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Supported lipid bilayers (SLBs) are commonly used as model systems mimicking biological membranes. Recently, we reported a new method to produce SLBs with incorporated membrane proteins, which is based on the application of peptide discs [Luchini et al., Analytical Chemistry, 2020, 92, 1081-1088]. Peptide discs are small discoidal particles composed of a lipid core and an outer belt of self-assembled 18A peptides. SLBs including membrane proteins can be formed by depositing the peptide discs on a solid support and subsequently removing the peptide by buffer rinsing. Here, we introduce a new variant of the 18A peptide, named dark peptide (d18A). d18A exhibits UV absorption at 214 nm, whereas the absorption at 280 nm is negligible. This improves sample preparation as it enables a direct quantification of the membrane protein concentration in the peptide discs by measuring UV absorption at 280 nm. We describe the application of the peptide discs prepared with d18A (dark peptide discs) to produce SLBs with a membrane protein, synaptobrevin 2 (VAMP2). The collected data showed the successful formation of SLBs with high surface coverage and incorporation of VAMP2 in a single orientation with the extramembrane domain exposed towards the bulk solvent. Compared to 18A, we found that d18A was more efficiently removed from the SLB. Our data confirmed the structural organisation of VAMP2 as including both alpha-helical and beta-sheet secondary structure. We further verified the orientation of VAMP2 in the SLBs by characterising the binding of VAMP2 with alpha-synuclein. These results point at the produced SLBs as relevant membrane models for biophysical studies as well as nanostructured biomaterials.

The antiapoptotic protein Bcl-xL is a major regulator of cell death and survival, but many aspects of its functions remain elusive. It is mostly localized in the mitochondrial outer membrane (MOM) owing to its C-terminal hydrophobic α-helix. In order to gain further information about its membrane organization, we set up a model system combining cell-free protein synthesis and nanodisc insertion. We found that, contrary to its proapoptotic partner Bax, neosynthesized Bcl-xL was spontaneously inserted into nanodiscs. The deletion of the C-terminal α-helix of Bcl-xL prevented nanodisc insertion. We also found that nanodisc insertion protected Bcl-xL against the proteolysis of the 13 C-terminal residues that occurs during expression of Bcl-xL as a soluble protein in E. coli. Interestingly, we observed that Bcl-xL increased the insertion of Bax into nanodiscs, in a similar way to that which occurs in mitochondria. Cell-free synthesis in the presence of nanodiscs is, thus, a suitable model system to study the molecular aspects of the interaction between Bcl-xL and Bax during their membrane insertion.

Analysis of proteins and complexes under native mass spectrometric (MS) and solution conditions was typically performed using the time-of-flight analyzers, due to their routine high m/z transmission and detection capabilities. However, over recent years, the ability of Orbitrap-based mass spectrometers to transmit and detect a range of high molecular weight species is well documented. Herein, we describe how a 15 Tesla Fourier transform ion cyclotron resonance (15 Tesla FT-ICR) instrument is more than capable of analyzing a wide range of ions in the high m/z scale (>5000), in both positive and negative instrument polarities, ranging from the inorganic ceasium iodide salt clusters; a humanized IgG1k monoclonal antibody (mAb; 148.2 kDa); a IgG1-mertansine mAb drug conjugate (148.5 kDa, drug-to-antibody ratio, DAR 2.26); a siRNA-IgG1 mAb conjugate (159.1 kDa; ribonucleic acid to antibody ratio, RAR 1); the membrane protein aquaporin-Z (97.2 kDa) liberated from a C8E4 detergent micelle; the empty MSP1D1-nanodisc (142.5 kDa) and the tetradecameric chaperone protein complex GroEL (806.2 kDa; GroEL dimer at 1.6 MDa). We also investigate different regions of the FT-ICR which impact ion transmission and desolvation. Finally, we demonstrate how the transmission of these species and resultant spectra are highly consistent with those previously generated on both quadrupole time-of-flight (Q-ToF) and Orbitrap instrumentation. This report serves as an impactful example of how FT-ICR mass analyzers are competitive to Q-ToFs and Orbitraps for high mass detection at high m/z.

Proteins embedded in biological membranes perform essential functions in all organisms, serving as receptors, transporters, channels, cell adhesion molecules, and other supporting cellular roles. These membrane proteins comprise ~30% of all human proteins and are the targets of ~60% of FDA-approved drugs, yet their extensive characterization using established biochemical and biophysical methods has continued to be elusive due to challenges associated with the purification of these insoluble proteins. In response, the development of nanodisc techniques, such as nanolipoprotein particles (NLPs) and styrene maleic acid polymers (SMALPs), allowed membrane proteins to be expressed and isolated in solution as part of lipid bilayer rafts with defined, consistent nanometer sizes and compositions, thus enabling solution-based measurements. Fluorescence correlation spectroscopy (FCS) is a relatively simple yet powerful optical microscopy-based technique that yields quantitative biophysical information, such as diffusion kinetics and concentrations, about individual or interacting species in solution. Here, we first summarize current nanodisc techniques and FCS fundamentals. We then provide a focused review of studies that employed FCS in combination with nanodisc technology to investigate a handful of membrane proteins, including bacteriorhodopsin, bacterial division protein ZipA, bacterial membrane insertases SecYEG and YidC, Yersinia pestis type III secretion protein YopB, yeast cell wall stress sensor Wsc1, epidermal growth factor receptor (EGFR), ABC transporters, and several G protein-coupled receptors (GPCRs).

Aquaporins are tetrameric integral membrane proteins that act as water channels, and can also permeabilize membranes to other solutes. The monomer appears to be the functional form despite all aquaporins being organized as tetramers, which therefore must provide a clear functional advantage. In addition to this quaternary organization, some aquaporins can act as adhesion molecules in membrane junctions, when tetramers located in opposing membranes interact via their extracellular domains. These stacked forms have been observed in a range of aquaporins, whether using lipidic membrane environments, in electron crystallography, or using detergent micelles, in single-particle cryo-electron microscopy (cryo-EM). In the latter technique, structural studies can be performed when the aquaporin is reconstituted into nanodiscs of lipids that are surrounded by a protein scaffold. During attempts to study E. coli Aquaporin Z (AqpZ), we have found that in some conditions these nanodiscs tend to form filaments that appear to be either thicker head-to-tail or thinner side-to-side stacks of nanodiscs. Nanodisc oligomerization was observed using orthogonal analytical techniques analytical ultra-centrifugation and mass photometry, although the nature of the oligomers (head-to-tail or side-to-side) could not be determined. Using the latter technique, the AqpZ tetramer itself formed oligomers of increasing size when solubilized only in detergent, which is consistent with multiple stacking of AqpZ tetramers. We observed images consistent with both of these filaments in negative staining EM conditions, but only thicker filaments in cryo-EM conditions. We hypothesize that the apparent nanodisc side-to-side arrangement that can only be visualized in negative staining conditions is related to artifacts due to the sample preparation. Filaments of any kind were not observed in EM when nanodiscs did not contain AqpZ, or after addition of detergent into the nanodisc cryo-EM preparation, at concentrations that did not disrupt nanodisc formation. To our knowledge, these filaments have not been observed in nanodiscs preparations of other membrane proteins. AqpZ, like other aquaporins has a charge asymmetry between the cytoplasmic (more positive) and the extracellular sides, which may explain the likely head-to-tail stacking observed, both in nanodisc preparations and also in detergent micelles. Ministry of Education (MOE) Published version This research and the APC were funded by grants awarded to J.T. by the Singapore Ministry of Education (MOE) under tier 1 grant number RG101/20.

