Reconstituted Membrane Research Articles

Structural and Spectroscopic Characterization of Supported Sarcoplasmic Reticulum Membranes on Solid Substrates.

Sarcoplasmic reticulum (SR) membranes from rabbit muscle were deposited on silicon substrates and characterized by the combination of spectral ellipsometry (SE), high energy specular X-ray reflectivity (XRR), specular neutron reflectivity (NR), and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Following the optimization of the preparative conditions by SE, the detailed structures in the direction perpendicular to the membrane were probed by XRR. ATR-FTIR data showed strong signals from amide I and amide II bands of the native SR membranes containing a large amount of Ca2+-ATPase, which could not be achieved by the reconstitution in artificial lipid membranes. The treatment with protease led to a significant decrease in the amide peaks, and the XRR data confirmed the modulation of the membrane structures. The obtained data show the potential of the in situ combination of reflectivity and vibrational spectroscopy of native supported membranes in order to unravel both structure and dynamics of complex biological membranes.

The exocyst in context.

The exocyst is a hetero-octameric complex involved in the exocytosis arm of cellular trafficking. Specifically, it tethers secretory vesicles to the plasma membrane, but it is also a main convergence point for many players of exocytosis: regulatory proteins, motor proteins, lipids and Soluble N-ethylmaleimide-sensitive factor Attachment Protein Receptor (SNARE) proteins are all connected physically by the exocyst. Despite extensive knowledge about its structure and interactions, the exocyst remains an enigma precisely because of its increasingly broad and flexible role across the exocytosis process. To solve the molecular mechanism of such a multi-tasking complex, dynamical structures with self, other proteins, and environment should be described. And to do this, interrogation within contexts increasingly close to native conditions is needed. Here we provide a perspective on how different experimental contexts have been used to study the exocyst, and those that could be used in the future. This review describes the structural breakthroughs on the isolated in vitro exocyst, followed by the use of membrane reconstitution assays for revealing in vitro exocyst functionality. Next, it moves to in situ cell contexts, reviewing imaging techniques that have been, and that ideally could be, used to look for near-native structure and organization dynamics. Finally, it looks at the exocyst structure in situ within evolutionary contexts, and the potential of structure prediction therein. From in vitro, to in situ, cross-context investigation of exocyst structure has begun, and will be critical for functional mechanism elucidation.

Biogenesis, engineering and function of membranes in the CO 2 -fixing pyrenoid.

Approximately one-third of global CO 2 assimilation is performed by the pyrenoid 1 , a liquid-like organelle found in most algae and some plants 2 . Specialized membranes are hypothesized to drive CO 2 assimilation in the pyrenoid by delivering concentrated CO 2 3,4 , but their biogenesis and function have not been experimentally characterized. Here, we show that homologous proteins SAGA1 and MITH1 mediate the biogenesis of the pyrenoid membrane tubules in the model alga Chlamydomonas reinhardtii and are sufficient to reconstitute pyrenoid-traversing membranes in a heterologous system, the plant Arabidopsis thaliana . SAGA1 localizes to the regions where thylakoid membranes transition into tubules and is necessary to initiate tubule formation. MITH1 localizes to the tubules and is necessary for their extension through the pyrenoid. Tubule-deficient mutants exhibit growth defects under CO 2 -limiting conditions, providing evidence for the function of membrane tubules in CO 2 delivery to the pyrenoid. Furthermore, these mutants form multiple aberrant condensates of pyrenoid matrix, indicating that a normal tubule network promotes the coalescence of a single pyrenoid. The reconstitution of pyrenoid-traversing membranes in a plant represents a key milestone toward engineering a functional pyrenoid into crops for improving crop yields. More broadly, our study demonstrates the functional importance of pyrenoid membranes, identifies key biogenesis factors, and paves the way for the molecular characterization of pyrenoid membranes across the tree of life.

Overcoming Protein Orientation Mismatch Enables Efficient Nanoscale Light-Driven ATP Production.

