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The production of functionally active membrane proteins (MPs) in an adequate membrane environment is a key step in structural biology. Polymer–lipid particles based on styrene and maleic acid (SMA) represent a promising type of membrane mimic, as they can extract properly folded MPs directly from their native lipid environment. However, the original SMA polymer is sensitive to acidic pH levels, which has led to the development of several modifications: SMA-EA, SMA-QA, and others. Here, we introduce a novel SMA derivative with a negatively charged taurine moiety, SMA-tau, and investigate the formation and characteristics of lipid–SMA-EA and lipid–SMA-tau membrane-mimicking particles. Our findings demonstrate that both polymers can form nanodiscs with a patch of lipid bilayer that can undergo phase transitions at temperatures close to those of the lipid bilayer membranes. Finally, we discuss the potential applications of these SMAs for NMR spectroscopy. [ABSTRACT FROM AUTHOR]

For the A2A adenosine receptor (A2AAR), a class A G-protein-coupled receptor (GPCR), reconstituted in n-dodecyl-β-D-maltoside (DDM)/‌‌‌‌‌cholesteryl hemisuccinate (CHS) mixed micelles, previous 19F-NMR studies revealed the presence of multiple simultaneously populated conformational states. Here, we study the influence of a different detergent, lauryl maltose neopentyl glycol (LMNG) in mixed micelles with CHS, and of lipid bilayer nanodiscs on these conformational equilibria. The populations of locally different substates are pronouncedly different in DDM/‌‌‌‌‌CHS and LMNG/‌‌‌‌‌CHS micelles, whereas the A2AAR conformational manifold in LMNG/‌‌‌‌‌CHS micelles is closely similar to that in the lipid bilayer nanodiscs. Considering that nanodiscs represent a closer match of the natural lipid bilayer membrane, these observations support that LMNG/‌‌‌‌‌CHS micelles are a good choice for reconstitution trials of class A GPCRs for NMR studies in solution. [ABSTRACT FROM AUTHOR]

Physical and chemical criteria of biocompatibility of the active polymer implants and stimuli-responsive scaffolds are considered. From the standpoint of the surface physics and controlled wetting, the possibilities of dynamic control of biocompatibility and adaptive changes in the implant properties in response to the signal from the surrounding tissues are considered. The basic properties of the active biocompatible and biomimetic implantable materials, which distinguish them from the passive implants, are summarized. The latter include: electrophysical and electrophysiological membrane biocompatibility (up to the analogy with biomembranes – the so-called Fendler’s “membrane mimetics”); excitability, that is, the ability to qualitatively change their state in response to the external stimulus; compatibility of the matching parameters and impedances of biomembranes and active implantable materials; the presence of the main types of the energy conversion characteristic of biomembranes (chemiosmotic, electrochemical, electromechanical, etc.); the ability to transport and release pharmaceuticals consistent with the parameters of the cellular microenvironment and regulated by its state. Due to the qualitative change in the biomedical aim of such implants (from replacing the natural function to its regeneration and maintenance), there is a possibility of implementing various new biologically relevant functions using these materials, such as the ability to sensing and actuation, based on their reactivity and signal / energy conversion capacity. Of particular interest is the adaptive realization of the above functions in a growing and developing organism during its ontogenesis.

In biological olfactory systems, olfactory receptors (ORs) can recognize and discriminate between thousands of volatile organic compounds with very high sensitivity and specificity. The superior properties of ORs have led to the development of OR‐based biosensors that have shown promising potential in many applications over the past two decades. In particular, newly designed technologies in gene synthesis, protein expression, solubilization, purification, and membrane mimetics for membrane proteins have greatly opened up the previously inaccessible industrial potential of ORs. In this review, gene design, expression and solubilization strategies, and purification and reconstitution methods available for modern industrial applications are examined, with a focus on ORs. The limitations of current OR production technology are also estimated, and future directions for further progress are suggested. [ABSTRACT FROM AUTHOR]

