Lipid Bilayer Environment Research Articles

Reversible Aggregation of Chlorophyll Derivative Induced by Phase Transition of Lipid.

Controlling the supramolecular organization of pigment molecules will provide innovative materials that exhibit variable optical properties. In nature, photosynthetic systems employ chlorophyllous supramolecules in which each pigment molecule is suitably organized in proteins, and their properties are adequately optimized by changing the structures of the surrounding amino acid residues. Here, we report a strategy for varying the aggregation behavior of a chlorophyll derivative by using a phase-transition phenomenon of lipid bilayers. Methyl pyropheophorbide a (MPP) was employed as a chlorophyllous pigment in our artificial system, and synthetic phosphatidylcholines with saturated acyl chain(s) were also used. The MPP molecules successfully accumulated within the lipid bilayer of liposomes without changing the vesicular structure. When the lipid bilayer was in a gel form (under the phase-transition temperature, Tm), the embedded MPP aggregated to yield a dimeric form showing red-shifted absorption bands and circular dichroism signals. When the solutions of MPP-containing liposomes were heated to higher temperatures than their Tm, MPP disaggregated to monomeric form as the absorption spectrum changed into its original fashion in dichloromethane. The reversible thermochromic (dis)aggregation of the MPP molecules had good cyclability. Additional careful examination of the phase transition in the MPP-lipid co-assemblies clarified that the critical temperatures of the MPP (dis)aggregation were in good agreement with the phase-transition temperatures of the pigment-containing bilayers. The reversible MPP aggregation in the lipid bilayers occurred in a wide range of temperatures (around 10-55 °C) by changing the length of the diacyl side chains of phospholipids. The reversible thermochromism of the chlorophyllous system was established by varying the nature of the surrounding lipid bilayer. This study can provide a useful strategy for making variable tetrapyrrolic aggregate systems induced by mild extrinsic stimuli.

Expanding the Types of Lipids Amenable to Native Mass Spectrometry of Lipoprotein Complexes.

Native mass spectrometry (MS) has become an important tool for the analysis of membrane proteins. Although detergent micelles are the most commonly used method for solubilizing membrane proteins for native MS, nanoscale lipoprotein complexes such as nanodiscs are emerging as a promising complementary approach because they solubilize membrane proteins in a lipid bilayer environment. However, prior native MS studies of intact nanodiscs have employed only a limited set of phospholipids that are similar in mass. Here, we extend the range of lipids that are amenable to native MS of nanodiscs by combining lipids with masses that are simple integer multiples of each other. Although these lipid combinations create complex distributions, overlap between resonant peak series allows interpretation of nanodisc spectra containing glycolipids, sterols, and cardiolipin. We also investigate the gas-phase stability of nanodiscs with these new lipids towards collisional activation. We observe that negative ionization mode or charge reduction stabilizes nanodiscs and is essential to preserving labile lipids such as sterols. These new approaches to native MS of nanodiscs will enable future studies of membrane proteins embedded in model membranes that more accurately mimic natural bilayers. Graphical Abstract.

Interfacial Binding Sites for Cholesterol on G Protein-Coupled Receptors

A docking procedure is described that allows the transmembrane surface of a G protein-coupled receptor (GPCR) to be swept rapidly for potential binding sites for cholesterol at the bilayer interfaces on the two sides of the membrane. The procedure matches 89% of the cholesterols resolved in published GPCR crystal structures, when cholesterols likely to be crystal packing artifacts are excluded. Docking poses are shown to form distinct clusters on the protein surface, the clusters corresponding to “greasy hollows” between protein ridges. Docking poses depend on the angle of tilt of the GPCR in the surrounding lipid bilayer. It is suggested that thermal motion could alter the optimal binding pose for a cholesterol molecule, with the range of binding poses within a cluster providing a guide to the range of thermal motions likely for a cholesterol within a binding site.

