Varying Lipid Composition Research Articles

System size effects on the free energy landscapes from molecular dynamics of phase-separating bilayers.

The "lipid raft" hypothesis proposes that cell membranes contain distinct domains of varying lipid compositions, where "rafts" of ordered lipids and cholesterol coexist with disordered lipid regions. Experimental and theoretical phase diagrams of model membranes have revealed multiple coexisting phases. Molecular dynamics (MD) simulations can also capture spontaneous phase separation of bilayers. However, these methods merely determine the signof the free energy change upon phase separation-whether or not it is favorable-but not the amplitude. Recently, we developed a workflow to compute the free energy of phase separation from MD simulations using the weighted ensemble method. However, while theoretical treatments generally focus on infinite systems and experimental measurements on mesoscopic to macroscopic systems, MD simulations are comparatively small. Therefore, if we are to put the results of these calculations into the appropriate context, we need to understand the effects the finite size of the simulation has on the computed free energy landscapes. In this study, we investigate this phenomenon by computing free energy profiles for a model phase-separating system as a function of system size, ranging from 324 to 10 110 lipids. The results suggest that, within the limits of statistical uncertainty, bulk-like behavior emerges once the systems contain roughly 4000 lipids.

Differential Effects of Soy Isoflavones on the Biophysical Properties of Model Membranes.

The effects that the main soy isoflavones, genistein and daidzein, have upon the biophysical properties of a model lipid bilayer composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or DOPC with cholesterol (4 to 1 mol ratio) have been investigated by transbilayer water permeability, differential scanning calorimetry, and confocal Raman microspectroscopy. Genistein is found to increase water permeability, decrease phase transition temperature, reduce enthalpy of transition, and induce packing disorder in the DOPC membrane with an increasing concentration. On the contrary, daidzein decreases water permeability and shows negligible impact on thermodynamic parameters and packing disorder at comparable concentrations. For a cholesterol-containing DOPC bilayer, both genistein and daidzein exhibit an overall less pronounced effect on transbilayer water permeability. Their respective differential abilities to modify the physical and structural properties of biomembranes with varying lipid compositions signify a complex and sensitive nature to isoflavone interactions, which depends on the initial state of bilayer packing and the differences in the molecular structures of these soy isoflavones, and provide insights in understanding the interactions of these molecules with cellular membranes.

Fragment-based approach to study fungicide-biomimetic membrane interactions.

In this study, the molecular interactions of the allylamine-type fungicide butenafine and a set of substructures ("fragments") with liposomes mimicking biological membranes were studied to gain a better understanding of the structural factors governing membrane affinity and perturbation. Specifically, drug/fragment-membrane interactions were investigated using an interdisciplinary approach involving micro differential scanning calorimetry, open-tubular capillary electrochromatography, nanoplasmonic sensing, and quartz crystal microbalance. By incubating the drug and the fragment compounds with liposomes with varying lipid composition or by externally adding the compounds to preformed liposomes, a detailed mechanistic picture on the underlying drug/fragment-membrane interactions was obtained. The nature and the degree of ionisation of polar head groups of the lipids had a major influence on the nature of drug-membrane interactions, and so had the presence and relative concentration of cholesterol within the membranes. The in-depth understanding of drug/fragment-membranes interactions established by the presented interdisciplinary fragment-based approach may be useful in guiding the design and early-stage evaluation of prospective antifungal drug candidates, and the discovery of agents with improved membrane penetrating characteristics in general.

Peptoid-based macrodiscs of variable lipid composition for structural studies of membrane proteins by oriented-sample solid-state NMR

Solid-state Nuclear Magnetic Resonance (NMR) in combination with magnetically aligned discoidal lipid mimics allows for studying the conformations of membrane proteins in planar, lipid-rich bilayer environments and at the physiological temperature. We have recently demonstrated the general applicability of macrodiscs composed of DMPC lipids and peptoid belts, which yield magnetic alignment and NMR spectroscopic resolution comparable or superior to detergent-containing bicelles. Here we report on a considerable improvement in the magnetic alignment and NMR resolution of peptoid-based macrodiscs consisting of a mixture of the zwitterionic and negatively charged lipids (DMPC/DMPG at the 85% to 15% molar ratio). The resulting linewidths are about 30% sharper due to the higher orientational order parameter likely arising from the stabilizing electrostatic repulsion between the discs. Moreover, highly aligned, detergent-free macrodiscs can be formed with a longer-chain lipid, DPPC. Interestingly, the spectra of Pf1 in the two lipid mimetics are almost indistinguishable, which would mean that the overall transmembrane helix tilt might be governed not only by the hydrophobic matching but also possibly by the interactions of the flanking lysine and arginine residues at the membrane interface.