Alcohol consumption and high-fat (HF) diets often coincide in Western society, resulting in synergistic negative effects on liver function. Although studies have analyzed the global protein expression in the context of alcoholic liver disease (ALD) and metabolic dysfunction-associated steatotic liver disease (MASLD), none has offered specific insights on liver dysregulation at the membrane proteome level. Membrane-specific profiling of metabolic and compensatory phenomena is usually overshadowed in conventional proteomic workflows. In this study, we use the Peptidisc method to isolate and compare the membrane protein (MP) content of the liver with its unique biological functions. From mice fed with an HF diet and ethanol in drinking water, we annotate over 1500 liver proteins with half predicted to have at least one transmembrane segment. Among them, we identify 106 integral MPs that are dysregulated compared to the untreated sample. Gene Ontology analysis reveals several dysregulated membrane-associated processes like lipid metabolism, cell adhesion, xenobiotic processing, and mitochondrial membrane formation. Pathways related to cholesterol and bile acid transport are also mutually affected, suggesting an adaptive mechanism to counter the upcoming steatosis of the liver model. Taken together, our Peptidisc-based profiling of the diet-dysregulated liver provides specific insights and hypotheses into the role of the transmembrane proteome in disease development, and flags desirable MPs for therapeutic and diagnostic targeting., (© 2024 The Author(s). PROTEOMICS published by Wiley‐VCH GmbH.)

G protein-coupled receptors (GPCRs) are a large membrane protein family found in higher organisms, including the human body. GPCRs mediate cellular responses to diverse extracellular stimuli and thus control key physiological functions, which makes them important targets for drug design. Signaling by GPCRs is related to the structure and dynamics of these proteins, which are modulated by extrinsic ligands as well as by intracellular binding partners such as G proteins and arrestins. Here, we review some basics of using nuclear magnetic resonance (NMR) spectroscopy in solution for the characterization of GPCR conformations and intermolecular interactions that relate to transmembrane signaling. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nanodisc technology is increasingly being used in structural, biochemical and biophysical studies of membrane proteins. The computational approaches have revealed many important features of nanodisc assembly, structures and dynamics. Therefore, we reviewed the application of computational approaches, especially molecular modeling and molecular dyncamics (MD) simulations, to characterize nanodiscs, including the structural models, assembly and disassembly, protocols for modeling, structural properties and dynamics, and protein-lipid interactions in nanodiscs. More amazing computational studies about nanodiscs are looked forward to in the future. [Display omitted] • Nanodiscs: a crucial biomembrane mimetic system. • Computational insights into nanodiscs. • Review of nanodisc-related computational efforts. • Shedding light on advancements and diverse perspectives of computational methods. [ABSTRACT FROM AUTHOR]

We leveraged variable-temperature 19F-NMR spectroscopy to compare the conformational equilibria of the human A 2A adenosine receptor (A 2A AR), a class A G protein-coupled receptor (GPCR), across a range of temperatures ranging from lower temperatures typically employed in 19F-NMR experiments to physiological temperature. A 2A AR complexes with partial agonists and full agonists showed large increases in the population of a fully active conformation with increasing temperature. NMR data measured at physiological temperature were more in line with functional data. This was pronounced for complexes with partial agonists, where the population of active A 2A AR was nearly undetectable at lower temperature but became evident at physiological temperature. Temperature-dependent behavior of complexes with either full or partial agonists exhibited a pronounced sensitivity to the specific membrane mimetic employed. Cellular signaling experiments correlated with the temperature-dependent conformational equilibria of A 2A AR in lipid nanodiscs but not in some detergents, underscoring the importance of the membrane environment in studies of GPCR function. [Display omitted] • Active A 2A AR population increases with increasing temperature in lipid nanodiscs • Active A 2A AR population exhibits different temperature dependence in detergents • Partial agonist complexes present a unique conformational state in nanodiscs • Temperature dependence of partial agonist equilibria depends on membrane mimetic Thakur and Ray et al. use 19F-NMR to compare the conformational equilibria of the A 2A adenosine receptor across different temperatures and with different membrane mimetics. They show that NMR data measured in lipid nanodiscs at physiological temperature correlate with signaling assays whereas data from detergent preparations does not. [ABSTRACT FROM AUTHOR]

Membrane proteins are key players of the cell. Their structure and the interactions they form with their lipid environment are required to understand their function. Here we explore liposomes as membrane mimetics for mass spectrometric analysis of peripheral membrane proteins and peptides. Liposomes are advantageous over other membrane mimetics in that they are easy to prepare, can be varied in size and composition, and are suitable for functional assays. We demonstrate that they dissociate into lipid clusters in the gas phase of a mass spectrometer while intact protein and protein–lipid complexes are retained. We exemplify this approach by employing different liposomes including proteoliposomes of two model peptides/proteins differing in size. Our results pave the way for the general application of liposomes for mass spectrometric analysis of membrane‐associated proteins. [ABSTRACT FROM AUTHOR]

