/HOC11S SAM mitigates the decrease in the attractive ion-pairing interactions resulting from a reduced χFcsurf. SLBs of ≳80% area coverage form on the FcC11S/HOC11S SAM for all the phospholipid types down to χFcsurf of at least 0.2, composition yielding a water contact angle (θW) of 44 ± 4°. By contrast, a greater number of ion-pairing interactions is required on the hydrophobic FcC11S/CH3C11S surface to drive the vesicle fusion process; bilayers or bilayer patches form at χFcsurf ≳ 0.6 (θW = 97 ± 3°). These findings will aid in tailoring the surface chemistry of redox-active modified surfaces to widen the conditions that yield supported lipid membranes.

Activity-based directed evolution of a membrane editor in mammalian cells.

Cellular membranes contain numerous lipid species, and efforts to understand the biological functions of individual lipids have been stymied by a lack of approaches for controlled modulation of membrane composition in situ. Here we present a strategy for editing phospholipids, the most abundant lipids in biological membranes. Our membrane editor is based on a bacterial phospholipase D (PLD), which exchanges phospholipid head groups through hydrolysis or transphosphatidylation of phosphatidylcholine with water or exogenous alcohols. Exploiting activity-dependent directed enzyme evolution in mammalian cells, we have developed and structurally characterized a family of 'superPLDs' with up to a 100-fold enhancement in intracellular activity. We demonstrate the utility of superPLDs for both optogenetics-enabled editing of phospholipids within specific organelle membranes in live cells and biocatalytic synthesis of natural and unnatural designer phospholipids in vitro. Beyond the superPLDs, activity-based directed enzyme evolution in mammalian cells is a generalizable approach to engineer additional chemoenzymatic biomolecule editors.

Interaction between Iron Nitrosyl Complexes and Phosphatidylcholine Membranes: A Fluorescence Study

Fluorescent probes: 8-anilino-1-naphthalenesulfonate (ANS), eosin Y, and pyrene were used to study the interaction between liposomes and three nitrosyl iron complexes that are promising anti-inflammatory agents and cardioprotectors. It was shown that the studied complexes compete with ANS molecules for the bonding sites in a bilayer of liposomes. The incorporation of the complexes into the lipid bilayer was also studied according to the quenching of eosin Y and pyrene fluorescence. The data suggest that iron nitrosyl complexes interact with the phospholipids headgroups and can penetrate deeper into the hydrophobic center of a lipid bilayer, where they affect the packing of fatty acid chains. The pronounced membranotropic properties of the complexes correlated with their ability to inhibit lipid peroxidation. Complexes with high constants of pyrene bonding are the most effective antioxidants.

Bacterial catabolism of membrane phospholipids links marine biogeochemical cycles.

In marine systems, the availability of inorganic phosphate can limit primary production leading to bacterial and phytoplankton utilization of the plethora of organic forms available. Among these are phospholipids that form the lipid bilayer of all cells as well as released extracellular vesicles. However, information on phospholipid degradation is almost nonexistent despite their relevance for biogeochemical cycling. Here, we identify complete catabolic pathways for the degradation of the common phospholipid headgroups phosphocholine (PC) and phosphorylethanolamine (PE) in marine bacteria. Using Phaeobacter sp. MED193 as a model, we provide genetic and biochemical evidence that extracellular hydrolysis of phospholipids liberates the nitrogen-containing substrates ethanolamine and choline. Transporters for ethanolamine (EtoX) and choline (BetT) are ubiquitous and highly expressed in the global ocean throughout the water column, highlighting the importance of phospholipid and especially PE catabolism in situ. Thus, catabolic activation of the ethanolamine and choline degradation pathways, subsequent to phospholipid metabolism, specifically links, and hence unites, the phosphorus, nitrogen, and carbon cycles.

