Lipid Headgroups Research Articles

Lysozyme Interaction with Phospholipid Nanodroplets Probed by Sum Frequency Scattering Vibrational Spectroscopy.

When a nanoparticle (NP) is introduced into a biological environment, its identity and interactions are immediately attributed to the dense layer of proteins that quickly covers the particle. The formation of this layer, dubbed the protein corona, is in general a combination of proteins interacting with the surface of the NP and a contest between other proteins for binding sites either at the surface of the NP or upon the dense layer. Despite the importance for surface engineering and drug development, the molecular mechanisms and structure behind interfacial biomolecule action have largely remained elusive. We use ultrafast sum frequency scattering (SFS) spectroscopy to determine the structure and the mode of action by which these biomolecules interact with and manipulate interfaces. The majority of work in the field of sum frequency generation has been done on flat model interfaces. This limits some important membrane properties such as membrane fluidity and dimensionality─important factors in biomolecule-membrane interactions. To move toward three-dimensional (3D) nanoscopic interfaces, we utilize SFS spectroscopy to interrogate the surface of 3D lipid monolayers, which can be used as a model lipid-based nanocarrier system. In this study, we have utilized SFS spectroscopy to follow the action of lysozyme. SFS spectra in the amide I region suggest that there is lysozyme at the interface and that the lysozyme induces an increased lipid monolayer order. The binding of lysozyme with the NP is demonstrated by an increase in acyl chain order determined by the ratio of the CH3 symmetric and CH2 symmetric peak amplitudes. Furthermore, the lipid headgroup orientation s-PO2- change strongly supports lysozyme insertion into the lipid layer causing lipid disruption and reorientation. Altogether, with SFS, we have made a huge stride toward understanding the binding and structure change of proteins within the protein corona.

Miscibility of Phosphatidylcholines in Bilayers: Effect of Acyl Chain Unsaturation

The miscibility of phospholipids in a hydrated bilayer is an issue of fundamental importance for understanding the organization of biological membranes. Despite research on lipid miscibility, its molecular basis remains poorly understood. In this study, all-atom MD simulations complemented by Langmuir monolayer and DSC experiments have been performed to investigate the molecular organization and properties of lipid bilayers composed of phosphatidylcholines with saturated (palmitoyl, DPPC) and unsaturated (oleoyl, DOPC) acyl chains. The experimental results showed that the DOPC/DPPC bilayers are systems exhibiting a very limited miscibility (strongly positive values of excess free energy of mixing) at temperatures below the DPPC phase transition. The excess free energy of mixing is divided into an entropic component, related to the ordering of the acyl chains, and an enthalpic component, resulting from the mainly electrostatic interactions between the headgroups of lipids. MD simulations showed that the electrostatic interactions for lipid like-pairs are much stronger than that for mixed pairs and temperature has only a slight influence on these interactions. On the contrary, the entropic component increases strongly with increasing temperature, due to the freeing of rotation of acyl chains. Therefore, the miscibility of phospholipids with different saturations of acyl chains is an entropy-driven process.

Unusual phosphatidylcholine lipid phase behavior in the ionic liquid ethylammonium nitrate

HypothesisThe forces that govern lipid self-assembly ionic liquids are similar to water, but their different balance can result in unexpected behaviour. ExperimentsThe self-assembly behaviour and phase equilibria of two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in the most common protic ionic liquid, ethylammonium nitrate (EAN) have been investigated as function of composition and temperature by small- and wide-angle X-ray scattering (SAXS/WAXS) and small-angle neutron scattering (SANS). FindingsBoth lipids form unusual self-assembly structures and show complex and unexpected phase behaviour unlike that seen in water; DSPC undergoes a gel Lβ to crystalline Lc phase transition on warming, while POPC forms worm-like micelles L1 upon dilution. This surprising phase behaviour is attributed to the large size of the EAN ions that solvate the lipid headgroup compared to water changing amphiphile packing. Weaker H-bonding between EAN and lipid headgroups also contributes. These results provide new insight for the design of lipid based nanostructured materials in ionic liquids with atypical properties.