Measuring the expression levels of membrane proteins (MPs) is crucial for understanding cell differentiation and tissue specificity, defining disease characteristics, identifying biomarkers, and developing therapeutics. While bottom-up proteomics addresses the need for accurately surveying the membrane proteome, the lower abundance and hydrophobic nature of MPs pose challenges in sample preparation. As MPs normally reside in the lipid bilayer, conventional extraction methods rely on detergents, introducing here a paradox - detergents prevent aggregation and facilitate protein processing, but themselves become contaminants that interfere with downstream analytical applications. Various detergent removal methods exist to mitigate this issue, including filter-aided sample preparation, SP3, suspension trapping, and membrane mimetics. This review delves into the fundamentals of each strategy, applications, merits, and limitations, providing insights into their effectiveness in MP research., (© 2024 The Author(s).)

Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present ‘on-gradient’ reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroides reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional ‘on-bead’ approach, we reconstituted Escherichia coli proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.

Membrane proteins, particularly those on the cell surface, play pivotal roles in diverse physiological processes, and their dysfunction is linked to a broad spectrum of diseases. Despite being crucial biomarkers and therapeutic drug targets, their low abundance and hydrophobic nature pose challenges in isolation and quantification, especially when extracted from tissues and organs. To overcome these hurdles, we developed the membrane-mimicking peptidisc, enabling the isolation of the membrane proteome in a water-soluble library conducive to swift identification through liquid chromatography with tandem mass spectrometry. This study applies the method across five mice organs, capturing between 200 and 450 plasma membrane proteins in each case. More than just membrane protein identification, the peptidisc is used to estimate the relative abundance across organs, linking cell-surface protein molecular functions to organ biological roles, thereby contributing to the ongoing discourse on organ specificity. This contribution holds substantial potential for unveiling new avenues in the exploration of biomarkers and downstream applications involving knowledge of the organ cell-surface proteome.

• The organization of biological membranes is affected by the protein and lipid composition. • Mass spectrometry is well-suited to identify lipids in biological membranes. • Mass spectrometry provides insights into lipid binding to membrane proteins and helps unravelling the binding modes. • Membrane mimics for studying membrane proteins in a native-like lipid environment are available. • Membrane mimics are compatible with mass spectrometry. Biological membranes are selectively permeable barriers important for cell organization and compartmentalization. Their organisation strongly depends on the lipids that constitute the lipid bilayer as well as the proteins that reside in the membrane. Unravelling the organisation of biological membranes is therefore of great importance to understand cellular function driven by integral and peripheral membrane proteins. Recent developments in mass spectrometry made it a powerful tool contributing to our present-day understanding of membrane composition and organisation. The two main deliverables of mass spectrometry are (i) the identification and quantification of the membrane components, and (ii) the analysis of their structural arrangements. In this review article, we first briefly discuss the aspects of membrane organization that are accessible through mass spectrometry. We then provide detailed insights into the various mass spectrometric strategies which help identifying lipids from membranes or membrane protein assemblies, unravelling the lipid binding modes in membrane proteins and uncovering their structural roles. We further discuss the growing interest in membrane mimetics providing membrane proteins with a native-like lipid environment for structural and functional studies and the possibilities of mass spectrometry to contribute in these experiments. [ABSTRACT FROM AUTHOR]

Over the past decade, the vast amount of information generated through structural and biophysical studies of GPCRs has provided unprecedented mechanistic insight into the complex signalling behaviour of these receptors. With this recent information surge, it has also become increasingly apparent that in order to reproduce the various effects that lipids and membranes exert on the biological function for these allosteric receptors, in vitro studies of GPCRs need to be conducted under conditions that adequately approximate the native lipid bilayer environment. In the first part of this review, we assess some of the more general effects that a membrane environment exerts on lipid bilayer-embedded proteins such as GPCRs. This is then followed by the consideration of more specific effects, including stoichiometric interactions with specific lipid subtypes. In the final section, we survey a range of different membrane mimetics that are currently used for in vitro studies, with a focus on NMR applications.