Adenosine triphosphate (ATP)-producing modules energized by light-driven proton pumps are powerful tools for the bottom-up assembly of artificial cell-like systems. However, the maximum efficiency of such modules is prohibited by the random orientation of the proton pumps during the reconstitution process into lipid-surrounded nanocontainers. Here, we overcome this limitation using a versatile approach to uniformly orient the light-driven proton pump proteorhodopsin (pR) in liposomes. pR is post-translationally either covalently or noncovalently coupled to a membrane-impermeable protein domain guiding orientation during insertion into preformed liposomes. In the second scenario, we developed a novel bifunctional linker, trisNTA-SpyTag, that allows for the reversible connection of any SpyCatcher-containing protein and a HisTag-carrying protein. The desired protein orientations are verified by monitoring vectorial proton pumping and membrane potential generation. In conjunction with ATP synthase, highly efficient ATP production is energized by the inwardly pumping population. In comparison to other light-driven ATP-producing modules, the uniform orientation allows for maximal rates at economical protein concentrations. The presented technology is highly customizable and not limited to light-driven proton pumps but applicable to many membrane proteins and offers a general approach to overcome orientation mismatch during membrane reconstitution, requiring little to no genetic modification of the protein of interest.

Factors influencing the detergent-free membrane protein isolation using synthetic nanodisc-forming polymers

The detergent-free isolation of membrane proteins using synthetic polymers is becoming the desired approach for functional and structural studies of membrane proteins. Since the expression levels for many membrane proteins are low and a high yield of functionalized reconstituted membrane proteins is essential for in vitro studies, it is crucial to optimize the experimental conditions for a given polymer to solubilize target membranes/proteins effectively. The factors that affect membrane solubilization and subsequently the isolation of a target membrane protein include polymer concentration, polymer charge, temperature, pH, and concentration of divalent metal ions. Therefore, it is important to have knowledge about the efficacy of different types of polymers in solubilizing cell membranes. In this study, we evaluate the efficacy of inulin-based non-ionic polymers in solubilizing E. coli membranes enriched with rat flavin mononucleotide binding-domain (FBD) of cytochrome-P450-reductase (CPR) and rabbit cytochrome-b5 (Cyt-b5) under various solubilization conditions. Our results show that a 1:1 (w/w) membrane:polymer ratio, low temperature, high pH and sub-millimolar concentration of metal ions favor the solubilization of E. coli membranes enriched with FBD or Cyt-b5. Conversely, the presence of excess divalent metal ions affected the final protein levels in the polymer-solubilized samples. We believe that the results from this study provide knowledge to assess and plan the use of non-ionic polymers in membrane protein studies.

Probing macromolecular crowding at the lipid membrane interface with genetically-encoded sensors.

Biochemical processes within the living cell occur in a highly crowded environment, where macromolecules, first of all proteins and nucleic acids, occupy up to 30 % of the volume. The phenomenon of macromolecular crowding is not an exclusive feature of the cytoplasm and can be observed in the densely protein-packed, nonhomogeneous cellular membranes and at the membrane interfaces. Crowding affects diffusional and conformational dynamics of proteins within the lipid bilayer, alters kinetic and thermodynamic properties of biochemical reactions, and modulates the membrane organization. Despite its importance, the non-invasive quantification of the membrane crowding is not trivial. Here, we developed the genetically-encoded fluorescence-based sensor for probing the macromolecular crowding at the membrane interfaces. Two sensor variants, both composed of fluorescent proteins and a membrane anchor, but differing by the flexible linker domains were characterized in vitro, and the procedures for the membrane reconstitution were established. Steric pressure induced by membrane-tethered synthetic and protein crowders altered the sensors' conformation, causing increase in the intramolecular Förster's resonance energy transfer. Notably, the effect of protein crowders only weakly correlated with their molecular weight, suggesting that other factors, such as shape and charge contribute to the crowding via the quinary interactions. Finally, measurements performed in inner membrane vesicles of E. coli validated the crowding-dependent dynamics of the sensors in the physiologically relevant environment. The sensors offer broad opportunities to study interfacial crowding in a complex environment of native membranes, and thus add to the toolbox of methods for studying membrane dynamics and proteostasis. This article is protected by copyright. All rights reserved.