Detergent headgroups control TolC folding in vitro

TolC is the trimeric outer membrane component of the efflux pump system in Escherichia coli that is responsible for antibiotic efflux from bacterial cells. Overexpression of efflux pumps has been reported to decrease susceptibility to antibiotics in a variety of bacterial pathogens. Reliable production of membrane proteins allows for the biophysical and structural characterization needed to better understand efflux and for the development of therapeutics. Preparation of recombinant protein for biochemical/structural studies often involves the production of proteins as inclusion body aggregates from which active proteins are recovered. Here, we find that the in vitro folding of TolC into its functional trimeric state from inclusion bodies is dependent on the headgroup composition of detergent micelles used. Nonionic detergent favors the formation of functional trimeric TolC, whereas zwitterionic detergents induce the formation of a non-native, oligomeric TolC fold. We also find that nonionic detergents with shorter alkyl lengths facilitate TolC folding. It remains to be seen whether the charges in lipid headgroups have similar effects on membrane insertion and folding in biological systems.

Interplay of lipid head group and packing defects in driving amyloid-beta–mediated myelin-like model membrane deformation

Accumulating evidence suggests that amyloid plaque associated myelin lipid loss as a result of elevated amyloid burden might also contribute to Alzheimer's disease. The amyloid fibrils though closely associated with lipids under physiological conditions, however, the progression of membrane remodeling events leading to lipid-fibril assembly remains unknown. Here we first reconstitute the interaction of Aβ-40 with myelin-like model membrane and show that the binding of Aβ-40 induces extensive tubulation. To look into the mechanism of membrane tubulation we chose a set of membrane conditions varying in lipid packing density and net charge that allows us to dissect the contribution of lipid specificity of Aβ-40 binding, aggregation kinetics, and subsequent changes in membrane parameters such as fluidity, diffusion, and compressibility modulus. We show that the binding of Aβ-40 depends predominantly on the lipid packing defect densities and electrostatic interactions and results in rigidification of the myelin-like model membrane during the early phase of amyloid aggregation. Furthermore, elongation of Aβ-40 into higher oligomeric and fibrillar species leads to eventual fluidization of the model membrane followed by extensive lipid membrane tubulation observed in the late phase. Taken together, our results capture mechanistic insights into snapshots of temporal dynamics of Aβ-40 - myelin-like model membrane interaction and demonstrate how short timescale, local phenomena of binding, and fibril-mediated load generation results in the consequent association of lipids with growing amyloid fibrils.

The Impact of Antimicrobial Peptides on the Acinetobacter baumannii Inner Membrane Is Modulated by Lipid Polyunsaturation.

The Gram-negative pathogen Acinetobacter baumannii is a primary contributor to nosocomial multi-drug-resistant (MDR) infections. To combat the rise of MDR infections, novel features of A. baumannii need to be considered for the development of new treatment options. One such feature is the preferential scavenging of exogenous lipids, including host-derived polyunsaturated fatty acids (PUFAs), for membrane phospholipid synthesis. These alterations in membrane composition impact both the lipid chemistry and the membrane biophysical properties. In this work we examine how antimicrobial peptides (AMPs) interact with the inner membranes of A. baumannii in the presence and absence of polyunsaturated phospholipids. Using coarse-grained molecular dynamics simulations of complex A. baumannii inner membrane models derived from lipidomes of bacteria grown in the presence and absence of PUFAs, we examine the impact of the adsorption of four prototypical AMPs (CAMEL, LL-37, pexiganan, and magainin-2) on the membrane biophysical properties. Our simulations reveal that the impact of AMP adsorption on the membrane biophysical properties was dependent on both the membrane composition and the specific AMP involved. Both lipid headgroup charge and tail unsaturation played important roles in driving the interactions that occurred both within the membrane and between the membrane and AMPs. The changes to the membrane biophysical properties also showed a complex relationship with the AMP's physical properties, such as AMP charge, chain length, and charge-to-mass ratio. Cumulatively, this work highlights the importance of studying AMPs using a complex membrane environment and provides insights into the mechanistic action of AMPs in polyunsaturated lipid-rich bacterial membranes.

Lipid-Specific Direct Translocation of the Cell-Penetrating Peptide NAF-144-67 across Bilayer Membranes.