The twin‐arginine translocase (Tat) transports folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. In Gram‐negative bacteria and chloroplasts, the translocon consists of three subunits, TatA, TatB, and TatC, of which TatA is responsible for the actual membrane translocation of the substrate. Herein we report on the structure, dynamics, and lipid interactions of a fully functional C‐terminally truncated ‘core TatA’ from Arabidopsis thaliana using solution‐state NMR. Our results show that TatA consists of a short N‐terminal transmembrane helix (TMH), a short connecting linker (hinge) and a long region with propensity to form an amphiphilic helix (APH). The dynamics of TatA were characterized using 15N relaxation NMR in combination with model‐free analysis. The TMH has order parameters characteristic of a well‐structured helix, the hinge is somewhat less rigid, while the APH has lower order parameters indicating structural flexibility. The TMH is short with a surprisingly low protection from solvent, and only the first part of the APH is protected to some extent. In order to uncover possible differences in TatA's structure and dynamics in detergent compared to in a lipid bilayer, fast‐tumbling bicelles and large unilamellar vesicles were used. Results indicate that the helicity of TatA increases in both the TMH and APH in the presence of lipids, and that the N‐terminal part of the TMH is significantly more rigid. The results indicate that plant TatA has a significant structural plasticity and a capability to adapt to local environments. [ABSTRACT FROM AUTHOR]

For detailed functional characterization, membrane proteins are usually studied in detergent. However, it is becoming clear that detergent micelles are often poor mimics of the lipid environment in which these proteins function. In this work we compared the catalytic properties of the membrane-embedded cytochrome c -dependent nitric oxide reductase ( c NOR) from Paracoccus ( P. ) denitrificans in detergent, lipid/protein nanodiscs, and proteoliposomes. We used two different lipid mixtures, an extract of soybean lipids and a defined mix of synthetic lipids mimicking the original P. denitrificans membrane. We show that the catalytic activity of detergent-solubilized c NOR increased threefold upon reconstitution from detergent into proteoliposomes with the P. denitrificans lipid mixture, and above two-fold when soybean lipids were used. In contrast, there was only a small activity increase in nanodiscs. We further show that binding of the gaseous ligands CO and O 2 are affected differently by reconstitution. In proteoliposomes the turnover rates are affected much more than in nanodiscs, but CO-binding is more significantly accelerated in liposomes with soybean lipids, while O 2 -binding is faster with the P. denitrificans lipid mix. We also investigated proton-coupled electron transfer during the reaction between fully reduced c NOR and O 2 , and found that the p K a of the internal proton donor was increased in proteoliposomes but not in nanodiscs. Taking our results together, the liposome-reconstituted enzyme shows significant differences to detergent-solubilized protein. Nanodiscs show much more subtle effects, presumably because of their much lower lipid to protein ratio. Which of these two membrane-mimetic systems best mimics the native membrane is discussed. [ABSTRACT FROM AUTHOR]

Small isotropic bicelles are versatile membrane mimetics, which, in contrast tomicelles, provide a lipid bilayer and are at the same time suitable for solution-state NMR studies. The lipid composition of the bilayer is flexible allowing for incorporation of various head groups and acyl chain types. In bicelles, lipids are solubilized by detergents, which are localized in the rimof the disk-shaped lipid bilayer. Bicelles have been characterized by a broad array of biophysical methods, pulsed-field gradient NMR (PFG NMR) being one of them. PFG NMR can readily be used to measure diffusion coefficients of macromolecules. It is thus employed to characterize bicelle size and morphology. Even more importantly, PFG NMR can be used to study the degree of protein association to membranes. Here, we present the advances that have been made in producing small, fast-tumbling isotropic bicelles from a variety of lipids and detergents, together with insights on the morphology of such mixtures gained from PFG NMR. Furthermore, we review approaches to study protein-membrane interaction by PFG NMR. [ABSTRACT FROM AUTHOR]

The crystal structure of a designed phospholipid-inspired amphiphilic phosphopeptide at 0.8 Å resolution is presented. The phosphorylated β-hairpin peptide crystallizes to form a lamellar structure that is stabilized by intra- and intermolecular hydrogen bonding, including an extended β-sheet structure, as well as aromatic interactions. This first reported crystal structure of a two-tailed peptidic bilayer reveals similarities in thickness to a typical phospholipid bilayer. However, water molecules interact with the phosphopeptide in the hydrophilic region of the lattice. Additionally, solid-state NMR was used to demonstrate correlation between the crystal structure and supramolecular nanostructures. The phosphopeptide was shown to self-assemble into semi-elliptical nanosheets, and solid-state NMR provides insight into the self-assembly mechanisms. This work brings a new dimension to the structural study of biomimetic amphiphilic peptides with determination of molecular organization at the atomic level. [ABSTRACT FROM AUTHOR]