Conformational investigation of the asymmetric periplasmic domains of E. coli LptB2FGC using SDSL CW EPR spectroscopy.

The majority of pathogenic Gram-negative bacteria benefit from intrinsic antibiotic resistance, attributed primarily to the lipopolysaccharide (LPS) coating of the bacterial envelope. To effectively coat the bacterial cell envelope, LPS is transported from the inner membrane by the LPS transport (Lpt) system, which comprises seven distinct Lpt proteins, LptA-G, that form a stable protein bridge spanning the periplasm to connect the inner and outer membranes. The driving force of this process, LptB2FG, is an asymmetric ATP binding cassette (ABC) transporter with a novel architecture and function that ejects LPS from the inner membrane and facilitates transfer to the periplasmic bridge. Here, we utilize site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy to probe conformational differences between the periplasmic domains of LptF and LptG. We show that LptC solely interacts with the edge β-strand of LptF and does not directly interact with LptG. We also quantify the interaction of periplasmic LptC with LptF. Additionally, we show that LPS cannot enter the protein complex externally, supporting the unidirectional LPS transport model. Furthermore, we present our findings that the presence of LPS within the LptB2FGC binding cavity and the membrane reconstitution environment affect the structural orientation of the periplasmic domains of LptF and LptG, but overall are relatively fixed with respect to one another. This study will provide insight into the structural asymmetry associated with the newly defined type VI ABC transporter class.

Tunable biomimetic bacterial membranes from binary and ternary lipid mixtures and their application in antimicrobial testing

The reconstruction of accurate yet simplified mimetic models of cell membranes is a very challenging goal of synthetic biology. To date, most of the research focuses on the development of eukaryotic cell membranes, while reconstitution of their prokaryotic counterparts has not been fully addressed, and the proposed models do not reflect well the complexity of bacterial cell envelopes. Here, we describe the reconstitution of biomimetic bacterial membranes with an increasing level of complexity, developed from binary and ternary lipid mixtures. Giant unilamellar vesicles composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE); PC and phosphatidylglycerol (PG); PE and PG; PE, PG and cardiolipin (CA) at varying molar ratios were successfully prepared by the electroformation method. Each of the proposed mimetic models focuses on reproducing specific membrane features such as membrane charge, curvature, leaflets asymmetry, or the presence of phase separation. GUVs were characterized in terms of size distribution, surface charge, and lateral organization. Finally, the developed models were tested against the lipopeptide antibiotic daptomycin. The obtained results showed a clear dependency of daptomycin binding efficiency on the amount of negatively charged lipid species present in the membrane. We anticipate that the models proposed here can be applied not only in antimicrobial testing but also serve as platforms for studying fundamental biological processes in bacteria as well as their interaction with physiologically relevant biomolecules.

Function Investigations and Applications of Membrane Proteins on Artificial Lipid Membranes.

Membrane proteins play an important role in key cellular functions, such as signal transduction, apoptosis, and metabolism. Therefore, structural and functional studies of these proteins are essential in fields such as fundamental biology, medical science, pharmacology, biotechnology, and bioengineering. However, observing the precise elemental reactions and structures of membrane proteins is difficult, despite their functioning through interactions with various biomolecules in living cells. To investigate these properties, methodologies have been developed to study the functions of membrane proteins that have been purified from biological cells. In this paper, we introduce various methods for creating liposomes or lipid vesicles, from conventional to recent approaches, as well as techniques for reconstituting membrane proteins into artificial membranes. We also cover the different types of artificial membranes that can be used to observe the functions of reconstituted membrane proteins, including their structure, number of transmembrane domains, and functional type. Finally, we discuss the reconstitution of membrane proteins using a cell-free synthesis system and the reconstitution and function of multiple membrane proteins.

Rapid and Highly Stable Membrane Reconstitution by LAiR Enables the Study of Physiological Integral Membrane Protein Functions.