The cell-penetrating peptide NAF-1 has recently emerged as a promising candidate for selective penetration and destruction of cancer cells. It displays numerous membrane-selective behaviors including cell-specific uptake and organelle-specific degradation. In this work, we explore membrane penetration and translocation of NAF-1 in model lipid bilayer vesicles as a function of lipid identity in zwitterionic phosphatidylcholine lipids mixed with anionic phosphatidylserine, phosphatidylglycerol, and phosphatidic acid lipids. By monitoring the digestion of NAF-1 using the protease trypsin located inside but not outside the vesicles, we determined that the translocation of NAF-1 was significantly enhanced by the presence of phosphatidic acid in the membrane compared to the other three anionic or zwitterionic lipids. These findings were correlated to fluorescence measurements of dansyl-labeled NAF-1, which revealed whether noncovalent interactions between NAF-1 and the bilayer were most stable either at the membrane/solution interface or within the membrane interior. Phosphatidic acid promoted interactions with fatty acid tails, while phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol stabilized interactions with polar lipid headgroups. Interfacial vibrational sum frequency spectroscopy experiments revealed that the phosphate moiety on phosphatidic acid headgroups was better hydrated than on the other three lipids, which helped to shuttle NAF-1 into the hydrophobic region. Our findings demonstrate that permeation does not depend on the net charge on phospholipid lipid headgroups in these model vesicles and suggest a model wherein NAF-1 crosses membranes selectively due to lipid-specific interactions at bilayer surfaces.

Location of dopamine in lipid bilayers and its relevance to neuromodulator function

Dopamine (DA) is a neurotransmitter that also acts as a neuromodulator, with both functions being essential to brain function. Here, we present the first experimental measurement of DA location in lipid bilayers using x-ray diffuse scattering, solid-state deuterium NMR, and electron paramagnetic resonance. We find that the association of DA with lipid headgroups as seen in electron density profiles leads to an increase of intermembrane repulsion most likely due to electrostatic charging. DA location in the lipid headgroup region also leads to an increase of the cross-sectional area per lipid without affecting the bending rigidity significantly. The order parameters measured by solid-state deuterium NMR decrease in the presence of DA for the acyl chains of PC and PS lipids, consistent with an increase in the area per lipid due to DA. Most importantly, these results support the hypothesis that three-dimensional diffusion of DA to target membranes could be followed by relatively more efficient two-dimensional diffusion to receptors within those membranes.

Dynamic Sorting of Mobile and Rigid Molecules in Biomembranes by Magic-Angle Spinning 13C NMR

Understanding the membrane dynamics of complex systems is essential to follow their function. As molecules in membranes can be in a rigid or mobile state depending on external (temperature, pressure) or internal (pH, domains, etc.) conditions, we propose an in-depth examination of NMR methods to filter highly mobile molecular parts from others that are in more restricted environments. We have thus developed a quantitative magic-angle spinning (MAS) 13C NMR approach coupled with cross-polarization (CP) and/or Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) on rigid and fluid unlabeled model membranes. We demonstrate that INEPT can detect only very mobile lipid headgroups in gel (solid-ordered) phases; the remaining rigid parts are only detected with CP. A direct correlation is established between the normalized line intensity as obtained by CP and the C-H (C-D) order parameters measured by wide-line 2H NMR or extracted from molecular dynamics: ICP/IDPeq ≈ 5|SCH|, indicating that when the order is greater than 0.2-0.3 (maximum value of 0.5 for chain CH2), only rigid parts can be filtered and detected using CP techniques. In very fluid (liquid-disordered) membranes, where there are many more active motions, both INEPT and CP detect resonances, with, however, a clear propensity of each technique to detect mobile and restricted molecular parts, respectively. Interestingly, the 13C NMR chemical shift of lipid hydrocarbon chains can be used to monitor order-disorder phase transitions and calculate the fraction of chain defects (rotamers) and the part of the transition enthalpy due to bond rotations (6-7 kJ·mol-1 for dimyristolphosphatidylcholine, DMPC). Cholesterol-containing membranes (liquid-ordered phases) can be dynamically contrasted as the rigid-body sterol is mainly detected by the CP technique, with a contact time of 1 ms, and the phospholipid by INEPT. Our work opens up a straightforward, robust, and cost-effective route for the determination of membrane dynamics by taking advantage of well-resolved conventional 13C NMR experiments without the need of isotopic labeling.

Selective Action of Antimalarial Hydroxychloroquine on the Packing of Phospholipids and Interfacial Water Associated with Lysosomal Model Membranes: A Vibrational Sum Frequency Generation Study.