Once removed from their natural environment, membrane proteins depend on membrane-mimetic systems to retain their native structures and functions. To this end, lipid-bilayer nanodiscs that are bounded by scaffold proteins or amphiphilic polymers such as styrene/maleic acid (SMA) copolymers have been introduced as alternatives to detergent micelles and liposomes for in vitro membrane-protein research. Herein, we show that an alternating diisobutylene/maleic acid (DIBMA) copolymer shows equal performance to SMA in solubilizing phospholipids, stabilizes an integral membrane enzyme in functional bilayer nanodiscs, and extracts proteins of various sizes directly from cellular membranes. Unlike aromatic SMA, aliphatic DIBMA has only a mild effect on lipid acyl-chain order, does not interfere with optical spectroscopy in the far-UV range, and does not precipitate in the presence of low millimolar concentrations of divalent cations. [ABSTRACT FROM AUTHOR]

Membrane proteins are one of the most challenging and attractive objects in modern structural biology, as they are targets for the majority of medicines. However, studies of membrane proteins are hindered by several obstacles, including their low ability to crystallize, highly dynamic behavior of some of their domains, and need for membrane-like environment. Although solution nuclear magnetic resonance (NMR) is a very powerful technique of structural biology in terms of the amount of provided data, it imposes several limitations on the object under investigation, with the main constraint being related to the size of the object. For this reason, the membrane mimetic has to form particles of small size and simultaneously to properly simulate the bilayer membrane to be applicable for solution NMR spectroscopy. Here we review the recent advances in the field of membrane mimetics for solution NMR studies, discuss the advantages and drawbacks of specific membrane-like environments, and formulate the criteria for the selection of proper environment for a particular membrane protein or domain. [ABSTRACT FROM AUTHOR]

Protein function strongly depends on temperature, which is related to temperature-dependent changes in the equilibria of protein conformational states. We leveraged variable-temperature 19 F-NMR spectroscopy to interrogate the temperature dependence of the conformational landscape of the human A 2A adenosine receptor (A 2A AR), a class A GPCR. Temperature-induced changes in the conformational equilibria of A 2A AR in lipid nanodiscs were markedly dependent on the efficacy of bound drugs. While antagonist complexes displayed only modest changes as the temperature rose, both full and partial agonist complexes exhibited substantial increases in the active state population. Importantly, the temperature-dependent response of complexes with both full and partial agonists exhibited a pronounced sensitivity to the specific membrane mimetic employed. In striking contrast to observations within lipid nanodiscs, in detergent micelles the active state population exhibited different behavior for A 2A AR complexes with both full and partial agonists. This underscores the importance of the protein environment in understanding the thermodynamics of GPCR activation., Competing Interests: Competing/conflicting interests Authors have no conflicts of interests.

Biological membranes are essential in providing the stability of membrane proteins in a functional state. Functionally stable homogeneous sample is required for biophysical electron paramagnetic resonance (EPR) studies of membrane proteins for obtaining pertinent structural dynamics of the protein. Significant progresses have been made for the optimization of the suitable membrane environments required for biophysical EPR measurements. However, no universal membrane mimetic system is available that can solubilize all membrane proteins suitable for biophysical EPR studies while maintaining the functional integrity. Great efforts are needed to optimize the sample condition to obtain better EPR data quality of membrane proteins that can provide meaningful information on structural dynamics. In this mini-review, we will discuss important aspects of membrane mimetics for biophysical EPR measurements and current progress with some of the recent examples. [Display omitted] • Biological membranes are required for the stability and functionality of membrane proteins. • Site-directed spin labeling EPR is used to probe structural dynamics of membrane proteins. • Recent progresses in membrane mimetics for EPR studies of membrane proteins are discussed. • Illustrative EPR studies of membrane proteins in different membrane mimetics are discussed. [ABSTRACT FROM AUTHOR]

Membrane proteins are a highly relevant class of biological molecules and comprise ∼60% of current drug targets. Before being analyzed by structural, biochemical, and biophysical methods, membrane proteins must first be extracted from cellular membranes - often using detergents. Detergent-extracted membrane proteins are amenable to analysis by structural, biochemical, and biophysical techniques. In certain cases, however, detergents can disturb native protein conformations and/or biological activity. This has led to the development of membrane mimetics, which stabilize membrane proteins in a native membrane-like environment that is water-soluble and detergent-free. This review provides an overview of recent developments in the membrane mimetic field, with a focus on nanodiscs, Saposin lipid nanoparticles (SapNPs), peptidiscs, and SMA lipid particles (SMALPs) - and highlights their utility for supporting biophysical, biochemical, and structural characterization of membrane proteins and complexes., (© 2023 The Author(s).)