Capric Acid and Myristic Acid Permeability Enhancers in Curved Liposome Membranes.

Liposomes are considered as advanced drug delivery systems for cancer treatment. A generation of pH-sensitive liposomes is being developed that use fatty acids (FAs) as a trigger for drug release in tumor tissues. However, FAs are also known to enhance permeability, and it is unclear whether FAs in liposomes may cause drug leakage or premature drug release. The passive permeability of the drug through the membrane of the liposome is thus a crucial factor for timely drug delivery. To investigate how the curvature and lipid composition of liposomes affect their passive permeability, coarse-grained molecular dynamics were performed. The permeability was determined with a counting method. Flat bilayers and three liposomes with varying diameters were studied, which had varying lipid compositions of dipalmitoylphosphatidylcholine, cholesterol, and deprotonated or neutral saturated FAs. The investigated permeants were water and two other small permeants, which have different free energy profiles (solubility) across the membrane. First, for the curvature effect, our results showed that curvature increases the water permeability by reducing the membrane thickness. The permeability increase for water is about a factor of 1.7 for the most curved membranes. However, a high curvature decreases permeability for permeants with free energy profiles that are a mix of wells and barriers in the headgroup region of the membrane. Importantly, the type of experimental setup is expected to play a dominant role in the permeability value, i.e., whether permeants are escaping or entering the liposomes. Second, for the composition effect, FAs decrease both the area per lipid (APL) and the membrane thickness, resulting in permeability increases of up to 55%. Cholesterol has a similar effect on the APL but has the opposite impact on membrane thickness and permeability. Therefore, FAs and cholesterol have opposing effects on permeability, with cholesterol's effect being slightly stronger in our simulated bilayers. As all permeability values were well within a factor of 2, and with liposomes usually being larger and less curved in experimental applications, it can be concluded that the passive drug release from a pH-sensitive liposome does not seem to be significantly affected by the presence of FAs.

Ex Vivo Drug Screening Assay with Artificial Membranes: Characterizing Cholesterol Desorbing Competencies of Beta-Cyclodextrins.

Despite advancements in contemporary therapies, cardiovascular disease from atherosclerosis remains a leading cause of mortality worldwide. Supported lipid bilayers (SLBs) are membrane interfaces that can be constructed with varying lipid compositions. Herein, we use a solvent-assisted lipid bilayer (SALB) construction method to build SLB membranes with varying cholesterol compositions to create a lipid-sterol interface atop a piezoelectric sensor. These cholesterol-laden SLBs were utilized to investigate the mechanisms of various cholesterol-lowering drug molecules. Within a flow-cell, membranes with varying cholesterol content were exposed to cyclodextrins 2-hydroxypropyl-beta-cyclodextrin (HPβCD) and methyl-beta-cyclodextrin (MβCD). Quartz-crystal microgravimetry with dissipation monitoring (QCM-D) enabled the collection of in vitro, real-time changes in relative areal mass and dissipation. We define the cholesterol desorbing competency of a cyclodextrin species via measures of the rate of cholesterol removal, the rate of the transfer of membrane-bound cholesterol to drug-complexed cholesterol, and the binding strength of the drug to the cholesterol-ladened membrane. Desorption data revealed distinct cholesterol removal kinetics for each cyclodextrin while also supporting a model for the lipid-cholesterol-drug interface. We report that MβCD removes a quantity of cholesterol 1.61 times greater, with a speed 2.12 times greater, binding affinity to DOPC lipid interfaces 1.97 times greater, and rate of internal cholesterol transfer 3.41 times greater than HPβCD.

Dual Functional Full-Color Carbon Dot-Based Organelle Biosensor Array for Visualization of Lipid Droplet Subgroups with Varying Lipid Composition in Living Cells.