Understanding the structural change of lysosomal membranes induced by hydroxychloroquine (HCQ) drug is essential as it has been considered as one of the probable mechanisms of its antimalarial action. In this context, vibrational sum frequency generation (VSFG) spectra of the O-H region of water and C-H of the hydrocarbon chain of negatively charged and zwitterionic phospholipids associated with the lysosomal membrane in the absence and presence of different concentrations of HCQ have been measured at the air/water interface. The interfacial water at the negatively charged and zwitterionic lipids gets restructured in the presence of HCQ; however, the mechanism of restructuring is different due to the charge of the head groups of lipids. Interestingly, the presence of HCQ leads to a disorder in the negatively charged lipids, irrespective of their chemical nature, mainly by creating the gauche defect in the hydrocarbon chain of the lipid. In contrast, the ordering of the zwitterionic lipid does not show any appreciable change with the addition of HCQ. The finding on the selectivity of HCQ in affecting the ordering of the lipid depending on its head group charge and restructuring of interfacial water may be useful in understanding the molecular level mechanism of the antimalarial action of HCQ.

PDK1:PKCα heterodimer association-dissociation dynamics in single-molecule diffusion tracks on a target membrane

Previous studies have documented the formation of a heterodimer between the two protein kinases PDK1 and PKCα on a lipid bilayer containing their target lipids. This work investigates the association-dissociation kinetics of this PDK1:PKCα heterodimer. The approach monitors the two-dimensional diffusion of single, membrane-associated PDK1 molecules for diffusivity changes as PKCα molecules bind and unbind. In the absence of PKCα, a membrane-associated PDK1 molecule exhibits high diffusivity (or large diffusion constant, D) because its membrane-contacting PH domain binds the target PIP3 lipid headgroup with little bilayer penetration, yielding minimal frictional drag against the bilayer. In contrast, membrane-associated PKCα contacts the bilayer via its C1A, C1B, and C2 domains, which each bind at least one target lipid with significant bilayer insertion, yielding a large frictional drag and low diffusivity. The present findings reveal that individual fluor-PDK1 molecules freely diffusing on the membrane surface undergo reversible switching between distinct high and low diffusivity states, corresponding to the PDK1 monomer and the PDK1:PKCα heterodimer, respectively. The observed single-molecule diffusion trajectories are converted to step length time courses, then subjected to two-state, hidden Markov modeling and dwell time analysis. The findings reveal that both the PDK1 monomer state and the PDK1:PKCα heterodimer state decay via simple exponential kinetics, yielding estimates of rate constants for state switching in both directions. Notably, the PDK1:PKCα heterodimer has been shown to competitively inhibit PDK1 phosphoactivation of AKT1, and is believed to play a tumor suppressor role by limiting excess activation of the highly oncogenic PDK1/AKT1/mTOR pathway. Thus, the present elucidation of the PDK1:PKCα association-dissociation kinetics has important biological and medical implications. More broadly, the findings illustrate the power of single-molecule diffusion measurements to reveal the kinetics of association-dissociation events in membrane signaling reactions that yield a large change in diffusive mobility.

Association of adenosine triphosphate with lipid headgroups measured by NMR spectroscopy.

Adenosine triphosphate (ATP) is the energy currency of biological processes because of its central role in energy conversion in biological cells. Given the complexity and the molecular disorder of the cellular environment, the mechanism of energy conversion into useful work still poses challenging questions in molecular biophysics. Using nuclear magnetic resonance spectroscopy and dynamic light scattering, we show that ATP molecules have a measurable affinity to phosphatidylcholine lipid headgroups. The adenosine and ribose groups of the ATP molecule insert into the headgroup region of the membrane as seen by 2D NOESY spectra. Based on 31P NMR measurements, the most likely orientation for the phosphate chain is away from the membrane surface into the water space and the interaction of ATP with lipid headgroups leads to electrostatic charging of membrane surfaces. The interaction of adenosine triphosphate with lipid membranes has relevance to biophysical and biochemical studies of energy conversion in biological cells. In particular, the affinity of ATP to lipid headgroups can affect the interaction with specialized receptors present in biological cells. The observed association of ATP with membrane surfaces primarily through the adenosine group allows phosphate chains to participate in hydrolysis reactions consistent with biochemical data.

Atomic-scale characterisation of dielectric properties and structured water molecules at the interface of lipid membranes.

Water molecules play a major role in determining the structure and function of biomolecules and their assembly. Hydration layers occurring at the interface of macromolecules strongly depend on their molecular charge distribution and dielectric polarization, which in turn determine their electrostatic and electrodynamic properties. At a greater scale, water molecules surrounding and permeating the cell membrane have been demonstrated organising on the cell membrane components themselves, i.e. lipids and transmembrane proteins. For instance, evidences showed that lipid head groups are supposed to be surrounded by more than one hydration layer, being the first layer formed by a tightly bound shell, and outer layers likely to be displaced due to protein-lipid interactions. Hence, it is of fundamental importance to understand the physical properties of interfacial water in order to gain information on the structure and function of biological systems.