The role of biological membranes as a target in biological radiation damage remains unclear. In the present article, we show that the structural, biochemical and biophysical changes of a simple biological membrane, i.e. human erythrocyte membranes, which is altered after exposure, could be studied by membrane mimetics and conventional NMR spectrometers. [ABSTRACT FROM AUTHOR]

The mechanism of action of antimicrobial peptides depends on physicochemical properties such as structure, concentration, and oligomerization. Here, we focused on the effect of dimerization on the mechanism of action of aurein 1.2 (AU). We designed a lysine-linked AU dimer, (AU) 2 K, and its interaction with membrane mimetics was studied using four biophysical techniques and molecular dynamics simulations. Circular dichroism and molecular dynamics studies showed that AU displayed a typical spectrum for disordered structures in aqueous solution whereas (AU) 2 K exhibited the typical spectrum of α-helices in a coiled-coil conformation, wherein helices are wrapped around each other. With the addition of large unilamellar vesicles (LUVs), AU adopted an α-helix structure whereas the coiled-coil structure of (AU) 2 K assumed an extended conformation. Carboxyfluorescein release experiments with LUVs showed that both peptides were able to permeabilize vesicles although the leakage response to increases in peptide concentration differed. Optical microscopy experiments showed that both peptides induced pore opening and the dimer eventually caused the vesicles to burst. Finally, calorimetric traces determined by isothermal titration calorimetry on the LUVs also showed significant differences in peptide–membrane interactions. Together, the results of our study demonstrated that dimerization changes the mechanism of action of AU. [ABSTRACT FROM AUTHOR]

Solute carrier (SLC) 26 1 1 SLC26: Solute-linked carrier 26. or sulfate permease (SulP) 2 2 SulP: Sulfate permease family. anion transporters, belong to a phylogenetically ancient family of secondary active transporters. Members of the family are involved in several human genetic diseases and cell physiological processes. Despite their importance, the substrates for transport by this family of proteins have been poorly characterized. In this study, recombinant Stm YchM/DauA, 3 3 Stm YchM/DauA: YchM/DauA from Salmonella typhimurium. a SulP from Salmonella typhimurium was purified to homogeneity and functionally characterized. StmYchM /DauA was found to be a dimer in solution as determined by size exclusion chromatography coupled to multiple angle light scattering. We report a functional characterization of the SulP proteins in two membrane mimetic systems and reveal a dual nature of anionic substrates for SulP. Stm YchM/DauA functionally incorporated into nanodiscs could bind fumarate with millimolar affinities ( K D = 4.6 ± 0.29 mM) as detected by intrinsic tryptophan fluorescence quench studies. In contrast, electrophysiological experiments performed in reconstituted liposomes indicate a strong bicarbonate transport in the presence of chloride but no detectable electrogenic fumarate transport. We hence suggest that while SulP acts as an electrogenic bicarbonate transporter, fumarate may serve as substrate under different conditions indicating multiple functions of SulP. [ABSTRACT FROM AUTHOR]

Supported lipid bilayers are used as a convenient model cell membrane system to study biologically important molecule–lipid interactions in situ. However, the lipid bilayer models are often simple and the acquired results with these models may not provide all pertinent information related to a real cell membrane. In this work, we use sum frequency generation (SFG) vibrational spectroscopy to study molecular-level interactions between the antimicrobial peptides (AMPs) MSI-594, ovispirin-1 G18, magainin 2 and a simple 1,2-dipalmitoyl-d62- sn -glycero-3-phosphoglycerol (dDPPG)/1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoglycerol (POPG) bilayer. We compared such interactions to those between the AMPs and a more complex dDPPG/ Escherichia coli ( E. coli ) polar lipid extract bilayer. We show that to fully understand more complex aspects of peptide–bilayer interaction, such as interaction kinetics, a heterogeneous lipid composition is required, such as the E. coli polar lipid extract. The discrepancy in peptide–bilayer interaction is likely due in part to the difference in bilayer charge between the two systems since highly negative charged lipids can promote more favorable electrostatic interactions between the peptide and lipid bilayer. Results presented in this paper indicate that more complex model bilayers are needed to accurately analyze peptide–cell membrane interactions and demonstrates the importance of using an appropriate lipid composition to study AMP interaction properties. [ABSTRACT FROM AUTHOR]