Impact of non-ionizable lipids and phase mixing methods on structural properties of lipid nanoparticle formulations

Lipid nanoparticles (LNPs) have been widely investigated for nucleic acid therapeutic delivery, and demonstrated their potential in enabling new mRNA vaccines. LNPs are usually formulated with multi-lipid components and the composition variables may impact their structural properties. Here, we investigated the impact of helper lipids on physicochemical properties of LNPs using a Design of Experiments (DoE) definitive screening design. Phospholipid head group, degree of unsaturation, ratio to cholesterol as well as PEG-lipid content were varied and a series of 14 LNPs were prepared by microfluidic- and solvent-injection mixing. Solvent-injection mixing by a robotic liquid handler yielded 50–225 nm nanoparticles with highly ordered, ∼5 nm inter-lamellar spacing as measured by small angle X-ray scattering (SAXS) and confirmed by cryo-transmission electron microscopy (cryo-EM).In contrast, microfluidic mixing resulted in less ordered, notably smaller (50–75 nm) and more homogenous nanoparticles. Significant impacts of the stealth-lipid DSPE-PEG2000 on nanoparticle size, polydispersity and encapsulation efficiency of an oligonucleotide cargo were observed in LNPs produced by both methods, while varying the phospholipid type and content had only marginal effect on these physicochemical properties. These findings suggest that from a physicochemical perspective, the design space for combinations of helper lipids in LNPs may be considerably larger than anticipated based on the conservative formulation composition of the currently FDA-approved LNPs, thereby opening opportunities for screening and optimization of novel LNP formulations.

The effect of methylated phosphatidylethanolamine derivatives on the ionization properties of signaling phosphatidic acid

Phosphatidylethanolamine (PE) and Phosphatidylcholine (PC) are the most abundant glycerophospholipids in eukaryotic membranes. The differences in the physicochemical properties of their headgroups have contrasting modulatory effects on their interaction with intracellular macromolecules. As such, their overall impact on membrane structure and function differs significantly. Enzymatic methylation of PE's amine headgroup produces two methylated derivatives namely monomethyl PE (MMPE) and dimethyl PE (DMPE) which have physicochemical properties that generally range between that of PE and PC. Additionally, their influence on membrane properties differs from both PE and PC. Although variations in headgroup methylation have been reported to affect signaling pathways, the direct influence that these differences exert on the ionization properties of signaling phospholipids have not been investigated. Here, we briefly review membrane function and structure that are mediated by the differences in headgroup methylation between PE, MMPE, DMPE and PC. In addition, using 31P MAS NMR, we investigate the effect of these four phospholipids on the ionization properties of the ubiquitous signaling anionic lipid phosphatidic acid (PA). Our results show that PA's ionization properties are differentially affected by changes in phospholipid headgroup methylation. This could have important implications for PA-protein binding and hence physiological functions in cells where signaling events lead to changes in abundance of methylated PE derivatives in the membrane.

A mechanistic understanding of the effects of polyethylene terephthalate nanoplastics in the zebrafish (Danio rerio) embryo

Plastic pollution, especially by nanoplastics (NPs), has become an emerging topic due to the widespread existence and accumulation in the environment. The research on bioaccumulation and toxicity mechanism of NPs from polyethylene terephthalate (PET), which is widely used for packaging material, have been poorly investigated. Herein, we report the first use of high-resolution magic-angle spinning (HRMAS) NMR based metabolomics in combination with toxicity assay and behavioural end points to get systems-level understanding of toxicity mechanism of PET NPs in intact zebrafish embryos. PET NPs exhibited significant alterations on hatching and survival rate. Accumulation of PET NPs in larvae were observed in liver, intestine, and kidney, which coincide with localization of reactive oxygen species in these areas. HRMAS NMR data reveal that PET NPs cause: (1) significant alteration of metabolites related to targeting of the liver and pathways associated with detoxification and oxidative stress; (2) impairment of mitochondrial membrane integrity as reflected by elevated levels of polar head groups of phospholipids; (3) cellular bioenergetics as evidenced by changes in numerous metabolites associated with interrelated pathways of energy metabolism. Taken together, this work provides for the first time a comprehensive system level understanding of toxicity mechanism of PET NPs exposure in intact larvae.