Herpesviruses deform membrane during capsid nuclear export by lipid ordering and protein scaffolding.

Herpesviruses are large viruses that infect nearly all vertebrates and some invertebrates and cause lifelong infections in most of the world's population. A critical step in their replication is the export of the viral capsids from the nucleus into the cytoplasm, which occurs by an unusual mechanism termed nuclear egress. Too large to fit through nuclear pores, capsids instead bud at the inner nuclear membrane to form perinuclear enveloped virions, which then fuse with the outer nuclear membrane, releasing the capsids into the cytoplasm. This process is mediated by the virus-encoded nuclear egress complex (NEC) that deforms the membrane around the capsid. To understand how the NEC generates negative membrane curvature, we reconstituted the membrane budding process in vitro using the NEC from herpes simplex virus 1, a prototypical herpesvirus that causes cold sores. To probe its mechanism, we employed a combination of confocal microscopy, electron spin resonance, and structural biology. We found that the NEC uses clusters of positively charged residues to insert into the lipid headgroups, which increases lipid order. We also found that the NEC oligomerizes into a membrane-bound hexagonal coat on the inner surface of the budded vesicles. We propose that the NEC combines lipid ordering and oligomerization to mold the membrane into a spherical shape. Whereas lipid ordering generates negative membrane curvature locally, the NEC oligomerization into a hexagonal scaffold is necessary to achieve negative membrane curvature over a large membrane area. Similar principles may underlie membrane deformation in other systems. Our findings provide a biophysical explanation for the phenomenon of virus-induced nuclear budding.

Role of lipid composition on human plasma membrane interactions with the Ebola virus matrix protein VP40.

The Ebola virus is a very dangerous zoonotic virus and its infection can cause hemorrhagic fever with high fatality rates. The Ebola virus matrix protein VP40 is the major protein responsible for the formation of the viral matrix by localizing and assembling it at the inner leaflet of the human plasma membrane (PM). This assembly leads to the formation of virus-like-particles (VLP) and eventual budding. Anionic lipids PI(4,5)P2 (PIP2) and Phosphatidylserine (PS) have been shown to facilitate VP40-membrane binding through electrostatic interactions with cationic VP40 residues. It has been determined that Phosphatidic Acid (PA), resulting from the enzyme Phospholipase-D (PLD), may also play an active role in this process. We performed coarse-grained molecular dynamics (CGMD) simulations to investigate the effects various lipids have on VP40-lipid interactions. We used a triple-dimer structure of VP40 and a PM composed of various lipid types. Contact analyses show interactions with specific cationic VP40 residues that facilitate most of the lipid interactions. Interaction strengths between VP40 residues and lipid headgroups are found to vary when PA was included in the PM compared to when it was not. The radial distribution functions also show differences in local lipid clustering between the two systems. These simulations provide new insights on VP40 interactions with the human PM and how different lipids may influence the overall membrane association and assembly of the VP40 dimers.

Involvement of alpha-subunit N-termini in the mechanism and regulation of the Na+,K+- and H+,K+-ATPases.

The cytoplasmic lysine-rich N-termini of the Na+,K+- and H+,K+-ATPases have been designated as R domains because of their possible role in regulation of ion pumping activity. Prior to the publication of any crystal structures of either protein, it was suggested, based on mutagenesis studies, that regulation of the Na+,K+-ATPase could involve the formation and breakage of a salt bridge between Lys30 of the N-terminus and Glu233 in the first M2-M3 cytoplasmic loop of the alpha subunit. In structural studies of the Na+,K+-ATPase, the N-terminus has never been able to be resolved, probably because it is intrinsically disordered. Nevertheless, after theoretically predicting the N-terminus conformation, and adding it to the structure, the distance between residues Lys30 and Glu233 makes it doubtful that a direct long-lived interaction between them is involved in pump regulation. As an alternative hypothesis, we propose that regulation could occur via an interaction of the positively charged lysine residues of the N-terminus with negatively charged lipid headgroups (notably phosphatidylserine) on the cytoplasmic surface of the surrounding membrane, and that such an interaction could potentially be regulated by an electrostatic switch mechanism, whereby serine and tyrosine residues of the N-terminus are phosphorylated by protein kinases. Phosphorylation of these residues would decrease the positive charge of the N-terminus, allowing its release from the membrane. Experimental and theoretical data will be presented which support this hypothesis.