Functional reintegration into lipid environments represents a major challenge for in vitro investigation of integral membrane proteins (IMPs). Here, we report a new approach, termed LMNG Auto-insertion Reintegration (LAiR), for reintegration of IMPs into lipid bilayers within minutes. The resulting proteoliposomes displayed an unprecedented capability to maintain proton gradients and long-term stability. LAiR allowed for monitoring catalysis of a membrane-bound, physiologically relevant polyisoprenoid quinone substrate by Escherichia coli cytochromes bo 3 (cbo 3) and bd (cbd) under control of the proton motive force. LAiR also facilitated bulk-phase detection and physiological assessment of the "proton leak" in cbo 3, a controversial catalytic state that previously was only approachable at the single-molecule level. LAiR maintained the multisubunit integrity and higher-order oligomeric states of the delicate mammalian F-ATP synthase. Given that LAiR can be applied to both liposomes and planar membrane bilayers and is compatible with IMPs and lipids from prokaryotic and eukaryotic sources, we anticipate LAiR to be applied broadly across basic research, pharmaceutical applications, and biotechnology.

Conformation-dependent diffusion properties of a transmembrane protein.

Fundamental cell physiology originates from conformational changes and diffusion dynamics of membrane proteins. However, studying the interplay between nanoscale protein conformations and membrane mechanics is inherently challenging in cell-based experiments. In this work, we investigate the diffusion dynamics of multidrug resistance ABC transporter BmrA as the function of protein conformations: apo (open) or post-hydrolytic (closed) upon in-vitro membrane reconstitution. We perform single-particle tracking measurements on BmrA reconstituted in giant unilamellar vesicles with controlled and varying membrane tensions. We observe a distinct conformation-dependent diffusion profile of BmrA which is inhibited upon increase in membrane tension. Further, experiments involving saturating levels of ATP indicate confinement of proteins to specific “hubs” indicating a new mechanism of protein clustering that competes with membrane tension, and protein enzymatic reactions. Our work gives insights into the intrinsic diffusion dynamics of an active membrane transporter, its interconnection with membrane mechanics, and conformational cycles; all possibly influencing the cell function.

Electrical recordings from dendritic spines of adult mouse hippocampus and effect of the actin cytoskeleton.

Dendritic spines (DS) are tiny protrusions implicated in excitatory postsynaptic responses in the CNS. To achieve their function, DS concentrate a high density of ion channels and dynamic actin networks in a tiny specialized compartment. However, to date there is no direct information on DS ionic conductances. Here, we used several experimental techniques to obtain direct electrical information from DS of the adult mouse hippocampus. First, we optimized a method to isolate DS from the dissected hippocampus. Second, we used the lipid bilayer membrane (BLM) reconstitution and patch clamping techniques and obtained heretofore unavailable electrical phenotypes on ion channels present in the DS membrane. Third, we also patch clamped DS directly in cultured adult mouse hippocampal neurons, to validate the electrical information observed with the isolated preparation. Electron microscopy and immunochemistry of PDS-95 and NMDA receptors and intrinsic actin networks confirmed the enrichment of the isolated DS preparation, showing open and closed DS, and multi-headed DS. The preparation was used to identify single channel activities and “whole-DS” electrical conductance. We identified NMDA and Ca2+-dependent intrinsic electrical activity in isolated DS and in situ DS of cultured adult mouse hippocampal neurons. In situ recordings in the presence of local NMDA, showed that individual DS intrinsic electrical activity often back-propagated to the dendrite from which it sprouted. The DS electrical oscillations were modulated by changes in actin cytoskeleton dynamics by addition of the F-actin disrupter agent, cytochalasin D, and exogenous actin-binding proteins. The data indicate that DS are elaborate excitable electrical devices, whose activity is a functional interplay between ion channels and the underlying actin networks. The data argue in favor of the active contribution of individual DS to the electrical activity of neurons at the level of both the membrane conductance and cytoskeletal signaling.