In situ visualization of lipid composition diversity in lipid droplets (LDs) is essential for decoding lipid metabolism and function. However, effective probes for simultaneously localizing and reflecting the lipid composition of LDs are currently lacking. Here, we synthesized full-color bifunctional carbon dots (CDs) that can target LDs as well as respond to the nuance in internal lipid compositions with highly sensitive fluorescence signals, due to lipophilicity and surface state luminescence. Combined with microscopic imaging, uniform manifold approximation and projection, and sensor array concept, the capacity of cells to produce and maintain LD subgroups with varying lipid composition was clarified. Moreover, in oxidative stress cells, LDs with characteristic lipid compositions were deployed around mitochondria, and the proportion of LD subgroups changed, which gradually disappeared when treated with oxidative stress therapeutics. The CDs demonstrate great potential for in situ investigation of the LD subgroups and metabolic regulations.

Characterization of phosphoinositide domains in asymmetric giant unilamellar vesicles.

Phosphoinositides (PIPs) have been shown to mediate a broad range of cellular events by attracting proteins to specific cellular sites at distinct time points. This spatiotemporal control of protein function is rooted in the rich chemical functionality of the phosphoinositide headgroup, which not only gives rise to selective interactions with proteins but also results in unique physicochemical properties. Several studies have been directed at detailing PIP properties in model symmetric lipid bilayers, however, biological membranes exhibit compositional asymmetry between bilayer leaflets. It is unclear to what extent PIP properties are altered in such an environment. In the last decade, methods have been introduced that enable the fabrication of asymmetric lipid vesicles. We have extended the scope of one of these methods – Ca2+ initiated hemifusion of a symmetric giant unilamellar vesicles (sGUV) with solid supported lipid bilayers (SLB) – to enable the fabrication of asymmetric giant unilamellar vesicles (aGUVs) that exhibit phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the outer leaflet. We found that using Ca2+ as the initiator for sGUV/SLB hemifusion leads to a large disparity of PI(4,5)P2 content in the formed aGUVs. We attribute this disparity to the formation of macroscopic Ca2+/PI(4,5)P2 domains, which leads to sGUVs interacting with SLB regions rich or poor in PI(4,5)P2. In contrast, when Mg2+ is used as initiator, no macroscopic domains form in the SLB and hence, the aGUVs show significantly higher compositional uniformity. We find that Mg2+ concentrations as low as 1 mM are sufficient to initiate the hemifusion process. Using this method to obtain aGUVs, we investigate phosphoinositide domain properties for varying lipid compositions in the leaflet opposite the PIP containing leaflet (raft vs. non-raft lipid mixtures) and we explore the impact of bivalent cations on the lateral morphology of the respective aGUVs.

Structuring and de-structuring of nanovectors from algal lipids. Part 1: physico-chemical characterization

Lipid nanocarriers are among the most employed systems for drug delivery purposes in several research and industrial sectors, since their favorable properties ensure broad applicability. The design and characterization of these nanosystems are of paramount importance to obtain controlled outcome, since the supramolecular structure and molecular interactions deeply impact the functionality of the resulting aggregates. The choice of the most appropriate formulation for the target of interest relies on in-depth physico-chemical characterization in order to optimize stability, loading rates and sustained release. Several supramolecular architectures suited for carrier development can be obtained from lipid building blocks, by varying lipid composition and packing parameter. In particular, cubosome and liposome aggregates are often used as drug vectors thanks to their high cargo capability and biocompatibility. Moreover, the possibility to employ lipids from natural sources i.e. biomasses to prepare nanosystems makes them especially attractive. In this work, two aggregate types were characterized and compared as drug vectors for poorly water-soluble antioxidants, particularly curcumin and two adjuvants (i.e. tocopherol and piperine). The nanovectors were obtained by extracting lipids from algal biomasses with different lipid composition, and characterized by advanced structural (DLS, SAXS, Cryo-TEM) techniques, spectroscopy (NMR) and calorimetry (ITC). Finally, the structural stability of both aggregate types was evaluated.

Lipid composition dependent binding of apolipoprotein E signal peptide: Importance of membrane cholesterol in protein trafficking

Soluble secretory and membrane proteins contain a short stretch of signal peptide (SP) at their N-terminal end, which gets cleaved after reaching the destination organelle. However, the importance of SP in protein trafficking is not fully understood. The lipid compositions of cellular organelles are highly heterogeneous, and the preference of SP toward a particular lipid composition might play a key role in unidirectional trafficking of protein. In order to understand the preference of Apolipoprotein E (ApoE) toward endoplasmic reticulum (ER), we have studied the interaction of its SP with membranes of varying lipid compositions. The importance of cholesterol is paramount as subcellular organelles contain differential amount of cholesterol; endoplasmic reticulum (ER) contains the least amount of cholesterol. We have utilized batteries of steady-state and time-resolved fluorescence techniques to understand the affinity of ApoE signal peptide toward membranes of varying lipid compositions. We observed that the ApoE signal peptide binds tightly with membranes devoid of cholesterol, and binding affinity reduces with increasing concentration of membrane cholesterol. Our results clearly suggest the importance of membrane composition in the unidirectional movement of ApoE toward ER. This property of SP can further be utilized for the development of organelle specific cargo delivery.