Dissecting the mechanisms of environment sensitivity of smart probes for quantitative assessment of membrane properties.

The plasma membrane, as a highly complex cell organelle, serves as a crucial platform for a multitude of cellular processes. Its collective biophysical properties are largely determined by the structural diversity of the different lipid species it accommodates. Therefore, a detailed investigation of biophysical properties of the plasma membrane is of utmost importance for a comprehensive understanding of biological processes occurring therein. During the past two decades several environment-sensitive probes have been developed and become popular tools to investigate membrane properties. Although these probes are assumed to report on membrane order in similar ways, their individual mechanisms remain to be elucidated. In this study, using model membrane systems, we characterized the probes Pro12A, NR12S and NR12A in depth and examined their sensitivity to parameters with potential biological implications, such as the degree of lipid saturation, double bond position and configuration (cis vs. trans), phospholipid headgroup and cholesterol content. Applying spectral imaging together with atomistic molecular dynamics simulations and time-dependent fluorescent shift analyses, we unravelled individual sensitivities of these probes to different biophysical properties, their distinct localizations and specific relaxation processes in membranes. Overall, Pro12A, NR12S and NR12A serve together as a toolbox with a wide range of applications allowing to select the most appropriate probe for each specific research question.

Small changes in lipid backbone linkages can lead to big changes in liquid-liquid phase separation in model membranes.

Cells can dynamically alter the spatial organization of lipids and proteins in the plasma membrane (PM) to regulate processes including cell signaling. The size and morphology of membrane domains, and how these depend on lipid composition, have drawn attention as potentially important variables in these processes. Model membrane studies have proven invaluable for elucidating the influence of phospholipid chains and headgroups, as well as cholesterol concentration, on membrane phase behavior. Following this approach, we have investigated simplified three- and four-component models for the PM outer leaflet to understand the role of the lipid backbone. Specifically, we replaced ester-linked DPPC and DOPC with their ether-linked counterparts to selectively change the membrane dipole potential of coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases, while titrating DOPC with POPC to control the line tension. Fluorescence imaging of GUVs gave insight into the macroscopic phase behavior, while FRET and cryo-EM of LUVs allowed us to probe nanoscale heterogeneity. Our results can be understood in terms of a model in which the tendency to minimize line tension is counteracted by dipole repulsion, which can in some cases result in stable arrays of small domains or modulated phases. These findings suggest that relatively small changes in the structure of the lipid backbone can influence domain formation and morphology in biomimetic membranes.

Polyunsaturated fatty acids and the effect of phospholipid headgroup on lipid rafts.

Omega-3 polyunsaturated fatty acids (n-3 PUFA) are commonly consumed in fish oil supplements for a variety of associated health benefits. Here we investigate the premise that there is an underlying mechanism by which n-3 PUFA incorporated into membrane phospholipids regulate the architecture and functionality of lipid rafts. We ran coarse-grained (CG) MD simulations on lipid mixtures comprised in 1:1:1 mol ratio of palmitoyl sphingomyelin (PSM) and cholesterol with 1-palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and with 1-palmitoyl-2-docoshexaenoylphosphatidylethanolamine (PDPE) or 1-palmitoyl-oleoylphosphatidylethanolamine (POPE). PSM and cholesterol are raft forming lipids that pack together tightly. PDPC and PDPE, which have polyunsaturated docosahexaenoic acid (DHA) at the sn-2 position, prefer a disordered non-raft environment. After 18 µs of simulation, visual inspection confirms that PSM and cholesterol cluster together more in the presence of PDPC than POPC. We attribute the increase in clustering to the greater difference in order parameter measured for the polyunsaturated phospholipid relative to PSM. The clustering of PSM and cholesterol appears less for PDPE and POPE, reflecting a smaller differential in order between PE and PSM that we ascribe to the smaller headgroup of PE and resulting intrinsically higher order.

Via conformational flexibility, triglycerides stabilize organic-water interfaces of changing area.