Plasticins (PTC) are dermaseptin-related antimicrobial peptides characterized by a large number of leucine and glycine residues arranged in GXXXG motifs that are often described to promote helix association within biological membranes. We report the structure and interaction properties of two plasticins, PTC-B1 from Phyllomedusa bicolor and a cationic analog of PTC-DA1 from Pachymedusa dacnicolor , which exhibit membrane-lytic activities on a broad range of microorganisms. Despite a high number of glycine, CD and NMR spectroscopy show that the two plasticins adopt mainly alpha-helical conformations in a wide variety of environments such as trifluoroethanol, detergent micelles and lipid vesicles. In DPC and SDS, plasticins adopt well-defined helices that lie parallel to the micelle surface, all glycine residues being located on the solvent-exposed face. Spectroscopic data and cross-linking experiments indicate that the GXXXG repeats in these amphipathic helices do not provide a strong oligomerization interface, suggesting a different role from GXXXG motifs found in transmembrane helices. [ABSTRACT FROM AUTHOR]

The M2 protein, a proton channel, from Influenza A has been structurally characterized by X-ray diffraction and by solution and solid-state NMR spectroscopy in a variety of membrane mimetic environments. These structures show substantial backbone differences even though they all present a left-handed tetrameric helical bundle for the transmembrane domain. Variations in the helix tilt influence drug binding and the chemistry of the histidine tetrad responsible for acid activation, proton selectivity and transport. Some of the major structural differences do not arise from the lack of precision, but instead can be traced to the influences of the membrane mimetic environments. The structure in lipid bilayers displays unique chemistry for the histidine tetrad, which binds two protons cooperatively to form a pair of imidazole-imidazolium dimers. The resulting interhistidine hydrogen bonds contribute to a three orders of magnitude enhancement in tetramer stability. Integration with computation has provided detailed understanding of the functional mechanism for proton selectivity, conductance and gating of this important drug target. [ABSTRACT FROM AUTHOR]

Abstract: Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid–protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K+ channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of 15N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems. [Copyright &y& Elsevier]

Abstract: The native environment of membrane proteins is complex and scientists have felt the need to simplify it to reduce the number of varying parameters. However, experimental problems can also arise from oversimplification which contributes to why membrane proteins are under-represented in the protein structure databank and why they were difficult to study by nuclear magnetic resonance (NMR) spectroscopy. Technological progress now allows dealing with more complex models and, in the context of NMR studies, an incredibly large number of membrane mimetics options are available. This review provides a guide to the selection of the appropriate model membrane system for membrane protein study by NMR, depending on the protein and on the type of information that is looked for. Beside bilayers (of various shapes, sizes and lamellarity), bicelles (aligned or isotropic) and detergent micelles, this review will also describe the most recent membrane mimetics such as amphipols, nanodiscs and reverse micelles. Solution and solid-state NMR will be covered as well as more exotic techniques such as DNP and MAOSS. [Copyright &y& Elsevier]

Abstract: We have identified a family of peptoids that inhibits in vitro the activity of the apoptosome, a macromolecular complex that activates mitochondrial-dependent apoptosis pathways. The analysis of peptide-based cell compatible delivery systems of the most active peptoid is presented. The active peptoid was then fused to cell penetrating peptides (CPP) as penetratin (PEN-peptoid) and HIV-1 TAT (TAT-peptoid). PEN-peptoid showed greater cell viability and as a consequence better efficiency as an apoptosis inhibitor than the TAT-peptoid. The intracellular trafficking of both inhibitors was studied by flow cytometry and confocal fluorescence microscopy. Finally, the influence of the cargo (peptoid) molecules on the conformational behavior of the CPP in buffers and in membrane mimetic environments was analyzed using circular dichroism (CD) spectroscopy. [Copyright &y& Elsevier]

LFB (FKCRRWQWRMKKLGA-HN2) is a 15-residue linear antimicrobial peptide derived from bovine lactoferricin, which has antimicrobial activity similar to that of the intact 25-residue disulfide-cyclized peptide. Previous alanine-scan studies, in which all of the residues in LFB were individually replaced with Ala, showed that the 2 tryptophan (Trp) residues of LFB were crucial to its antimicrobial activity. When either Trp6 or Trp8 was replaced with Ala (LFBA6 and LFBA8, respectively), these 2 peptides were almost devoid of antimicrobial activity. We determined the structures of LFB, LFBA6, and LFBA8 bound to membrane-mimetic SDS micelles using NMR spectroscopy, and studied their interactions with different phospholipid-model membranes. The membrane interactions of LFB exhibited little correlation with its antimicrobial activity, suggesting that the mechanism of action of LFB involves intracellular targets. However, the much higher antimicrobial activity of LFB compared with LFBA6 and LFBA8 might result, in part, from the formation of energetically favorable cation–π interactions observed only in LFB. Information about the importance of Arg and Trp cation–π interactions will provide insight for the future design of potent antimicrobial peptidomimetics. [ABSTRACT FROM AUTHOR]