Membrane Interactivity Shared by Receptor- Acting Drugs

Background: Although lipids have been regarded as a passive component to constitute biomembranes, they can also play an important role in modulation of the activity or function of membrane-embedded proteins like receptors. Membrane lipids are presumed to be one of additional sites of action for receptor-acting drugs because their broad pharmacological spectra are not necessarily interpretable by the direct action on receptors. In order to obtain novel insights into the drug target and mechanism, we reviewed the membrane interactivity of different classes of drugs to act on representative receptors.
 Methods: A search of the scientific articles published between 1979 and 2018 was carried out by using PubMed/MEDLINE, Google Scholar and ACS Publications. The relevant research papers published in recognized international journals and on-line journals in English were preferred, but the review articles of specific importance were also included, although non-English language citations were excluded. Collected articles were reviewed by title, abstract and text for relevance with preference to more recent publications.
 Results: Results of the literature search indicate that membrane interactivity is shared by various drugs that act on α- and β-adrenergic, muscarinic and nicotinic acetylcholine, γ-aminobutyric acid type A, N-methyl-D-aspartate, opioid and transient receptor potential vanilloid type-1 receptors. These receptor agonists and antagonists not only interact with receptor proteins but also would structure-specifically interact with membrane lipids to affect receptors by modifying the lipid bilayer environments surrounding them with the resultant conformational change of receptor proteins.
 Conclusion: The structure-specific membrane interaction is pharmacologically contributable to diverse effects of receptor-acting drugs.

Sensing Ability and Formation Criterion of Fluid Supported Lipid Bilayer Coated Graphene Field-Effect Transistors.

Supported lipid bilayers (SLBs) have been widely used to provide native environments for membrane protein studies. In this study, we utilized graphene field-effect transistors (GFETs) coated with a fluid SLB to perform label-free detection of membrane-associated ligand-receptor interactions in their native lipid bilayer environment. It is known that the analyte-binding event needs to occur within the Debye length for it to be significantly sensed by an FET sensor. However, the thickness of a lipid bilayer is around 4-5-nm-thick, which is larger than the Debye length of a solution with physiologically relevant ionic strength. There is thus a question of whether an FET sensor can detect the binding event above the bilayer. In this study, we show how the existence of an SLB can influence the effective detection distance and the formation criterion of a fluid and continuous SLB on a graphene surface. We discovered that the water intercalation between the graphene and the underlying silica substrate hinders the SLB formation but is required for the stable electrical recording by a GFET. To verify the existence of a fluid SLB on graphene, which was previously complicated by the graphene fluorescence quenching effect, we developed a modified fluorescence recovery after photobleaching method. In addition, our results showed that SLB coated GFETs can quantitatively detect ligand binding onto the receptors embedded in the SLBs. The comparison of our experimental data with a theoretical model shows that the contribution of the SLB acyl chain hydrophobic region to the screening effect can be negligible and, therefore, that the effective detection region can extend beyond the SLB.

The hydrodynamic motion of Nanodiscs.

We present a fluorescence-based methodology for monitoring the rotational dynamics of Nanodiscs. Nanodiscs are nano-scale lipid bilayers surrounded by a helical membrane scaffold protein (MSP) that have found considerable use in studying the interactions between membrane proteins and their lipid bilayer environment. Using a long-lifetime Ruthenium label covalently attached to the Nanodiscs, we find that Nanodiscs of increasing diameter, made by varying the number of helical repeats in the MSP, display increasing rotational correlation times. We also model our system using both analytical equations that describe rotating spheroids and numerical calculations performed on atomic models of Nanodiscs. Using these methods, we observe a linear relationship between the experimentally determined rotational correlation times and those calculated from both analytical equations and numerical solutions. This work sets the stage for accurate, label-free quantification of protein-lipid interactions at the membrane surface.

Antidepressants are modifiers of lipid bilayer properties.

The two major classes of antidepressants, tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs), inhibit neurotransmitter reuptake at synapses. They also have off-target effects on proteins other than neurotransmitter transporters, which may contribute to both desired changes in brain function and the development of side effects. Many proteins modulated by antidepressants are bilayer spanning and coupled to the bilayer through hydrophobic interactions such that the conformational changes underlying their function will perturb the surrounding lipid bilayer, with an energetic cost (ΔG def) that varies with changes in bilayer properties. Here, we test whether changes in ΔG def caused by amphiphilic antidepressants partitioning into the bilayer are sufficient to alter membrane protein function. Using gramicidin A (gA) channels to probe whether TCAs and SSRIs alter the bilayer contribution to the free energy difference for the gramicidin monomer⇔dimer equilibrium (representing a well-defined conformational transition), we find that antidepressants alter gA channel activity with varying potency and no stereospecificity but with different effects on bilayer elasticity and intrinsic curvature. Measuring the antidepressant partition coefficients using isothermal titration calorimetry (ITC) or cLogP shows that the bilayer-modifying potency is predicted quite well by the ITC-determined partition coefficients, and channel activity is doubled at an antidepressant/lipid mole ratio of 0.02-0.07. These results suggest a mechanism by which antidepressants could alter the function of diverse membrane proteins by partitioning into cell membranes and thereby altering the bilayer contribution to the energetics of membrane protein conformational changes.