Natamycin sequesters ergosterol and interferes with substrate transport by the lysine transporter Lyp1 from yeast

Natamycin is a polyene macrolide, widely employed to treat fungal keratitis and other yeast infections as well as to protect food products against fungal molds. In contrast to other polyene macrolides, such as nystatin or amphotericin B, natamycin does not form pores in yeast membranes, and its mode of action is not well understood. Here, we have employed a variety of spectroscopic methods, computational modeling, and membrane reconstitution to study the molecular interactions of natamycin underlying its antifungal activity. We find that natamycin forms aggregates in an aqueous solution with strongly altered optical properties compared to monomeric natamycin. Interaction of natamycin with model membranes results in a concentration-dependent fluorescence increase which is more pronounced for ergosterol- compared to cholesterol-containing membranes up to 20 mol% sterol. Evidence for formation of specific ergosterol-natamycin complexes in the bilayer is provided. Using nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopy, we find that natamycin sequesters sterols, thereby interfering with their well-known ability to order acyl chains in lipid bilayers. This effect is more pronounced for membranes containing the sterol of fungi, ergosterol, compared to those containing mammalian cholesterol. Natamycin interferes with ergosterol-dependent transport of lysine by the yeast transporter Lyp1, which we propose to be due to the sequestering of ergosterol, a mechanism that also affects other plasma membrane proteins. Our results provide a mechanistic explanation for the selective antifungal activity of natamycin, which can set the stage for rational design of novel polyenes in the future.

Tetraspanin CD53 controls Tcell immunity through regulation of CD45RO stability, mobility, and function.

T cells depend on the phosphatase CD45 to initiate Tcell receptor signaling. Although the critical role of CD45 in Tcells is established, the mechanisms controlling function and localization in the membrane are not well understood. Moreover, the regulation of specific CD45 isoforms in Tcell signaling remains unresolved. By using unbiased mass spectrometry, we identify the tetraspanin CD53 as a partner of CD45 and show that CD53 controls CD45 function and Tcell activation. CD53-negative Tcells (Cd53-/-) exhibit substantial proliferation defects, and Cd53-/- mice show impaired tumor rejection and reduced IFNγ-producing Tcells compared with wild-type mice. Investigation into the mechanism reveals that CD53 is required for CD45RO expression and mobility. In addition, CD53 is shown to stabilize CD45 on the membrane and is required for optimal phosphatase activity and subsequent Lck activation. Together, our findings reveal CD53 as a regulator of CD45 activity required for Tcell immunity.

Engineering Functional Membrane-Membrane Interfaces by InterSpy.

Engineering synthetic interfaces between membranes has potential applications in designing non-native cellular communication pathways and creating synthetic tissues. Here, InterSpy is introduced as a synthetic biology tool consisting of a heterodimeric protein engineered to form and maintain membrane-membrane interfaces between apposing synthetic as well as cell membranes through the SpyTag/SpyCatcher interaction. The inclusion of split fluorescent protein fragments in InterSpy allows tracking of the formation of a membrane-membrane interface and reconstitution of functional fluorescent protein in the space between apposing membranes. First, InterSpy is demonstrated by testing split protein designs using a mammalian cell-free expression (CFE) system. By utilizing co-translational helix insertion, cell-free synthesized InterSpy fragments are incorporated into the membrane of liposomes and supported lipid bilayers with the desired topology. Functional reconstitution of split fluorescent protein between the membranes is strictly dependent on SpyTag/SpyCatcher. Finally, InterSpy is demonstrated in mammalian cells by detecting fluorescence reconstitution of split protein at the membrane-membrane interface between two cells each expressing a component of InterSpy. InterSpy demonstrates the power of CFE systems in the functional reconstitution of synthetic membrane interfaces via proximity-inducing proteins. This technology may also prove useful where cell-cell contacts and communication are recreated in a controlled manner using minimal components.

Single-molecule phospholipase A2 becomes processive on melittin-induced membrane deformations

While it is established that the topology of lipid membranes plays an important role in biochemical processes, few direct observations exist regarding how the membranes are actively restructured and its consequences on subsequent reactions. In this work, we investigated how the two major components of bee venom, melittin and phospholipase A2 (PLA2), achieve activation by such membrane remodeling. Their membrane-disrupting functions have been reported to increase when both are present, but the mechanism of this synergism had not been established. Using membrane reconstitution, we found that melittin can form large-scale membrane deformities upon which PLA2 activity is 25-fold higher. Tracking of single-molecule PLA2 revealed that its processive behavior on these deformities underlies the enhanced activity. These results show how melittin and PLA2 work synergistically to enhance the lytic effects of the bee venom. More broadly, they also demonstrate how the membrane topology may be actively altered to modulate cellular membrane-bound reactions.