Determining small-molecule permeation through lipid membranes.

The passive permeability of cell membranes is of key importance in biology, biomedical research and biotechnology as it determines the extent to which various molecules such as drugs, products of metabolism, and toxins can enter or leave the cell unaided by dedicated transport proteins. The quantification of passive solute permeation is possible with radio-isotope distribution experiments, spectroscopic measurements and molecular dynamics simulations. This protocol describes stopped-flow fluorimetry measurements performed on lipid vesicles and living yeast cells to estimate the osmotic permeability of water and solutes across (bio)membranes. Encapsulation of the fluorescent dye calcein into lipid vesicles allows monitoring of volume changes upon osmotic shifts of the medium via (de)quenching of the fluorophore, which we interpret using a well-defined physical model that takes the dynamics of the vesicles into account to calculate the permeability coefficients of solutes. We also present analogous procedures to probe weak acid and base permeability in vesicles and cells by using the read-out of encapsulated or expressed pH-sensitive probes. We describe the preparation of synthetic vesicles of varying lipid composition and determination of vesicle size distribution by dynamic light scattering. Data on membrane permeation are obtained using either conventional or stopped-flow kinetic fluorescence measurements on instruments available in most research institutes and are analyzed with a suite of user-friendly MATLAB scripts ( https://doi.org/10.5281/zenodo.6511116 ). Collectively, these procedures provide a comprehensive toolbox for determining membrane permeability coefficients in a variety of experimental systems, and typically take 2-3 d.

A comparative study of interfacial environments in lipid nanodiscs and vesicles

Membrane protein conformations and dynamics are driven by the protein-lipid interactions occurring within the local environment of the membrane. These environments remain challenging to accurately capture in structural and biophysical experiments using bilayers. Consequently, there is an increasing need for realistic cell-membrane mimetics for in vitro studies. Lipid nanodiscs provide certain advantages over vesicles for membrane protein studies. Nanodiscs are increasingly used for structural and spectroscopic characterization of membrane proteins. Despite the common use of nanodiscs, the interfacial environments of lipids confined to a ∼10-nm diameter area have remained relatively underexplored. Here, we use ultrafast two-dimensional infrared spectroscopy and temperature-dependent infrared absorption measurements of the ester carbonyls to compare the interfacial hydrogen bond structure and dynamics in lipid nanodiscs of varying lipid compositions and sizes with ∼100-nm vesicles. We examine the effects of lipid composition and nanodisc size. We found that nanodiscs and vesicles share largely similar lipid-water H-bond environments and interfacial dynamics. Differences in measured enthalpies of H-bonding suggest that H-bond dynamics in nanodiscs are modulated by the interaction between the annular lipids and the scaffold protein.

Structural basis for Rab6 GTPase activation by the Ric1‐Rgp1 complex

Membrane trafficking is a highly coordinated process in eukaryotes essential for maintaining lipid homeostasis and sorting proteins to the proper location inside cells. At the hub of this dynamic set of pathways, the Golgi complex directs much of this sorting through vesicle mediated transport. Vesicles of varying lipid composition loaded with different protein cargo are in constant flux through the Golgi and precise coordination of these processes requires communication between compartments. Rab GTPases are small signaling proteins that act as molecular switches to regulate vesicular trafficking. Rab6 functions at the Golgi and is important for endosome to Golgi retrograde transport in both yeast and metazoans and for anterograde exit from the TGN in metazoans. The Ric1‐Rgp1 protein complex is a highly conserved guanine nucleotide exchange factor (GEF) required for activation of Rab6. In mammalian cells Ric1‐Rgp1 is known to be regulated by the Rab33 GTPase, but it remains unresolved how the activity of the complex is regulated and how it interacts with Rab6. Furthermore, very little is known about the structure and domain organization of Ric1‐Rgp1. We have used cryo‐EM to determine the high‐resolution structure of the yeast Ric1‐Rgp1‐Rab6(Ypt6) complex, representing the key intermediate of the nucleotide exchange reaction. This structure reveals the overall architecture of the complex and has enabled us to identify the specific interactions that govern activation of Rab6. Ongoing studies aim to test the physiological significance of these interactions and determine the orientation of this complex on the Golgi membrane surface.