Lipid- and Cholesterol-Mediated Timescale-Specific Modulation of Membrane Protein Dynamics

Membrane protein function fundamentally depends on the surrounding lipid bilayer. In the past few years, it has become clear that this fundamental relationship is intricate and multilayered, going far beyond simple hydrophobic interactions (e.g. Autzen et al, Science, 2018). It has also become clear that mechanisms of membrane protein activation by temperature exist, likely involving lipid dynamics as a regulatory factor (e.g. Gao et al, Nature, 2016). Hence, atomic insights into lipid-mediated modulation of membrane protein dynamics would provide new insights with the potential to fundamentally extend our understanding on lipid-protein interdependencies. Although highly anticipated, such insights remained elusive due to its intrinsic difficulty. To elucidate the impact of lipid dynamics on a membrane protein, we reconstituted the outer membrane protein X (OmpX) into a saturated, unsaturated and cholesterol-containing lipid-bilayer using the nanodisc technology. We then recorded timescale-specific NMR relaxation experiments with atomic resolution of both the membrane protein and lipids within the same nanodisc. The relaxation data revealed that lipid order, modified either biochemically or biophysically, changes the dynamics of the immersed membrane protein in a specific and timescale-dependent manner. A temperature-dependent dynamics analysis furthermore suggests a direct coupling between lipid and protein dynamics in the picosecond, microsecond and millisecond timescale, caused by the lipid's trans-gauche isomerization, the rotational diffusion of lipids and the fluidity of the lipid phase, respectively. These observations evidence a direct modulatory capability of the membrane to regulate protein function through lipid dynamics. Strikingly, the available timescales for such coupling covers at least 6 orders of magnitude, providing a substantially larger repertoire to regulate functions of immersed proteins, as compared to its aqueous counterpart.

Cryo-EM Structures Reveal Bilayer Remodeling during Ca2+ Activation of a TMEM16 Scramblase

The plasma membranes of eukaryotic cells are organized asymmetrically for cellular function. At rest, polar and charged lipids are sequestered to the inner leaflet by the activity of ATP-driven pumps. Activation of a specialized class of membrane proteins – phospholipid scramblases – causes rapid collapse of this asymmetry and externalization of negatively charged phosphatidylserine molecules. As a result, extracellular signaling networks, controlling processes such as apoptosis, blood coagulation, membrane fusion and repair, are activated. The TMEM16 family of membrane proteins includes phospholipid scramblases and Cl- channels, all of which are Ca2+-dependent. Prior structural and functional analyses of the fungal TMEM16 scramblase from Nectria haematococca identified a membrane-exposed hydrophilic groove that serves as the lipid translocation pathway. In the TMEM16A channel, this pathway is sealed from the membrane, thus preventing lipid access and enabling ion permeation. However, the mechanisms underlying Ca2+-dependent gating of TMEM16 scramblases, and their effects on the surrounding lipid bilayer, remain poorly understood. Here we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs. Differences between the Ca2+-free and Ca2+-bound states reveal that Ca2+ binding induces a global rearrangement of the transmembrane and cytosolic regions, which causes the lipid pathway to open. Further, comparison of these structures and a third in the presence of a long-chain inhibitory lipid, together with functional experiments, reveal that scramblases cause profound remodeling of the surrounding membrane. Specifically, scramblase activation causes the bilayer to thin at the open lipid pathway and the outer leaflet to bend towards the center of the bilayer at the dimer interface. We propose a model in which trans-bilayer lipid movement is enabled by these membrane and protein rearrangements and provide a general mechanistic framework for phospholipid scrambling.

EPR Spectroscopic Studies of the Voltage Sensor Domain (Q1-VSD) of Human KCNQ1 Potassium Ion Channel in Lipid Bilayers

KCNQ1 is a voltage gated potassium ion channel expressed in several tissues of the body and modulated by another membrane protein KCNE1 (E1). Q1 is involved in the repolarization phase of cardiac action potential. Q1/E1 interaction slows down the activation kinetics required for proper channel and heart function. Mutations in Q1 can cause Long-QT syndrome resulting in atrial fibrillation, sudden infant death syndrome, cardiac arrhythmias and congenital deafness. Q1-VSD is important as the channel is activated by a change in voltage, which is sensed by the VSD. Around 40% of the >200 reported disease-related mutations arise from the amino acid substitutions in the VSD, making structural and functional studies of Q1-VSD important. The structure of a membrane protein varies from micelles to lipid bilayer environment. Site-directed spin labeling (SDSL) EPR is a powerful structural biology technique to study the structural dynamics and topology of membrane proteins in lipid bilayers. In this study, the structural dynamics and topology of several residues of Q1-VSD in the POPC/POPG vesicles were studied using CW-EPR line shape analysis and CW power saturation techniques. Spin labeled mutants F130C and F232C were found to be located inside the lipid bilayer, while Q147C and F222C were found to be solvent accessible. The study provides novel insights to the structural details of Q1-VSD reconstituted in POPC/POPG vesicles for more advanced structural and functional studies.

Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase.

The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid, nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several TMEM16 family members have been identified as Ca2+-activated scramblases, but the mechanisms underlying their Ca2+-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here, we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs. These structures reveal that Ca2+-dependent activation of the scramblase entails global rearrangement of the transmembrane and cytosolic domains. These structures, together with functional experiments, suggest that activation of the protein thins the membrane near the transport pathway to facilitate rapid transbilayer lipid movement.

Polar Interactions Play an Important Role in the Energetics of the Main Phase Transition of Phosphatidylcholine Membranes.

Conformational changes of membrane proteins are accompanied by deformation in the surrounding lipid bilayer. To gain insight into the energetics of membrane deformation, the phase behavior of dimyristoylphosphatidylcholine (DMPC) membranes in the presence of the dipole potential, ψd, modifiers was investigated by differential scanning calorimetry. 7-Ketocholesterol, which weakens ψd and reduces membrane-perpendicular dipole–dipole repulsion, causes a discrete second peak on the high-temperature side of the main transition, whereas 6-ketocholestanol, which strengthens ψd and increases membrane-perpendicular dipole–dipole repulsion, merely produces a shoulder. Measurements on pure DMPC vesicles showed that the observed temperature profile could not be explained by a single endothermic process, that is, breaking of van der Waals forces between hydrocarbon chains alone. Removal of NaCl from the buffer caused an increase in the main transition temperature and the appearance of an obvious shoulder, implicating polar interactions. Consideration of the phosphatidylcholine (PC) head group dipole moment indicates direct interactions between PC dipoles that are unlikely to account for the additional process. It seems more likely that the breaking of an in-plane hydrogen-bonded network consisting of hydrating water dipoles together with zwitterionic lipid head groups is responsible. The evidence presented supports the idea that the breaking of van der Waals forces between lipid tails required for the main phase transition of PC membranes is coupled to partial breaking of a hydrogen-bonded network at the membrane surface.

Probing the Dynamics and Structural Topology of the Reconstituted Human KCNQ1 Voltage Sensor Domain (Q1-VSD) in Lipid Bilayers Using Electron Paramagnetic Resonance Spectroscopy.

KCNQ1 (Kv7.1 or KvLQT1) is a potassium ion channel protein found in the heart, ear, and other tissues. In complex with the KCNE1 accessory protein, it plays a role during the repolarization phase of the cardiac action potential. Mutations in the channel have been associated with several diseases, including congenital deafness and long QT syndrome. Nuclear magnetic resonance (NMR) structural studies in detergent micelles and a cryo-electron microscopy structure of KCNQ1 from Xenopus laevis have shown that the voltage sensor domain (Q1-VSD) of the channel has four transmembrane helices, S1-S4, being overall structurally similar with other VSDs. In this study, we describe a reliable method for the reconstitution of Q1-VSD into (POPC/POPG) lipid bilayer vesicles. Site-directed spin labeling electron paramagnetic resonance spectroscopy was used to probe the structural dynamics and topology of several residues of Q1-VSD in POPC/POPG lipid bilayer vesicles. Several mutants were probed to determine their location and corresponding immersion depth (in angstroms) with respect to the membrane. The dynamics of the bilayer vesicles upon incorporation of Q1-VSD were studied using31P solid-state NMR spectroscopy by varying the protein:lipid molar ratios confirming the interaction of the protein with the bilayer vesicles. Circular dichroism spectroscopic data showed that the α-helical content of Q1-VSD is higher for the protein reconstituted in vesicles than in previous studies using DPC detergent micelles. This study provides insight into the structural topology and dynamics of Q1-VSD reconstituted in a lipid bilayer environment, forming the basis for more advanced structural and functional studies.

Reconstitution of the Rhodopsin-Transducin Complex into Lipid Nanodiscs.