Saposins are lipid transfer proteins required for the degradation of sphingolipids in the lysosome. These small proteins bind lipids by transitioning from a closed, monomeric state to an open conformation exposing a hydrophobic surface that binds and shields hydrophobic lipid tails from the aqueous environment. Saposins form a range of multimeric assemblies to encompass these bound lipids and present them to hydrolases in the lysosome. This lipid-binding property of human saposin A has been exploited to form lipoprotein nanodiscs suitable for structural studies of membrane proteins. Here we present the crystal structure of a unique tetrameric assembly of murine saposin A produced serendipitously, following modifications of published protocols for making lipoprotein nanodiscs. The structure of this new saposin oligomer highlights the diversity of tertiary arrangement that can be adopted by these important lipid transfer proteins.

Magnetomechanical therapy is one of the most perspective directions in tumor microsurgery. According to the analysis of recent publications, it can be concluded that a nanoscalpel could become an instrument sufficient for cancer microsurgery. It should possess the following properties: (1) nano- or microsized; (2) affinity and specificity to the targets on tumor cells; (3) remote control. This nano- or microscalpel should include at least two components: (1) a physical nanostructure (particle, disc, plates) with the ability to transform the magnetic moment to mechanical torque; (2) a ligand—a molecule (antibody, aptamer, etc.) allowing the scalpel precisely target tumor cells. Literature analysis revealed that the most suitable nanoscalpel structures are anisotropic, magnetic micro- or nanodiscs with high-saturation magnetization and the absence of remanence, facilitating scalpel remote control via the magnetic field. Additionally, anisotropy enhances the transmigration of the discs to the tumor. To date, four types of magnetic microdiscs have been used for tumor destruction: synthetic antiferromagnetic P-SAF (perpendicular) and SAF (in-plane), vortex Py, and three-layer non-magnetic–ferromagnet–non-magnetic systems with flat quasi-dipole magnetic structures. In the current review, we discuss the biological effects of magnetic discs, the mechanisms of action, and the toxicity in alternating or rotating magnetic fields in vitro and in vivo. Based on the experimental data presented in the literature, we conclude that the targeted and remotely controlled magnetic field nanoscalpel is an effective and safe instrument for cancer therapy or theranostics.

MPV17 is an integral inner mitochondrial membrane protein, whose loss-of-function is linked to the hepatocerebral form of the mitochondrial-DNA-depletion syndrome, leading to a tissue-specific reduction of mitochondrial DNA and organ failure in infants. Several disease-causing mutations in MPV17 have been identified and earlier studies with reconstituted protein suggest that MPV17 forms a high conductivity channel in the membrane. However, the molecular and structural basis of the MPV17 functionality remain only poorly understood. In order to make MPV17 accessible to high-resolution structural studies, we here present an efficient protocol for its high-level production in E. coli and refolding into detergent micelles. Using biophysical and NMR methods, we show that refolded MPV17 in detergent micelles adopts a compact structure consisting of six membrane-embedded α-helices. Furthermore, we demonstrate that MPV17 forms oligomers in a lipid bilayer that are further stabilized by disulfide-bridges. In line with these findings, MPV17 could only be inserted into lipid nanodiscs of 8–12 nm in diameter if intrinsic cysteines were either removed by mutagenesis or blocked by chemical modification. Using this nanodisc reconstitution approach, we could show that disease-linked mutations in MPV17 abolish its oligomerization properties in the membrane. These data suggest that, induced by oxidative stress, MPV17 can alter its oligomeric state from a properly folded monomer to a disulfide-stabilized oligomeric pore which might be required for the transport of metabolic DNA precursors into the mitochondrial matrix to compensate for the damage caused by reactive oxygen species.

Over the past decade, the structural biology of membrane proteins (MPs) has taken a new turn thanks to epoch-making technical progress in single-particle electron cryo-microscopy (cryo-EM) as well as to improvements in sample preparation. The present analysis provides an overview of the extent and modes of usage of the various types of surfactants for cryo-EM studies. Digitonin, dodecylmaltoside, protein-based nanodiscs, lauryl maltoside-neopentyl glycol, glyco-diosgenin, and amphipols (APols) are the most popular surfactants at the vitrification step. Surfactant exchange is frequently used between MP purification and grid preparation, requiring extensive optimization each time the study of a new MP is undertaken. The variety of both the surfactants and experimental approaches used over the past few years bears witness to the need to continue developing innovative surfactants and optimizing conditions for sample preparation. The possibilities offered by novel APols for EM applications are discussed.

Bacteria respond to physical forces perceived as mechanical stress as part of their comprehensive environmental sensing strategy. Histidine kinases can then funnel diverse environmental stimuli into changes in gene expression through a series of phosphorelay reactions. Because histidine kinases are most often embedded in the inner membrane, they can be sensitive to changes in membrane tension that occurs, for example, in response to osmotic stress, or when deformation of the cell body occurs upon encountering a surface before forming biofilms, or inside the host in response to shear stress in the kidney, intestine, lungs, or blood stream. A summary of our recent work that links the histidine kinase EnvZ to mechanical changes in the inner membrane is provided and placed in a context of other bacterial systems that respond to mechanical stress.

1,4‐Dihydropyridines (DHP), the most commonly used antihypertensives, function by inhibiting the L‐type voltage‐gated Ca2+ (Cav) channels. DHP compounds exhibit chirality‐specific antagonistic or agonistic effects. The structure of rabbit Cav1.1 bound to an achiral drug nifedipine reveals the general binding mode for DHP drugs, but the molecular basis for chiral specificity remained elusive. Herein, we report five cryo‐EM structures of nanodisc‐embedded Cav1.1 in the presence of the bestselling drug amlodipine, a DHP antagonist (R)‐(+)‐Bay K8644, and a titration of its agonistic enantiomer (S)‐(−)‐Bay K8644 at resolutions of 2.9–3.4 Å. The amlodipine‐bound structure reveals the molecular basis for the high efficacy of the drug. All structures with the addition of the Bay K8644 enantiomers exhibit similar inactivated conformations, suggesting that (S)‐(−)‐Bay K8644, when acting as an agonist, is insufficient to lock the activated state of the channel for a prolonged duration., High‐resolution cryo‐EM structures of nanodisc‐embedded Cav1.1 in complex with antagonists Levamlodipine, (R)‐(+)‐Bay K8644, and dual agonist/antagonist (S)‐(−)‐Bay K8644 reveal the molecular basis of the allosteric modulation of the voltage‐gated Ca2+ channel Cav1.1 by dihydropyridine (DHP) drugs. Advanced structural understanding of the stereo‐selective mode of action of DHP will facilitate drug discovery targeting Cav channels.