Interactions between polymyxin B and various bacterial membrane mimics: A molecular dynamics study

Polymyxin B (PMB) is clinically used as a last-line therapy against life-threatening Gram-negative “superbugs”. However, thorough understanding of the membrane actions of PMB at a molecular level is still lacking. In this work, a variety of bacterial membrane mimics with varying lipid compositions were built, and their interactions with PMB were systematically investigated using coarse-grained molecular dynamics simulation. PMB demonstrated characteristic preference to specific lipid species during its interaction with different membrane systems, such as the rough mutant lipipolysacchrides (Re LPS) preference in an outer membrane (OM) or the cardiolipin and POPG affinity in an inner membrane (IM). As a result of the lipid-specific actions, complicated membrane interaction states of PMB were observed, including adsorption on the OM surface. In contrast, for the IM or a mutative OM containing “impurity lipids” like POPE, POPG or lipid A, it could insert into the membrane via its acyl chain. Such actions of PMB influence the structure and lipid mobility of the membrane. In particular, the OM-bound PMB breaks the synchronous movement of Re LPS molecules in the outer leaflet and makes them diffuse more randomly, while its insertion into IM blocks the phospholipid diffusion and makes the membrane more homogeneous in the trajectory space. Our results provide insight into the action mechanism of PMB at a membrane level and a foundation for developing novel and safer polymyxin strategies for better clinical use.

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.

Sub-Diffraction Limited Imaging of Lipid Domains using Ratiometric STED

Cellular membranes are comprised of heterogeneously distributed lipid domains that are important for cellular functions. While recent literature has greatly augmented the importance of the spatial separation and the structural features of membrane rafts, the biophysical techniques available for studying membrane nanodomains still remain sparse. Studying lipid clustering and domain formation at a sub-diffraction level continues to be a challenge despite great advances in the field of super-resolution microscopy. In this study, we report the use of a commercially available solvatochromic dye (Nile Red) as a lipid domain sensor in conjunction with stimulated emission depletion (STED) microscopy. By maintaining a fixed concentration of constantly exchanging fluorophores in bulk, we all but eliminate photobleaching from the STED depletion laser, an issue that is commonly associated with STED microscopy. We further leverage the solvatochromic nature of the dye, by monitoring its spectral shift to report on the lipid order changes as a result of changes in lipid composition. As a proof of concept, we performed ratio metric STED imaging on surface-immobilized large unilamellar vesicles (LUVs) prepared with varying lipid compositions to test the validity of our approach. Our results demonstrate that our simple, yet novel approach provides spatial resolution at a <100nm level, while simultaneously reporting on the lipid order of the immobilized LUVs. We anticipate that this technique will be applicable to a wide range of biological systems, and will yield valuable insights into the spatial and chemical organization of lipid rafts in cell membranes. This work was supported by the NIH R01 AI150453 grant to GBM.

Immune cell populations in the broiler ileum exhibit differential cytokine profiles in response to lipid source and peroxidation

Used restaurant oil offers a sustainable and affordable energy source in broiler diets but variable lipid composition and the presence of harmful peroxidation products may alter intestinal immunity. The objective of this study was to evaluate the effects of feeding different lipid sources with variable peroxidation statuses on immune cell populations producing interleukin-6 (IL6) and interferon-γ (IFNG) in the broiler ileum. Two hundred broilers were fed diets with 5% inclusion of control or peroxidized palm, soybean, flaxseed or fish oil in a 4 × 2 factorial treatment design. At 21d, 2 birds/ treatment were euthanized for ileum collection and immune cell populations were analyzed by RNAscope- in situ hybridization (ISH). Ileal production of IL6 increased 85.8% by feeding peroxidized flaxseed oil while IFNG-producing cells were increased 55.1-59.9% by feeding either control or peroxidized soybean oil (P ≤ 0.05). Feeding peroxidized lipid generally reduced CD3+ T cells not producing either IL6 or IFNG by 14.9-39.0% (P ≤ 0.05). Overall, these results suggest that IL6 and IFNG have differential responses to lipid source and peroxidation while lipid peroxidation negatively impacts T cell presence in the broiler chicken ileum. Inflammatory outcomes observed in broilers fed peroxidized flaxseed oil suggest that yellow grease containing this type of oil may detrimentally impact broilers, while soybean oil generally contributes to intestinal inflammation regardless of heat exposure<br>