Transmembrane proteins, such as G protein-coupled receptors (GPCR), require solubilization in detergents prior to purification. The recent development of novel detergents has allowed for the stabilization of GPCRs, which typically have a high degree of structural flexibility and are otherwise subject to denaturation. However, the detergent micelle environment is still very different from the native lipid membrane and the activity of GPCRs can be profoundly affected by interactions with annular lipid molecules. Moreover, GPCRs are often palmitoylated at their intracellular side, and a lipid bilayer environment would allow for proper orientation of these lipid modifications. Therefore, a reconstituted lipid bilayer environment would best mimic the physiological receptor microenvironment for biophysical studies of GPCRs and nanodiscs provide a methodology to address this aim. Nanodiscs are lipid bilayer discs stabilized by amphipathic membrane scaffolding proteins (MSP) where detergent-solubilized transmembrane proteins can be incorporated into them through a self-assembly process. Here we present a method for reconstituting the purified detergent-solubilized rhodopsin-transducin complex, the GPCR-G protein complex in visual phototransduction, into nanodiscs. The resulting complex incorporated into lipid nanodiscs can be used in biophysical studies including small-angle X-ray scattering and electron microscopy. This method is applicable to integral membrane proteins that mediate protein lipidation, including the zDHHC-family of S-acyltransferases and membrane-bound O-acyltransferases.

How non-enveloped viruses hijack host machineries to cause infection.

Viruses must navigate the complex endomembranous network of the host cell to cause infection. In the case of a non-enveloped virus that lacks a surrounding lipid bilayer, endocytic uptake from the plasma membrane is not sufficient to cause infection. Instead, the virus must travel within organelle membranes to reach a specific cellular destination that supports exposure or arrival of the virus to the cytosol. This is achieved by viral penetration across a host endomembrane, ultimately enabling entry of the virus into the nucleus to initiate infection. In this review, we discuss the entry mechanisms of three distinct non-enveloped DNA viruses-adenovirus (AdV), human papillomavirus (HPV), and polyomavirus (PyV)-highlighting how each exploit different intracellular transport machineries and membrane penetration apparatus associated with the endosome, Golgi, and endoplasmic reticulum (ER) membrane systems to infect a host cell. These processes not only illuminate a highly-coordinated interplay between non-enveloped viruses and their host, but may provide new strategies to combat non-enveloped virus-induced diseases.

Chemical Additives Enable Native Mass Spectrometry Measurement of Membrane Protein Oligomeric State within Intact Nanodiscs.

Membrane proteins play critical biochemical roles but remain challenging to study. Recently, native or nondenaturing mass spectrometry (MS) has made great strides in characterizing membrane protein interactions. However, conventional native MS relies on detergent micelles, which may disrupt natural interactions. Lipoprotein nanodiscs provide a platform to present membrane proteins for native MS within a lipid bilayer environment, but previous native MS of membrane proteins in nanodiscs has been limited by the intermediate stability of nanodiscs. It is difficult to eject membrane proteins from nanodiscs for native MS but also difficult to retain intact nanodisc complexes with membrane proteins inside. Here, we employed chemical reagents that modulate the charge acquired during electrospray ionization (ESI). By modulating ESI conditions, we could either eject the membrane protein complex with few bound lipids or capture the intact membrane protein nanodisc complex-allowing measurement of the membrane protein oligomeric state within an intact lipid bilayer environment. The dramatic differences in the stability of nanodiscs under different ESI conditions opens new applications for native MS of nanodiscs.

Characterizing the structure of styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) using RAFT polymerization for membrane protein spectroscopic studies.

Membrane proteins play an important role in maintaining the structure and physiology of an organism. Despite their significance, spectroscopic studies involving membrane proteins remain challenging due to the difficulties in mimicking their native lipid bilayer environment. Membrane mimetic systems such as detergent micelles, liposomes, bicelles, nanodiscs, lipodisqs have improved the solubility and folding properties of the membrane proteins for structural studies, however, each mimetic system suffers from its own limitations. In this study, using three different lipid environments, vesicles were titrated with styrene-maleic acid (StMA) copolymer leading to a homogeneous SMALP system (∼10 nm) at a weight ratio of 1:1.5 (vesicle: StMA solution). A combination of Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) was used to characterize these SMALPs. We used a controlled synthesis mechanism to synthesize StMA based block copolymers called reversible addition-fragmentation chain transfer polymerization (RAFT) SMALPs. Incorporation of the Voltage Sensor Domain of KCNQ1 (Q1-VSD) into RAFT SMALPs indicates that this is a promising application of this system to study membrane proteins using different biophysical techniques. V165C in Q1-VSD corresponding to the hydrophobic region was incorporated into the SMALP system. Continuous Wave-Electron Paramagnetic Resonance (CW-EPR) line shape analysis showed line shape broadening, exposing a lower rigid component and a faster component of the spin label.