Cardiovascular disease, the leading cause of mortality worldwide, is primarily caused by atherosclerosis, which is characterized by lipid and inflammatory cell accumulation in blood vessels and carotid intima thickening. Although disease management has improved significantly, new therapeutic strategies focused on accelerating atherosclerosis regression must be developed. Atherosclerosis models mimicking in vivo-like conditions provide essential information for research and new advances toward clinical application. New nanotechnology-based therapeutic opportunities have emerged with apoA-I nanoparticles (recombinant/reconstituted high-density lipoproteins, rHDL) as ideal carriers to deliver molecules and the discovery that microRNAs participate in atherosclerosis establishment and progression. Here, a therapeutic strategy to improve cholesterol efflux is developed based on a two-step administration of rHDL consisting of a first dose of antagomiR-33a-loaded rHDLs to induce adenosine triphosphate-binding cassette transporters A1 overexpression, followed by a second dose of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine rHDLs, which efficiently remove cholesterol from foam cells. A triple-cell 2D-atheroma plaque model reflecting the cellular complexity of atherosclerosis is used to improve efficiency of the nanoparticles in promoting cholesterol efflux. The results show that sequential administration of rHDL potentiates cholesterol efflux indicating that this approach may be used in vivo to more efficiently target atherosclerotic lesions and improve prognosis of the disease., This work was supported by Ministerio De Ciencia e Innovación; Proyectos De Generación De Conocimiento 2021 (Ref: PID2021-127056OB-I00). U.G.-G. was supported by Fundación Biofísica Bizkaia. A.B.-V. was supported by Programa de especialización de Personal Investigador Doctor en la UPV/EHU (2019) 2019–2020. S.J.-B. and A.L.-S. were supported by a grant Programa Investigador en Formación (2017–2018) and (2019–2020), Gobierno Vasco, respectively. A.L.-S. was partially supported by Fundación Biofísica Bizkaia.

Integral membrane proteins (IMPs) fulfill important physiological functions by providing cell–environment, cell–cell and virus–host communication; nutrients intake; export of toxic compounds out of cells; and more. However, some IMPs have obliterated functions due to polypeptide mutations, modifications in membrane properties and/or other environmental factors—resulting in damaged binding to ligands and the adoption of non-physiological conformations that prevent the protein from returning to its physiological state. Thus, elucidating IMPs’ mechanisms of function and malfunction at the molecular level is important for enhancing our understanding of cell and organism physiology. This understanding also helps pharmaceutical developments for restoring or inhibiting protein activity. To this end, in vitro studies provide invaluable information about IMPs’ structure and the relation between structural dynamics and function. Typically, these studies are conducted on transferred from native membranes to membrane-mimicking nano-platforms (membrane mimetics) purified IMPs. Here, we review the most widely used membrane mimetics in structural and functional studies of IMPs. These membrane mimetics are detergents, liposomes, bicelles, nanodiscs/Lipodisqs, amphipols, and lipidic cubic phases. We also discuss the protocols for IMPs reconstitution in membrane mimetics as well as the applicability of these membrane mimetic-IMP complexes in studies via a variety of biochemical, biophysical, and structural biology techniques.

There is an urgent need to find new antibacterial agents to combat bacterial infections, including agents that inhibit novel, hitherto unexploited targets in bacterial cells. Amongst novel targets are two-component signal transduction systems (TCSs) which are the main mechanism by which bacteria sense and respond to environmental changes. TCSs typically comprise a membrane-embedded sensory protein (the sensor histidine kinase, SHK) and a partner response regulator protein. Amongst promising targets within SHKs are those involved in environmental signal detection (useful for targeting specific SHKs) and the common themes of signal transmission across the membrane and propagation to catalytic domains (for targeting multiple SHKs). However, the nature of environmental signals for the vast majority of SHKs is still lacking, and there is a paucity of structural information based on full-length membrane-bound SHKs with and without ligand. Reasons for this lack of knowledge lie in the technical challenges associated with investigations of these relatively hydrophobic membrane proteins and the inherent flexibility of these multidomain proteins that reduces the chances of successful crystallisation for structural determination by X-ray crystallography. However, in recent years there has been an explosion of information published on (a) methodology for producing active forms of full-length detergent-, liposome- and nanodisc-solubilised membrane SHKs and their use in structural studies and identification of signalling ligands and inhibitors; and (b) mechanisms of signal sensing and transduction across the membrane obtained using sensory and transmembrane domains in isolation, which reveal some commonalities as well as unique features. Here we review the most recent advances in these areas and highlight those of potential use in future strategies for antibiotic discovery. This Review is part of a Special Issue entitled “Interactions of Bacterial Molecules with Their Ligands and Other Chemical Agents” edited by Mary K. Phillips-Jones.

As a membrane-mimetic system, detergent micelles are popularly used to extract membrane proteins from lipid environments and to maintain their solubility and stability in an aqueous medium. However, many membrane proteins encapsulated in conventional detergents tend to undergo structural degradation during extraction and purification, thus necessitating the development of new agents with enhanced properties. In the current study, two classes of new amphiphiles are introduced, resorcinarene-based glucoside and maltoside amphiphiles (designated RGAs and RMAs, respectively), for which the alkyl chains are facially segregated from the carbohydrate head groups. Of these facial amphiphiles, two RGAs (RGA-C11 and RGA-C13) conferred markedly enhanced stability to four tested membrane proteins compared to a gold-standard conventional detergent. The relatively high water solubility and micellar stability of the RGAs compared to the RMAs, along with their generally favourable behaviours for membrane protein stabilisation described here, are likely to be, at least in part, a result of the high conformational flexibility of these glucosides. This study suggests that flexibility could be an important factor in determining the suitability of new detergents for membrane protein studies.

Cellular membranes and associated proteins play critical physiological roles in organisms from all life kingdoms. In many cases, malfunction of biological membranes triggered by changes in the lipid bilayer properties or membrane protein functional abnormalities lead to severe diseases. To understand in detail the processes that govern the life of cells and to control diseases, one of the major tasks in biological sciences is to learn how the membrane proteins function. To do so, a variety of biochemical and biophysical approaches have been used in molecular studies of membrane protein structure and function on the nanoscale. This review focuses on electron paramagnetic resonance with site-directed nitroxide spin-labeling (SDSL EPR), which is a rapidly expanding and powerful technique reporting on the local protein/spin-label dynamics and on large functionally important structural rearrangements. On the other hand, adequate to nanoscale study membrane mimetics have been developed and used in conjunction with SDSL EPR. Primarily, these mimetics include various liposomes, bicelles, and nanodiscs. This review provides a basic description of the EPR methods, continuous-wave and pulse, applied to spin-labeled proteins, and highlights several representative applications of EPR to liposome-, bicelle-, or nanodisc-reconstituted membrane proteins.

Single-particle cryo-electron microscopy has become an indispensable technique in structural biology. In particular when studying membrane proteins, it allows the use of membrane-mimicking tools, which can be crucial for a comprehensive understanding of the structure-function relationship of the protein in its native environment. In this chapter we focus on the application of nanodiscs and use our recent studies on the TMEM16 family as an example.