Phosphatidylethanolamine-phosphatidylserine binding synergy of seven coagulation factors revealed using Nanodisc arrays on silicon photonic sensors

Blood coagulation is regulated through protein–protein and protein–lipid interactions that occur at the sub-endothelium following vascular damage. Soluble clotting proteins bind to membrane components in a phosphatidylserine (PS) dependent manner to assemble multi-protein complexes that regulate clot formation; however, PS is of limited abundance physiologically. In this manuscript, we investigate synergy between PS and phosphatidylethanolamine (PE)—a lipid of much higher abundance naturally. Using a label-free, silicon photonic technology, we constructed arrays of Nanodiscs having variable lipid composition and probed the binding interactions of seven different clotting factors with GLA domains that have never been studied in tandem experiments before. The factors studied were prothrombin, activated factor VII, factor IX, factor X, activated protein C, protein S, and protein Z. Equilibrium dissociation constants (Kd) for each coagulation factor binding to Nanodiscs with unique compositions of PE and PS were determined. While all factors showed greater binding affinities in the presence of PS and PE, the most dramatic improvements in binding were observed when PS quantities were lowest. This demonstrates that synergy is effective in promoting coagulation factor binding under physiological lipid compositions, as opposed to the artificially high PS content probed in most in vitro activity studies.

Membrane Thinning Induces Sorting of Lipids and the Amphipathic Lipid Packing Sensor (ALPS) Protein Motif.

Heterogeneities (e.g., membrane proteins and lipid domains) and deformations (e.g., highly curved membrane regions) in biological lipid membranes cause lipid packing defects that may trigger functional sorting of lipids and membrane-associated proteins. To study these phenomena in a controlled and efficient way within molecular simulations, we developed an external field protocol that artificially enhances packing defects in lipid membranes by enforcing local thinning of a flat membrane region. For varying lipid compositions, we observed strong thinning-induced depletion or enrichment, depending on the lipid's intrinsic shape and its effect on a membrane's elastic modulus. In particular, polyunsaturated and lysolipids are strongly attracted to regions high in packing defects, whereas phosphatidylethanolamine (PE) lipids and cholesterol are strongly repelled from it. Our results indicate that externally imposed changes in membrane thickness, area, and curvature are underpinned by shared membrane elastic principles. The observed sorting toward the thinner membrane region is in line with the sorting expected for a positively curved membrane region. Furthermore, we have demonstrated that the amphipathic lipid packing sensor (ALPS) protein motif, a known curvature and packing defect sensor, is effectively attracted to thinner membrane regions. By extracting the force that drives amphipathic molecules toward the thinner region, our thinning protocol can directly quantify and score the lipid packing sensing of different amphipathic molecules. In this way, our protocol paves the way toward high-throughput exploration of potential defect- and curvature-sensing motifs, making it a valuable addition to the molecular simulation toolbox.

Hydrophobic matching of HIV-1 Vpu transmembrane helix-helix interactions is optimized for subcellular location

The HIV-1 accessory protein Vpu mediates the downregulation of several host cell proteins, an activity that is critical for viral replication in vivo. As the first step in directing cell-surface proteins to internal cellular compartments, and in many cases degradation, Vpu binds a subset of its target proteins through their transmembrane domains. Each of the known targets of Vpu are synthesized in the ER, and must traverse the different membrane environments found along the secretory pathway, thus it is important to consider how membrane composition might influence the interactions between Vpu and its targets. We have used Förster resonance energy transfer (FRET) to measure the oligomerization of Vpu with the transmembrane domains of target proteins in model membranes of varying lipid composition. Our data show that both lipid bilayer thickness and acyl chain order can significantly influence monomer-oligomer equilibria within the Vpu-target system. Changes in oligomerization levels were found to be non-specific with no single Vpu-target interaction being favored under any condition. Our analysis of the influence of the membrane environment on the strength of helix-helix interactions between Vpu and its targets in vitro suggests that the strength of Vpu-target interactions in vivo will be partially dependent on the membrane environment found in specific membrane compartments.