The role of triglycerides (TGs) in both synthetic and biological membranes has long been the subject of study, involving metabolism, disease, and colloidal synthesis. TGs have been found to be critical components for successful liposomal encapsulation via double emulsion (Water/Oil/Water), which this work attempts to explain. TGs can occupy multiple positions in a biological membranes. The glycerol backbone can sit at the membrane's interface with water, adjacent to phospholipid headgroups (“surface” or “M” conformation), typically with relatively low (∼3%) solubility. TGs can also occupy “oil” phases, where the glycerol backbone is isolated from water, in either mid-membrane positions or lipoprotein-style phospholipid-coated TG droplets. Using 13C-Nuclear magnetic resonance spectroscopy (NMR) to determine the degree of hydration of the TG backbone, it was revealed that TGs transition from surface to oil phases as the organic solvent is removed via evaporation. A new, transitional TG backbone position has been identified, with a level of hydration between surface and oil. These results suggest that TGs are able to temporarily coat and stabilize the large water/organic interfaces present just after emulsification. As the chloroform evaporates and interfaces shrink, the TGs recede into mid-membrane spaces or to bud off into separate coated oil droplets, which are confirmed via transmission electron microscopy. These TG-rich droplets can be removed via high-speed centrifugation. Encapsulation efficiency is found to be inversely related to both chain saturation and length, indicating that membrane fluidization is a key property arising from the presence of TGs. Beyond clarification of a mechanism for high-efficiency liposomal encapsulation, these results implicate TGs as components that are able to stabilize biological membrane transitions involving changing interfacial area. This role for TGs may be of use in designing drug delivery systems and investigation of membrane transitions in life sciences.

Dynamics transitions of the KvAP voltage sensor during lipid-dependent gating.

In response to changes in membrane potential, the S4 helix of VSD undergoes a structural transition from resting/‘Down’ to activated/‘Up’ conformation, which is an extremely important step for the functioning of a voltage-gated ion channel. It is well known that the presence of phospholipid headgroups are an absolute requirement for the normal functioning of Kv channels whereas channels reconstituted in lipids lacking the phosphate headgroups renders the channel nonfunctional. Interestingly, apart from voltage-dependent gating, the gating of KvAP can also be influenced by the change in membrane lipid composition in a voltage-independent manner (i.e., lipid-dependent gating). Here, we have performed differential lipid reconstitution using liposomes composed of phospholipids (POPC/POPG) to stabilize the activated/‘Up’ conformation and using liposomes with a mixture of phospholipids and non-phospholipids (DOPC/cholesterol) to trap the isolated KvAP-VSD in its ‘Down’ conformation. The results, using site-directed NBD fluorescence on the entire S4 helix, show that the moving part of the KvAP sensor adopts a relaxed dynamics in its ‘Down’ state whereas a restricted dynamics is observed in its ‘Up’ state. Further, a variation in hydration dynamics is observed when the S4 transitions between the two functional states, which not only reduces the relative discrete population of S4 conformations, but also alters the lifetime distribution peaks of the S4 in ‘Down’ conformation. This strongly suggests that the structural dynamics and conformational heterogeneity of the S4 is significantly altered which correlates with the change in conformation of the sensor during gating in membranes. Importantly, our ‘Down’ state results strongly indicate a bending/breakage from the middle of the otherwise straight S4-helix in ‘Up’ conformation. Overall, our results highlight the impact of altered lipid-protein interface that has a strong influence on the structural dynamics of the functionally-important conformations of the KvAP sensor.

Ab initio prediction and characterization of membrane binding of HIV-1 broadly neutralizing antibodies by multi-scale simulations.