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug targets. Yet, a major barrier to their characterization and exploitation in academic or industrial settings is that most biochemical, biophysical, and drug screening strategies require these proteins to be in a water-soluble state. Our laboratory recently developed the peptidisc, a membrane mimetic offering a “one-size-fits-all” approach to the problem of membrane protein solubility. We present here a streamlined protocol that combines protein purification and peptidisc reconstitution in a single chromatographic step. This workflow, termed PeptiQuick, allows for bypassing dialysis and incubation with polystyrene beads, thereby greatly reducing exposure to detergent, protein denaturation, and sample loss. When PeptiQuick is performed with biotinylated scaffolds, the preparation can be directly attached to streptavidin-coated surfaces. There is no need to biotinylate or modify the membrane protein target. PeptiQuick is showcased here with the membrane receptor FhuA and antimicrobial ligand colicin M, using biolayer interferometry to determine the precise kinetics of their interaction. It is concluded that PeptiQuick is a convenient way to prepare and analyze membrane protein-ligand interactions within one day in a detergent-free environment.

Membrane proteins are important players in signal transduction and the exchange of metabolites within or between cells. Thus, this protein class is the target of around 60 % of currently marketed drugs, emphasizing their essential biological role. Besides functional assays, structural and dynamical investigations on this protein class are crucial to fully understanding their functionality. Even though X-ray crystallography and electron microscopy are the main methods to determine structures of membrane proteins and their complexes, NMR spectroscopy can contribute essential information on systems that (a) do not crystallize and (b) are too small for EM. Furthermore, NMR is a versatile tool for monitoring functional dynamics of biomolecules at various time scales. A crucial aspect of such studies is the use of a membrane mimetic that resembles a native environment and thus enables the extraction of functional insights. In recent decades, the membrane protein NMR community has moved from rather harsh detergents to membrane systems having more native-like properties. In particular, most recently phospholipid nanodiscs have been developed and optimized mainly for solution-state NMR but are now also being used for solid-state NMR spectroscopy. Nanodiscs consist of a patch of a planar lipid bilayer that is encircled by different (bio-) polymers to form particles of defined and tunable size. In this review, we provide an overview of available membrane mimetics, including nanodiscs, amphipols and bicelles, that are suitable for high-resolution NMR spectroscopy and describe how these advanced membrane mimetics can facilitate NMR studies on the structure and dynamics of membrane proteins. Since the stability of membrane proteins depends critically on the chosen membrane mimetic, we emphasize the importance of a suitable system that is not necessarily developed for solution-state NMR applications and hence requires optimization for each membrane protein. However, lipid-based membrane mimetics offer the possibility of performing NMR experiments at elevated temperatures and studying ligand and partner protein complexes as well as their functional dynamics in a realistic membrane environment. In order to be able to make an informed decision during the selection of a suitable membrane system, we provide a detailed overview of the available options for various membrane protein classes and thereby facilitate this often-difficult selection process for a broad range of desired NMR applications. (C) 2019 Elsevier B.V. All rights reserved.

Membrane proteins play important roles in health and disease. Despite their importance, the study of membrane proteins has been significantly limited by the difficulties inherent to their successful expression, purification, and stabilization once they have been extracted from the cell membrane. In addition, expression of human membrane proteins commonly requires the use of expensive and/or time-consuming eukaryotic systems, hence their successful expression in bacteria will be obviously beneficial for experimental research. Furthermore, since lipids can have critical effects on the activity of membrane proteins and given the composition similarities between the inner mitochondrial membrane and the bacterial plasma membrane, production of mitochondrial membrane proteins in E. coli represents a logical choice. Here, we present a novel protocol to produce a human mitochondrial ATP-Binding Cassette (ABC) transporter in E. coli. The function of the three known human mitochondrial ABC transporters is not fully understood, but X-ray crystallography models of ABCB10 produced in insect cells are available. We have successfully expressed and purified ABCB10 from E. coli. The yield is close to that of another bacterial ABC transporter routinely produced in our laboratory under similar conditions. In addition, we can efficiently reconstitute detergent purified ABCB10 into lipid nanodiscs. Measurements of ATPase activity of ABCB10 produced in E. coli show an ATP hydrolysis rate similar to other human ABC transporters. This novel protocol facilitates the production of this human mitochondrial transporter for biochemical, structural, and functional analysis, and can likely be adjusted for production of other mitochondrial transporters.

International audience; The structure, dynamics and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane, and reconstituted in a suitable membrane-mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co-polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes, forming nanosize discoidal proteolipid particles, also referred to as native nanodiscs. Here we show, for the first time, that high-resolution solid-state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified with the 2 x 34 kDa-large bacterial cation diffusion facilitator CzcD from Cupriavidus metallidurans CH34.

The lipid composition of the cellular membrane plays an important role in a number of biological processes including the binding of membrane-active peptides. Characterization of membrane binding remains challenging, due to the technical limitations associated with the use of standard biophysical techniques and available membrane models. Here, we investigate the lipid binding properties of two membrane-active peptides, VSTx1, a well characterized ion-channel inhibitor, identified from spider venom, that preferentially binds to anionic lipid mixtures, and AA139 an antimicrobial b-hairpin peptide with uncharacterised lipid binding properties, currently in pre-clinical development. The lipid binding properties of these peptides are elucidated using nanodiscs formed by both linear and circularized (sortase-mediated) forms of a membrane scaffold protein (MSP1D1 Delta H5). We find that nanodiscs formed by circularized MSPs-in contrast to those formed by linear MSPs-are sufficiently stable under sample conditions typically used for biophysical measurements (including lipid composition, a range of buffers, temperatures and concentrations). Using these circularized nanodiscs, we are able to extract detailed thermodynamic data using isothermal titration calorimetry (ITC) as well as atomic resolution mapping of the lipid binding interfaces of our isotope labeled peptides using solution-state, heteronuclear, nuclear magnetic resonance (NMR) spectroscopy. This represents a novel and general approach for elucidating the thermodynamics and molecular interface of membrane-active peptides toward flat lipid bilayers of variable composition. Our approach is validated by first determining the thermodynamic parameters and binding interface of VSTx1 toward the lipid bilayer, which shows good agreement with previous studies using lipid micelles and liposomes. The method is then applied to AA139, where the membrane binding properties are unknown. This characterization, involved solving the high-resolution structure of AA139 in solution using NMR spectroscopy and the development of a suitable expression system for isotope labeling. AA139 was found to bind exclusively to anionic membranes with moderate affinity (K-d similar to low mu M), and was found to have a lipid binding interface involving the termini of the beta-hairpin structure. The preference of AA139 for anionic lipids supports a role for membrane binding in the mode-of-action of this peptide, which is also consistent with its higher inhibitory activity against bacterial cells compared to mammalian cells. The described approach is a powerful method for investigation of the membrane binding properties of this important class of molecules.

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergent removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. In addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.