G-Protein Coupled Receptor Protein Synthesis on a Lipid Bilayer Using a Reconstituted Cell-Free Protein Synthesis System.

Membrane proteins are important drug targets which play a pivotal role in various cellular activities. However, unlike cytosolic proteins, most of them are difficult-to-express proteins. In this study, to synthesize and produce sufficient quantities of membrane proteins for functional and structural analysis, we used a bottom-up approach in a reconstituted cell-free synthesis system, the PURE system, supplemented with artificial lipid mimetics or micelles. Membrane proteins were synthesized by the cell-free system and integrated into lipid bilayers co-translationally. Membrane proteins such as the G-protein coupled receptors were expressed in the PURE system and a productivity ranging from 0.04 to 0.1 mg per mL of reaction was achieved with a correct secondary structure as predicted by circular dichroism spectrum. In addition, a ligand binding constant of 27.8 nM in lipid nanodisc and 39.4 nM in micelle was obtained by surface plasmon resonance and the membrane protein localization was confirmed by confocal microscopy in giant unilamellar vesicles. We found that our method is a promising approach to study the different classes of membrane proteins in their native-like artificial lipid bilayer environment for functional and structural studies.

Generation of membrane proteins in polymer-based lipoparticles as flow cytometry antigens

A major obstacle in the study of membrane proteins is the challenge in generating purified targets in a stable and native conformation. Potentially destabilizing detergents are often used to extract targets out of their native membranes. One approach to overcome these destabilization issues is to present targets in a lipid bilayer environment. While numerous lipid bilayer presentation approaches such as Nanodiscs and liposomes have been used through the years, most still require a detergent purified protein intermediate. Styrene-maleic acid lipoparticles (SMALPs) have been developed to directly solubilize membranes without the use of detergents and present membrane proteins in a native lipid bilayer environment. These discoidal lipid bilayer particles have been evaluated on several membrane protein classes (e.g. ion channels, transporters) and applications (e.g. spectroscopy, electron microscopy). Here, cannabinoid receptor 1 (CB1), a class A G-protein coupled receptor, was incorporated into a SMALP (CB1-SMALP) and shown to retain its native fold through binding studies with a CB1-specific conformational antibody. The binding studies were conducted in yeast and mammalian display formats using fluorescence-activated cell sorting (FACS). Compared to detergent preparations, CB1-SMALP also exhibited a marked increase in thermostability. This study provides the framework for a platform to generate full-length membrane protein screening antigens for flow cytometry-based studies.

Candida albicans biofilm-induced vesicles confer drug resistance through matrix biogenesis.

Cells from all kingdoms of life produce extracellular vesicles (EVs). Their cargo is protected from the environment by the surrounding lipid bilayer. EVs from many organisms have been shown to function in cell–cell communication, relaying signals that impact metazoan development, microbial quorum sensing, and pathogenic host–microbe interactions. Here, we have investigated the production and functional activities of EVs in a surface-associated microbial community or biofilm of the fungal pathogen Candida albicans. Crowded communities like biofilms are a context in which EVs are likely to function. Biofilms are noteworthy because they are encased in an extracellular polymeric matrix and because biofilm cells exhibit extreme tolerance to antimicrobial compounds. We found that biofilm EVs are distinct from those produced by free-living planktonic cells and display strong parallels in composition to biofilm matrix material. The functions of biofilm EVs were delineated with a panel of mutants defective in orthologs of endosomal sorting complexes required for transport (ESCRT) subunits, which are required for normal EV production in diverse eukaryotes. Most ESCRT-defective mutations caused reduced biofilm EV production, reduced matrix polysaccharide levels, and greatly increased sensitivity to the antifungal drug fluconazole. Matrix accumulation and drug hypersensitivity of ESCRT mutants were reversed by addition of wild-type (WT) biofilm EVs. Vesicle complementation showed that biofilm EV function derives from specific cargo proteins. Our studies indicate that C. albicans biofilm EVs have a pivotal role in matrix production and biofilm drug resistance. Biofilm matrix synthesis is a community enterprise; prior studies of mixed cell biofilms have demonstrated extracellular complementation. Therefore, EVs function not only in cell–cell communication but also in the sharing of microbial community resources.