Rare but potent broadly neutralizing antibodies (bnAbs) engage the HIV virion membrane and the membrane proximal exposed region (MPER) peptide during neutralization. A preliminary lipid mediated binding step is suggested to increase antigen avidity but it remains unclear how initial membrane association occurs and thus what features are essential to mature bnAbs against a lipid-antigen epitope. In this study, we derived computational methods to investigate biophysical features of MPER bnAbs driving membrane association. We performed microsecond atomistic simulations of Fabs docked in a lipid bilayer. Phospholipid binding in CDR loops is predicted in all 4 replicates of PGZL1, 4E10 and 10E8 where a phospholipid headgroup position closely matches the phosphate position in lipid-soaked x-ray crystallography structures (mean RMSD 0.6, 0.7, 2.7 Å respectively). Interaction profiles also highlight how framework regions FRH3 (PGZL1 and 4E10) or FRL3 (10E8) significantly associate with lipids. We then conducted fixed velocity pulling simulations for 4E10 and mutants with reduced affinity for lipid vesicles in vitro. Max force calculates differences in energy required to dissociate Fabs, establishing a bona fide tool to approximate membrane affinities. We expanded this platform to track evolution of binding affinity with inferred germline, intermediate, and mature PGZL1 variants that show increasing binding strengths. To reduce bias from pre-docked membrane conformations, we generated coarse grained models with a Fab initiated above a bilayer. Spontaneous membrane binding events (>1 µs) are detected in over half of the 18 independent 14 µs bnAb trajectories but only in 2 of 18 replicates for negative control BSA. Altogether we present a computational toolkit to biophysically evaluate residue-specific and surface level features of membrane binding antibodies and differentiate membrane affinities, facilitating future studies of complete MPER or other auto-reactive bnAb maturation pathways.

The membrane activity of the antimicrobial peptide caerin 1.1 is pH dependent.

Histidine-rich antimicrobial peptides (AMPs) are an important class of membrane-active peptides that can provide a scaffold to induce pH-sensitive interactions with cell membranes. The Australian tree frog AMP, caerin 1.1 (Cae-1), has three histidine residues, which modify the interactions with neutral and negatively charged lipid systems. NMR and MD simulations showed that in an acidic environment in comparison to physiological conditions, Cae-1 induced greater perturbation of the lipid dynamics and water penetration into the membrane interior. Lipid packing, chemical environment, and dynamics of the phospholipid headgroup were monitored using 31P solid-state NMR. In particular, the CODEX technique revealed that Cae-1 has a large impact on the slow re-orientation of the lipids. 2H solid-state NMR showed that Cae-1 ordered the acyl chains across the hydrophobic core. Interestingly, titration of the paramagnetic Mn2+ into the lipid-AMP systems resulted in extensive quenching of the 13C solid-state NMR signals of the lipid headgroup and acyl chain carbons. These results supported the MD results that showed Cae-1 was mainly inserted within the lipid bilayer structure of both neutral and negatively charged membranes, with the charged residues dragging the water and phosphate groups inwards. This could be an early step in the mechanism of membrane disruption by histidine-rich AMPs and indicated that Cae-1 is potentially forming toroidal pores in bilayers of neutral and negatively charged lipid membranes, especially under acidic conditions.

Cytosol-Exposed Lysine Residues Affect the Cytosol-Membrane Distribution and Serine-129 Phosphorylation of Alpha-Synuclein

Familial Parkinson’s disease and other neurodegenerative diseases known as synucleinopathies are strongly associated with α-Synuclein (αS) missense mutations. One of these mutations, E46K, had been hypothesized to increase the electrostatic attraction of αS to neuronal vesicle membranes due to positively charged lysine that attracts negatively charged phospholipid head groups. Here, we confirm the biochemical mechanism of E46K through four compound mutants, each with three replacements with lysine on a cytosol-exposed position of the αS alpha helix. We show that the cytosolic αS to membrane-bound αS ratios are significantly lower, and that the phosphorylation rates of serine-129-a pathological marker-are pronouncedly higher for the mutants than for wild type. This experiment addresses the previous knowledge gap in the understanding of basic amino acid replacements in cytosol-exposed positions of αS. Importantly, the validated effect of cytosol-exposed lysine residues has implications for exploring the mechanism of pathogenesis of αS mutants in familial Parkinson’s disease.