Purpose of Review Advances in technology have expanded telemedicine opportunities covering medical practice, research, and education. This is of particular importance in movement disorders (MDs), where the combination of disease progression, mobility limitations, and the sparse distribution of MD specialists increase the difficulty to access. In this review, we discuss the prospects, challenges, and strategies for telemedicine in MDs.Recent Findings Telemedicine for MDs has been mainly evaluated in Parkinson's disease (PD) and compared to in-office care is cost-effective with similar clinical care, despite the barriers to engagement. However, particular groups including pediatric patients, rare MDs, and the use of telemedicine in underserved areas need further research.Summary Interdisciplinary telemedicine and tele-education for MDs are feasible, provide similar care, and reduce travel costs and travel time compared to in-person visits. These benefits have been mainly demonstrated for PD but serve as a model for further validation in other movement disorders.

Graphical abstract, Membrane proteins are generally unstable in detergents. Therefore, biochemical and biophysical studies of membrane proteins in lipidic environments provides a near native-like environment suitable for membrane proteins. However, manipulation of proteins embedded in lipid bilayer has remained difficult. Methods such as nanodiscs and lipid cubic phase have been developed for easy manipulation of membrane proteins and have yielded significant insights into membrane proteins. Traditionally functional reconstitution of receptors in nanodiscs has been studied with radioligands. We present a simple and faster method for studying the functionality of reconstituted membrane proteins for routine characterization of protein batches after initial optimization of suitable conditions using radioligands. The benefits of the method are • Faster and generic method to assess functional reconstitution of membrane proteins. • Adaptable in high throughput format (≥96 well format). • Stability measurement in near-native lipid environment and lipid dependent melting temperatures.

Interactions between membrane proteins and lipids are often crucial for structure and function yet difficult to define because of their dynamic and heterogeneous nature. Here, we use mass spectrometry to demonstrate that membrane protein oligomers ejected from nanodiscs in the gas phase retain large numbers of lipid interactions. The complex mass spectra that result from gas‐phase dissociation were assigned using a Bayesian deconvolution algorithm together with mass defect analysis, allowing us to count individual lipid molecules bound to membrane proteins. Comparison of the lipid distributions measured by mass spectrometry with molecular dynamics simulations reveals that the distributions correspond to distinct lipid shells that vary according to the type of protein–lipid interactions. Our results demonstrate that nanodiscs offer the potential for native mass spectrometry to probe interactions between membrane proteins and the wider lipid environment.

Solute transport via ATP binding cassette (ABC) importers involves receptor-mediated substrate binding, which is followed by ATP-driven translocation of the substrate across the membrane. How these steps are exactly initiated and coupled, and how much ATP it takes to complete a full transport cycle, are subject of debate. Here, we reconstitute the ABC importer GlnPQ in nanodiscs and in proteoliposomes and determine substrate-(in)dependent ATP hydrolysis and transmembrane transport. We determined the conformational states of the substrate-binding domains (SBDs) by single-molecule Forster resonance energy transfer measurements. We find that the basal ATPase activity (ATP hydrolysis in the absence of substrate) is mainly caused by the docking of the closed-unliganded state of the SBDs onto the transporter domain of GlnPQ and that, unlike glutamine, arginine binds both SBDs but does not trigger their closing. Furthermore, comparison of the ATPase activity in nanodiscs with glutamine transport in proteoliposomes shows that the stoichiometry of ATP per substrate is close to two. These findings help understand the mechanism of transport and the energy coupling efficiency in ABC transporters with covalently linked SBDs, which may aid our understanding of Type I ABC importers in general.

Autophosphorylating histidine kinase CheA is central to signaling in bacterial chemotaxis. The kinase donates its phosphoryl group to two response regulators, CheY that controls flagellar rotation and thus motility and CheB, crucial for sensory adaptation. As measured by coupled CheY phosphorylation, incorporation into signaling complexes activates the kinase ∼1000-fold and places it under control of chemoreceptors. By the same assay, receptors modulate kinase activity ∼100-fold as a function of receptor ligand occupancy and adaptational modification. These changes are the essence of chemotactic signaling. Yet, the enzymatic properties affected by incorporation into signaling complexes, by chemoreceptor ligand binding or by receptor adaptational modification are largely undefined. To investigate, we performed steady-state kinetic analysis of autophosphorylation using a liberated kinase phosphoryl-accepting domain, characterizing kinase alone, in isolated core signaling complexes and in small arrays of core complexes assembled in vitro with receptors contained in isolated native membranes. Autophosphorylation in signaling complexes was measured as a function of ligand occupancy and adaptational modification. Activation by incorporation into signaling complexes and modulation in complexes by ligand occupancy and adaptational modification occurred largely via changes in the apparent catalytic rate constant (kcat ). Changes in the autophosphorylation kcat accounted for most of the ∼1000-fold kinase activation in signaling complexes observed for coupled CheY phosphorylation, and the ∼100-fold inhibition by ligand occupancy or modulation by adaptational modification. Our results indicate no more than a minor role in kinase control for simple sequestration of the autophosphorylation substrate. Instead they indicate direct effects on the active site.

The haem‐containing mono‐oxygenase cytochrome P450 3A4 (CYP3A4) and its redox partner NADPH‐dependent cytochrome P450 oxidoreductase (CPR) are among the most important enzymes in human liver for metabolizing drugs and xenobiotic compounds. They are membrane‐bound in the endoplasmic reticulum (ER). How ER colocalization and the complex ER phospholipid composition influence enzyme activity are not well understood. CPR and CYP3A4 were incorporated into phospholipid bilayer nanodiscs, both singly, and together in a 1 : 1 ratio, to investigate the significance of membrane insertion and the influence of varying membrane composition on steady‐state reaction kinetics. Reaction kinetics were analysed using a fluorimetric assay with 7‐benzyloxyquinoline as substrate for CYP3A4. Full activity of the mono‐oxygenase system, with electron transfer from NADPH via CPR, could only be reconstituted when CPR and CYP3A4 were colocalized within the same nanodiscs. No activity was observed when CPR and CYP3A4 were each incorporated separately into nanodiscs then mixed together, or when soluble forms of CPR were mixed with preassembled CYP3A4‐nanodiscs. Membrane integration and colocalization are therefore essential for electron transfer. Liver microsomal lipid had an enhancing effect compared with phosphatidylcholine on the activity of CPR alone in nanodiscs, and a greater enhancing effect on the activity of CPR‐CYP3A4 nanodisc complexes, which was not matched by a phospholipid mixture designed to mimic the ER composition. Furthermore, liver lipid enhanced redox coupling within the system. Thus, natural ER lipids possess properties or include components important for enhanced catalysis by CPR‐CYP3A4 nanodisc complexes. Our findings demonstrate the importance of using natural lipid preparations for the detailed analysis of membrane protein activity.