The Ionic Selectivity of Lysenin Channels in Open and Sub-Conducting States.

The electrochemical gradients established across cell membranes are paramount for the execution of biological functions. Besides ion channels, other transporters, such as exogenous pore-forming toxins, may present ionic selectivity upon reconstitution in natural and artificial lipid membranes and contribute to the electrochemical gradients. In this context, we utilized electrophysiology approaches to assess the ionic selectivity of the pore-forming toxin lysenin reconstituted in planar bilayer lipid membranes. The membrane voltages were determined from the reversal potentials recorded upon channel exposure to asymmetrical ionic conditions, and the permeability ratios were calculated from the fit with the Goldman–Hodgkin–Katz equation. Our work shows that lysenin channels are ion-selective and the determined permeability coefficients are cation and anion-species dependent. We also exploited the unique property of lysenin channels to transition to a stable sub-conducting state upon exposure to calcium ions and assessed their subsequent change in ionic selectivity. The observed loss of selectivity was implemented in an electrical model describing the dependency of reversal potentials on calcium concentration. In conclusion, our work demonstrates that this pore-forming toxin presents ionic selectivity but this is adjusted by the particular conduction state of the channels.

Fluorescence Approaches for Characterizing Ion Channels in Synthetic Bilayers.

Ion channels are membrane proteins that play important roles in a wide range of fundamental cellular processes. Studying membrane proteins at a molecular level becomes challenging in complex cellular environments. Instead, many studies focus on the isolation and reconstitution of the membrane proteins into model lipid membranes. Such simpler, in vitro, systems offer the advantage of control over the membrane and protein composition and the lipid environment. Rhodopsin and rhodopsin-like ion channels are widely studied due to their light-interacting properties and are a natural candidate for investigation with fluorescence methods. Here we review techniques for synthesizing liposomes and for reconstituting membrane proteins into lipid bilayers. We then summarize fluorescence assays which can be used to verify the functionality of reconstituted membrane proteins in synthetic liposomes.

Analysis of the mechanosensor channel functionality of TACAN.

Mechanosensitive ion channels mediate transmembrane ion currents activated by mechanical forces. A mechanosensitive ion channel called TACAN was recently reported. We began to study TACAN with the intent to understand how it senses mechanical forces and functions as an ion channel. Using cellular patch-recording methods, we failed to identify mechanosensitive ion channel activity. Using membrane reconstitution methods, we found that TACAN, at high protein concentrations, produces heterogeneous conduction levels that are not mechanosensitive and are most consistent with disruptions of the lipid bilayer. We determined the structure of TACAN using single-particle cryo-electron microscopy and observed that it is a symmetrical dimeric transmembrane protein. Each protomer contains an intracellular-facing cleft with a coenzyme A cofactor, confirmed by mass spectrometry. The TACAN protomer is related in three-dimensional structure to a fatty acid elongase, ELOVL7. Whilst its physiological function remains unclear, we anticipate that TACAN is not a mechanosensitive ion channel.

Polymer Nanodiscs and Their Bioanalytical Potential.

Membrane proteins (MPs) play a pivotal role in cellular function and are therefore predominant pharmaceutical targets. Although detailed understanding of MP structure and mechanistic activity is invaluable for rational drug design, challenges are associated with the purification and study of MPs. This review delves into the historical developments that became the prelude to currently available membrane mimetic technologies before shining a spotlight on polymer nanodiscs. These are soluble nanosized particles capable of encompassing MPs embedded in a phospholipid ring. The expanding range of reported amphipathic polymer nanodisc materials is presented and discussed in terms of their tolerance to different solution conditions and their nanodisc properties. Finally, the analytical scope of polymer nanodiscs is considered in both the demonstration of basic nanodisc parameters as well as in the elucidation of structures, lipid-protein interactions, and the functional mechanisms of reconstituted membrane proteins. The final emphasis is given to the unique benefits and applications demonstrated for native nanodiscs accessed through a detergent free process.