Evidence for long-term potentiation in lipid membranes.

Biological supramolecular assemblies, such as phospholipid bilayer membranes, have been used to demonstrate signal processing via short-term synaptic plasticity (STP) in the form of paired pulse facilitation (PPF) and depression (PPD), thus showing that model membranes have the potential to emulate the brain's efficiency and flexible cognitive capabilities. However, STP memory states in lipid bilayers are volatile and cannot be stored or accessed over relevant periods of time, a central requirement for learning. Using droplet interface bilayers (DIBs) composed of lipids and hexadecane, we apply an electrical stimulation protocol featuring repetitive voltage cycling in the form of a sinusoidal waveform. We show that DIBs displaying memcapacitance can also exhibit long-term synaptic plasticity in the form of long-term potentiation (LTP) associated with capacitive energy storage in the phospholipid bilayer. The time scales for the physical changes associated with LTP range between minutes to hours and are substantially longer than previous STP studies, where stored energy dissipated after only a few seconds. While STP behavior is the result of changes in bilayer geometry associated with reversible changes in bilayer area and thickness, LTP is the result of molecular and structural changes due to the zwitterionic lipid headgroups and the dielectric properties of the lipid bilayer that result from the buildup of an increasingly asymmetric charge distribution between the bilayer interfaces.

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.

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

Antimicrobial peptides are an important class of membrane-active peptides that can provide alternatives or complements to classic antibiotics. Among the many classes of AMPs, the histidine-rich family is of particular interest since they may induce pH-sensitive interactions with cell membranes. The AMP caerin 1.1 (Cae-1), from Australian tree frogs, has three histidine residues, and thus we studied the pH dependence of its interactions with model cell membranes. Using NMR spectroscopy and molecular dynamics simulations, we showed that Cae-1 induced greater perturbation of the lipid dynamics and water penetrations within the membrane interior in an acidic environment compared with physiological conditions. Using 31P solid-state NMR, the packing, chemical environment, and dynamics of the lipid headgroup were monitored. 2H solid-state NMR showed that Cae-1 ordered the acyl chains of the hydrophobic core of the bilayer. These results supported the molecular dynamics data, which showed that Cae-1 was mainly inserted within the lipid bilayer for both neutral and negatively charged membranes, with the charged residues pulling the water and phosphate groups inward. This could be an early step in the mechanism of membrane disruption by histidine-rich antimicrobial peptides and indicated that Cae-1 acts via a transmembrane mechanism in bilayers of neutral and anionic phospholipid membranes, especially in acidic conditions.

Lipid packing is disrupted in copolymeric nanodiscs compared with intact membranes

Discoidal lipid-protein nanoparticles known as nanodiscs are widely used tools in structural and membrane biology. Amphipathic, synthetic copolymers have recently become an attractive alternative to membrane scaffold proteins for the formation of nanodiscs. Such copolymers can directly intercalate into, and form nanodiscs from, intact membranes without detergents. Although these copolymer nanodiscs can extract native membrane lipids, it remains unclear whether native membrane properties are also retained. To determine the extent to which bilayer lipid packing is retained in nanodiscs, we measured the behavior of packing-sensitive fluorescent dyes in various nanodisc preparations compared with intact lipid bilayers. We analyzed styrene-maleic acid (SMA), diisobutylene-maleic acid (DIBMA), and polymethacrylate (PMA) as nanodisc scaffolds at various copolymer-to-lipid ratios and temperatures. Measurements of Laurdan spectral shifts revealed that dimyristoyl-phosphatidylcholine (DMPC) nanodiscs had increased lipid headgroup packing compared with large unilamellar vesicles (LUVs) above the lipid melting temperature for all three copolymers. Similar effects were observed for DMPC nanodiscs stabilized by membrane scaffolding protein MSP1E1. Increased lipid headgroup packing was also observed when comparing nanodiscs with intact membranes composed of binary mixtures of 1-palmitoyl-2-oleoyl-phosphocholine (POPC) and di-palmitoyl-phosphocholine (DPPC), which show fluid-gel-phase coexistence. Similarly, Laurdan reported increased headgroup packing in nanodiscs for biomimetic mixtures containing cholesterol, most notable for relatively disordered membranes. The magnitudes of these ordering effects were not identical for the various copolymers, with SMA being the most and DIBMA being the least perturbing. Finally, nanodiscs derived from mammalian cell membranes showed similarly increased lipid headgroup packing. We conclude that nanodiscs generally do not completely retain the physical properties of intact membranes.