This work was supported by funds from the St Andrews EM facility and the University of St Andrews. CP is a Royal Society of Edinburgh (RSE) Personal Research Fellow, funded by the Scottish Government. CP is also supported by a Tenovus Scotland Award (T15/41). JML was supported by a Programme grant from the Wellcome Trust (Number 045404). Synthetic and naturally occurring lipid-rich nanoparticles are of wide ranging importance in biomedicine. They include liposomes, bicelles, nanodiscs, exosomes and virus particles. The quantitative study of these particles requires methods for high-resolution visualization of the whole population. One powerful imaging method is cryo-EM of vitrified samples, but this is technically demanding, requires specialized equipment, provides low contrast and does not reveal all particles present in a population. Another approach is classical negative stain-EM, which is more accessible but is difficult to standardize for larger lipidic structures, which are prone to artifacts of structure collapse and contrast variability. A third method uses embedment in methylcellulose films containing uranyl acetate as a contrasting agent. Methylcellulose embedment has been widely used for contrasting and supporting cryosections but only sporadically for visualizing lipid rich vesicular structures such as endosomes and exosomes. Here we present a simple methylcellulose-based method for routine and comprehensive visualization of synthetic lipid rich nanoparticles preparations, such as liposomes, bicelles and nanodiscs. It combines a novel double-staining mixture of uranyl acetate (UA) and tungsten-based electron stains (namely phosphotungstic acid (PTA) or sodium silicotungstate (STA)) with methylcellulose embedment. While the methylcellulose supports the delicate lipid structures during drying, the addition of PTA or STA to UA provides significant enhancement in lipid structure display and contrast as compared to UA alone. This double staining method should aid routine structural evaluation and quantification of lipid rich nanoparticles structures. Publisher PDF

Conformational equilibria of G-protein–coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron–electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH+ form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of ∼16 min at 20 °C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners.

Highlights • Uranyl acetate/tungsten double stains are proposed for imaging lipid rich nanoparticle in TEM. • Combined with methylcellulose embedment, the technique enhances membrane contrast. • The technique works for liposomes, nanodiscs and bicelles. • The double staining should improve quantification of lipid rich nanoparticles., Synthetic and naturally occurring lipid-rich nanoparticles are of wide ranging importance in biomedicine. They include liposomes, bicelles, nanodiscs, exosomes and virus particles. The quantitative study of these particles requires methods for high-resolution visualization of the whole population. One powerful imaging method is cryo-EM of vitrified samples, but this is technically demanding, requires specialized equipment, provides low contrast and does not reveal all particles present in a population. Another approach is classical negative stain-EM, which is more accessible but is difficult to standardize for larger lipidic structures, which are prone to artifacts of structure collapse and contrast variability. A third method uses embedment in methylcellulose films containing uranyl acetate as a contrasting agent. Methylcellulose embedment has been widely used for contrasting and supporting cryosections but only sporadically for visualizing lipid rich vesicular structures such as endosomes and exosomes. Here we present a simple methylcellulose-based method for routine and comprehensive visualization of synthetic lipid rich nanoparticles preparations, such as liposomes, bicelles and nanodiscs. It combines a novel double-staining mixture of uranyl acetate (UA) and tungsten-based electron stains (namely phosphotungstic acid (PTA) or sodium silicotungstate (STA)) with methylcellulose embedment. While the methylcellulose supports the delicate lipid structures during drying, the addition of PTA or STA to UA provides significant enhancement in lipid structure display and contrast as compared to UA alone. This double staining method should aid routine structural evaluation and quantification of lipid rich nanoparticles structures.

There is a strong demand for techniques that allow the fabrication of biocompatible porous nanoparticles for drug delivery applications. In this work, a new method to fabricate size- and shape-controlled porous silicon (pSi) nanodiscs is described. The process relies on a combination of colloidal lithography and metal-assisted chemical etching. Height and diameter of the pSi nanodiscs can be easily adjusted. The nanodiscs are degradable in physiological milieu and are nontoxic to mammalian cells. In order to highlight the potential of the pSi nanodiscs in drug delivery, an in vitro investigation that involved loading of nanodiscs with the anticancer agent camptothecin and functionalization of the nanodisc periphery with an antibody that targets receptors on the surface of neuroblastoma cells is carried out. The thus-prepared nanocarriers are found to selectively attach to and kill cancer cells Refereed/Peer-reviewed

TonB-dependent membrane receptors from bacteria have been analyzed in detergent-containing solution, an environment that may influence the role of ligand in inducing downstream interactions. We report reconstitution of FhuA into a membrane mimetic: nanodiscs. In contrast to previous results in detergent, we show that binding of TonB to FhuA in nanodiscs depends strongly on ferricrocin. The stoichiometry of interaction is 1:1 and the binding constant KD is ~200nM; an equilibrium affinity that is ten-fold lower than reported in detergent. FhuA in nanodiscs also forms a high-affinity binding site for colicin M (KD ~3.5nM), while ferricrocin renders FhuA refractory to colicin binding. Together, these results demonstrate the importance of the ligand in regulating receptor interactions and the advantages of nanodiscs to study β-barrel membrane proteins in a membrane-like environment.

For membrane protein studies, nanodiscs have been shown to hold great potential in terms of preparing soluble samples while maintaining a lipid environment. Here, we describe the differences in lipid order and protein dynamics in MSP1 nanodiscs compared to lamellar preparations by solid-state NMR. For DMPC, an increase of the dipolar C-H lipid acyl chain order parameters in nanodiscs is observed in both gel- and liquid crystalline phases. Incorporating proteorhodopsin in these nanodiscs resulted in a significantly longer rotating frame spin-lattice relaxation time for 13C leerzeichen and better cross polarisation efficiency due to restricted protein dynamics. A comparison of 13C–13C correlation spectra revealed no structural differences. The incorporation of proteorhodopsin into nanodiscs has been optimised with respect to detergent and to protein/scaffold protein/lipid stoichiometries. Its functional state was probed by time-resolved optical spectroscopy revealing only minor differences between lamellar and nanodisc preparations. Our observations show remarkable dynamic effects between membrane proteins, lipids and scaffold protein. The potential use of nanodiscs for solid-state NMR applications is discussed.