Abstract: Suppressor of a groEL mutation protein E (SugE) is a small multidrug resistance (SMR) homologue. In comparison with other SMR proteins, SugE promotes bacterial resistance to a narrow range of quaternary ammonium compounds (QACs). Isothermal titration calorimetry was used to study the binding of QACs to Escherichia coli SugE in different membrane mimetic environments. In this study, the binding stoichiometry of SugE to drug was found to be 1:1, and the binding of SugE to drug was observed with the dissociation constant (K D) in the micromolar range for each of the drugs in the membrane mimetic environments explored. This interaction appears to be enthalpy-driven with enthalpies of 8–12kcal/mol for each of the drugs. These results are similar to those found with drug binding to the SMR protein EmrE in an earlier study. [Copyright &y& Elsevier]

Abstract: The hexapeptide Ac-RRWWRF-NH[sub 2] has earlier been identified as a potent antimicrobial peptide by screening synthetic combinatorial hexapeptide libraries. In this study, it was found that this peptide had a large influence on the thermotropic phase behavior of model membranes containing the negatively charged headgroup phosphatidylglycerol, a major component of bacterial membranes. In contrast, differential scanning calorimetry showed that it had little effect on model membranes containing the zwitterionic phosphatidylcholine headgroup, the main component of erythrocyte membranes. This behavior is consistent with its biological activity and with its affinity to these membranes as determined by titration calorimetry, implying that peptide–lipid interactions play an important role in this process. The structure of this peptide bound to membrane-mimetic sodium dodecyl sulfate (SDS) and dodecylphosphocholine micelles has been determined using conventional two-dimensional nuclear magnetic resonance methods. It forms a marked amphipathic structure in SDS with its hydrophobic residues on one side of the structure and with the positively charged residues on the other side. This amphipathic structure may allow this peptide to penetrate deeper into the interfacial region of negatively charged membranes, leading to local membrane destabilization. Knowledge about the importance of electrostatic interactions of Arg and the role of Trp residues as a membrane interface anchor will provide insight into the future design of potent antimicrobial peptidomimetics. [ABSTRACT FROM AUTHOR]

EmrE is a member of the small multidrug resistance family of proteins and functions as a efflux transporter of lipophilic cations. This integral membrane protein is composed of 110 amino acids and is very hydrophobic with small loops exposed to the aqueous environment. This protein has been purified in a variety of ways including extraction with chloroform:methanol mixtures. This study explored culture growth and induction conditions, the parameters of organic solvent extraction, running conditions of a lipophilic column for lipid removal, as well as solubilization conditions. Optimal expression and purification protocols are crucial to the characterization goals of this protein. The experiments presented here led to an improvement in the yield of pure EmrE obtained by organic solvent extraction methods at a level of 0.9±0.2 mg/L of culture. This is on the order of a 60% improvement over previous reports. [Copyright &y& Elsevier]

Thereby they help to further our understanding of MP structure and activity and so aid rational drug design. Amphipathic copolymers, membrane mimetics, membrane proteins, nanodiscs, styrene maleic acid lipid particles Keywords: amphipathic copolymers; membrane mimetics; membrane proteins; nanodiscs; styrene maleic acid lipid particles EN amphipathic copolymers membrane mimetics membrane proteins nanodiscs styrene maleic acid lipid particles 1 1 1 09/13/21 20210909 NES 210909 Synthetic polymer nanodiscs, structures that arise from amphipathic copolymers that capture membrane proteins (MPs) and surrounding phospholipids, offer a convenient detergent-free solution to the challenges associated with MP purification and analysis. [Extracted from the article]

Cover Picture: Liposomes as Carriers of Membrane-Associated Proteins and Peptides for Mass Spectrometric Analysis (Angew. Keywords: liposomes; mass spectrometry; membrane mimetics; peripheral membrane proteins; protein-lipid interactions EN liposomes mass spectrometry membrane mimetics peripheral membrane proteins protein-lipid interactions 10997 10997 1 05/06/21 20210510 NES 210510 B Liposomes as carriers b of proteins and peptides enable investigation of lipid clusters, protein oligomers and protein-lipid interactions in mass spectrometric analysis. Liposomes, mass spectrometry, membrane mimetics, peripheral membrane proteins, protein-lipid interactions. [Extracted from the article]