1 INTRODUCTION 1 1.1 CHANNELRHODOPSIN 2 2 1.1.1 The photocycle intermediates 3 1.1.2 The Channel Activity – Electrophysiological Measurements 5 1.1.3 Structural determinants of pore opening 6 1.2 THE AIMS OF THIS THESIS 8 2 MATERIAL AND METHODS 9 2.1 MATERIAL 9 2.1.1 Chemicals 9 2.1.2 Plasmids 10 2.1.3 Strains 10 2.1.4 Buffers 11 2.1.5 Media 12 2.1.6 Equipment 13 2.2 BIOCHEMICAL METHODS 14 2.2.1 Pichia pastoris/pPIC9K expression system 14 2.2.1.1 Mutation of the recombinant ChR2 DNA constructs 16 2.2.1.2 Transformation of Pichia pastoris with pPIC9k-ChR2 constructs and selection of multicopy clones 19 2.2.1.3 Expression of ChR2 in Pichia pastoris 21 2.2.1.4 ChR2 purification 22 2.2.2 Labeling 24 2.2.2.1 Spin labeling with MTSL 24 2.2.3 Nanodisc reconstitution 24 2.2.3.1 Expression and purification of MSP1D1in E. coli 24 2.2.3.2 Reconstitution of ChR2 in DMPC nanodiscs 25 2.3 BIOPHYSICAL METHODS 26 2.3.1 Spectroscopic methods 26 2.3.1.1 Optical spectroscopy 26 2.3.1.2 Resonance spectroscopy 30 2.3.1.3 Spin labeling efficiency 39 2.3.2 Electrophysiological measurements 40 3 RESULTS 41 3.1 GENETIC ENGINEERING: DESIGNING CYSTEINE-REDUCED VARIANTS 41 3.2 ION CONDUCTANCE OF THE CYSTEINE-REDUCED VARIANTS 47 3.3 CYSTEINE-REDUCED VARIANTS IN DETERGENT ENVIRONMENT 50 3.3.1 Photostability 50 3.3.2 Accumulation of the open channel (P3530) 51 3.3.3 Helical movements 56 3.4 CYSTEINE-REDUCED VARIANTS IN MEMBRANE ENVIRONMENT 58 3.4.1 Nanodisc Reconstitution 58 3.4.1.1 Optimization of the reconstitution procedure 60 3.4.1.2 Optimization of ChR2 reconstitution 62 3.4.1.3 ChR2 nanodiscs characterization 66 3.4.2 Photostability 69 3.4.3 Accumulation of the open channel (P3530) 70 3.4.4 Helical movements upon channel opening 77 3.4.4.1 Helix B movements 77 3.4.4.2 Helix F movements 83 3.4.4.3 Tracing distances beyond helix B and F: Introducing additional cysteines 86 3.5 THE Y196F VARIANT: LOCALIZING A DANGLING WATER 89 3.5.1 Results 90 3.6 THE T159C VARIANT: A FUNCTIONAL VARIANT WITH IMPROVED EXPRESSION 96 3.6.1 Results 97 4 DISCUSSION 100 4.1 NEW CYSTEINE-REDUCED VARIANTS FOR SIDE DIRECTED SPIN LABELING 100 4.1.1.1 Functionality in detergent environment 100 4.1.1.2 Membrane environment 102 4.1.1.3 Cysteine functionality 103 4.2 HELICAL MOVEMENTS 106 5 CONCLUSION 112 6 OUTLOOK 113 REFERENCES 116 ACKNOWLEDGEMENTS 124 APPENDIX 125 ABSTRACT 125 KURZZUSAMMENFASSUNG 126 SELBSTSTÄNDIGKEITSERKLÄRUNG 127 CURRICULUM VITAE 128 LIST OF TABLES 130 LIST OF FIGURES 133 LIST OF ABBREVIATIONS 142, Channelrhodopsins (ChRs) are ion channels that regulate phototaxis of green algae. The functional unit comprises a retinal chromophore embedded in seven transmembrane helices (7TM). Up to now, ChRs are the only light gated ion channels found in nature. After the discovery in 2002 the ChRs were soon exploited as a tool to trigger nerve cell action potentials. A new field of research, named optogenetics, was established. Since ChRs undergo a cyclic reaction triggered by light they can be investigated by a large set of different biophysical techniques and hence are ideal candidates to study the structure/function relationship on molecular level. In this work three functional ChR2 cysteine-variants were engineered to enable the site specific labeling of the cytosolic end of helix B and additionally helix F or C. The labeling positions can be investigated in the ground state as well as in the accumulated open state, P3. The variants were spin-labeled and subjected to pulsed electron double resonance (PELDOR) measurements. Thereby, an outward displacement of the cytosolic side of helix B and F in the open state could be observed. The helix B displacement persist also in the desensitized state, P4. These results strengthen the hypothesis of a prolongation of the pore between helices A, B, C and G by the observed outward movement of helix B in the open state. However, as suggested earlier, the major structural changes are to some extent decoupled from the on-gating, since the outward displacement of helix B persists in the desensitized state. Furthermore, we set the stage for the quantitative determination of the helix F movement, which is currently underway. The results of this thesis will help to guide the developments and benchmark future open state models., Kanalrhodopsine (KR) sind Ionenkanäle, die die Phototaxis von Grünalgen steuern. Die funktionale Einheit umfasst ein Retinal-Chromophor, das in sieben Transmembranhelices eingebettet ist. Kanalrhodopsine sind die einzigen, natürlichen, lichtgesteuerten Ionenkanäle, die bisher entdeckt wurden. Nach ihrer Entdeckung 2002 wurde bald erkannt, dass mit ihnen Aktionspotentiale in Nervenzellen durch Licht ausgelöst werden können. Da KR eine zyklische Reaktion durchlaufen, die nichtinvasiv durch Licht ausgelöst wird und größere strukturelle Veränderungen umfasst, eignen sie sich ideal um an ihnen Struktur-Funktions-Zusammenhänge biophysikalisch zu ergründen. In dieser Arbeit wurden drei aktive KR-Cysteinvarianten hergestellt, die spezifisch an dem zytosolischen Ende der Helix B und zusätzlich an Helix C oder F markiert werden können. Die markierten Positionen können im Grundzustand oder im akkumulierten offenen Zustand, P3, untersucht werden. Die KR-Varianten wurden spinmarkiert und in gepulsten Elektronendoppelresonanzmessungen untersucht. Die Messungen belegen eine Auswärtsbewegung der zytosolischen Seite von Helix B und F im offenen Zustand. Auch im desensitivierten Zustand, P4, bleibt Helix B nach außen versetzt. Die Ergebnisse untermauern die Hypothese einer Tunnelöffnung zwischen den Helices A, B, C und G. Gleichzeitig deuten die Resultate darauf hin, dass die großen Konformationsänderungen, wie bereits vermutet, zu einem gewissen Grad von der Tunnelöffnung entkoppelt sind, da Helix B auch im desensitivierten Zustand nach außen versetzt bleibt. Weiterhin wurde die Grundlage für eine quantitative Bestimmung der Helix F Bewegung bereitet, welche ein weiterhin laufendes Projekt ist. Die Ergebnisse dieser Arbeit werden helfen Modelle des offenen Kanals zu entwickeln und zu beurteilen.

Membrane proteins (MPs) are prevalent drug discovery targets involved in many cell processes. Despite their high potential as drug targets, the study of MPs has been hindered by limitations in expression, purification and stabilization in order to acquire thermodynamic and kinetic parameters of small molecules binding. These bottlenecks are grounded on the mandatory use of detergents to isolate and extract MPs from the cell plasma membrane and the coexistence of multiple conformations, which reflects biochemical versatility and intrinsic instability of MPs. In this work ,we set out to define a new strategy to enable surface plasmon resonance (SPR) measurements on a thermostabilized and truncated version of the human adenosine (A2A) G-protein-coupled receptor (GPCR) inserted in a lipid bilayer nanodisc in a label- and detergent-free manner by using a combination of affinity tags and GFP-based fluorescence techniques. We were able to detect and characterize small molecules binding kinetics on a GPCR fully embedded in a lipid environment. By providing a comparison between different binding assays in membranes, nanodiscs and detergent micelles, we show that nanodiscs can be used for small molecule binding studies by SPR to enhance the MP stability and to trigger a more native-like behaviour when compared to kinetics on A2A receptors isolated in detergent. This work provides thus a new methodology in drug discovery to characterize the binding kinetics of small molecule ligands for MPs targets in a lipid environment.

Studies of the membrane proteins suggest their close interaction with the lipid surroundings. Membrane proteins and their activities are affected by the composition and structure of the lipid bilayer, therefore adequate surroundings for studied protein are crucial for the model membrane to ensure its biological relevance. In recent years nanodiscs which are small fragments of lipid bilayer stabilised by derivatives of apolipoprotein, called membrane scaffold protein (MSP), have been established as alternative tool in structural and functional studies of membrane proteins. In this study, the influence MSP of different length on structure and dynamics of DMPC and POPC bilayer was investigated and compared to bilayer present in liposomes. EPR spectroscopy technique using different PC-based spin probes was employed to show cholesterol-like organising effect of MSPs on lipid bilayer, thus giving a better insight into the nanodiscs model membrane structure, and its possible implications in the research of membrane protein applications.

Integral membrane proteins often present daunting challenges for biophysical characterization, a fundamental issue being how to select a surfactant that will optimally preserve the individual structure and functional properties of a given membrane protein. Bacterial reaction centers offer a rare opportunity to compare the properties of an integral membrane protein in different artificial lipid/surfactant environments with those in the native bilayer. Here, we demonstrate that reaction centers purified using a styrene maleic acid copolymer remain associated with a complement of native lipids and do not display the modified functional properties that typically result from detergent solubilization. Direct comparisons show that reaction centers are more stable in this copolymer/lipid environment than in a detergent micelle or even in the native membrane, suggesting a promising new route to exploitation of such photovoltaic integral membrane proteins in device applications.

Mari Numata,1 Yelena V Grinkova,2 James R Mitchell,1 Hong Wei Chu,1 Stephen G Sligar,2 Dennis R Voelker1 1Department of Medicine, Program in Cell Biology, National Jewish Health, Denver, CO, USA; 2Department of Biochemistry, University of Illinois, Urbana, IL, USA Abstract: There is increasing interest in the application of nanotechnology to solve the difficult problem of therapeutic administration of pharmaceuticals. Nanodiscs, composed of a stable discoidal lipid bilayer encircled by an amphipathic membrane scaffold protein that is an engineered variant of the human Apo A-I constituent of high-density lipoproteins, have been a successful platform for providing a controlled lipid composition in particles that are especially useful for investigating membrane protein structure and function. In this communication, we demonstrate that nanodiscs are effective in suppressing respiratory syncytial viral (RSV) infection both in vitro and in vivo when self-assembled with the minor pulmonary surfactant phospholipid palmitoyloleoylphosphatidylglycerol (POPG). Preparations of nanodiscs containing POPG (nPOPG) antagonized interleukin-8 production from Beas2B epithelial cells challenged by RSV infection, with an IC50 of 19.3 µg/mL. In quantitative in vitro plaque assays, nPOPG reduced RSV infection by 93%. In vivo, nPOPG suppressed inflammatory cell infiltration into the lung, as well as IFN-γ production in response to RSV challenge. nPOPG also completely suppressed the histopathological changes in lung tissue elicited by RSV and reduced the amount of virus recovered from lung tissue by 96%. The turnover rate of nPOPG was estimated to have a half-time of 60–120 minutes (m), based upon quantification of the recovery of the human Apo A-I constituent. From these data, we conclude that nPOPG is a potent antagonist of RSV infection and its inflammatory sequelae both in vitro and in vivo. Keywords: nanodiscs, therapeutic delivery, anti-viral, innate immunity, phospholipids

Nanodiscs (NDs) enable the analysis of membrane proteins (MP) in natural lipid bilayer environments. In combination with cell-free (CF) expression, they could be used for the co-translational insertion of MPs into defined membranes. This new approach allows the characterization of MPs without detergent contact and it could help to identify effects of particular lipids on catalytic activities. Association of MPs with different ND types, quality of the resulting MP/ND complexes as well as optimization parameters are still poorly analyzed. This study describes procedures to systematically improve CF expression protocols for the production of high quality MP/ND complexes. In order to reveal target dependent variations, the co-translational ND complex formation with the bacterial proton pump proteorhodopsin (PR), with the small multidrug resistance transporters SugE and EmrE, as well as with the Escherichia coli MraY translocase was studied. Parameters which modulate the efficiency of MP/ND complex formation have been identified and in particular effects of different lipid compositions of the ND membranes have been analyzed. Recorded force distance pattern as well as characteristic photocycle dynamics indicated the integration of functionally folded PR into NDs. Efficient complex formation of the E. coli MraY translocase was dependent on the ND size and on the lipid composition of the ND membranes. Active MraY protein could only be obtained with ND containing anionic lipids, thus providing new details for the in vitro analysis of this pharmaceutically important protein.

Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs). These bilayer constructs are fully soluble in aqueous environments and hold great promise as a model system to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time, yet limited studies have been conducted to examine this phenomenon. Understanding the source of this heterogeneity may lead to methods to mitigate heterogeneity or to control NLP size, which may be important for tailoring NLPs for specific membrane proteins. Here, we have used atomic force microscopy, ion mobility spectrometry, and transmission electron microscopy to quantify NLP size distributions on the single-particle scale, specifically focusing on assemblies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a recombinant apolipoprotein E variant containing the N-terminal 22 kDa fragment (E422k). Four discrete sizes of E422k/DMPC NLPs were identified by all three techniques, with diameters centered at approximately 14.5, 19, 23.5, and 28 nm. Computer simulations suggest that these sizes are related to the structure and number of E422k lipoproteins surrounding the NLPs and particles with an odd number of lipoproteins are consistent with the double-belt model, in which at least one lipoprotein adopts a hairpin structure.

B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) is a member of the Bcl-2 protein family having a pivotal role in triggering cell commitment to apoptosis. Bax is latent and monomeric in the cytosol but transforms into its lethal, mitochondria-embedded oligomeric form in response to cell stress, leading to the release of apoptogenic factors such as cytochrome C. Here, we dissected the structural correlates of Bax membrane insertion while oligomerization is halted. This strategy was enabled through the use of nanometer-scale phospholipid bilayer islands (nanodiscs) the size of which restricts the reconstituted system to single Bax-molecule activity. Using this minimal reconstituted system, we captured structural correlates that precede Bax homo-oligomerization elucidating previously inaccessible steps of the core molecular mechanism by which Bcl-2 family proteins regulate membrane permeabilization. We observe that, in the presence of BH3 interacting domain death agonist (Bid) BH3 peptide, Bax monomers induce the formation of ~3.5-nm diameter pores and significantly distort the phospholipid bilayer. These pores are compatible with promoting release of ions as well as proteinaceous components, suggesting that membrane-integrated Bax monomers in the presence of Bid BH3 peptides are key functional units for the activation of the cell demolition machinery.

PLoS one 11(3), e0151051 (2016). doi:10.1371/journal.pone.0151051, Published by PLoS, Lawrence, Kan.

The D2 dopamine receptor (DRD2) is a therapeutic target for Parkinson's disease(1)and antipsychotic drugs(2). DRD2 is activated by the endogenous neurotransmitter dopamine and synthetic agonist drugs such as bromocriptine(3), leading to stimulation of G(i)and inhibition of adenylyl cyclase. Here we used cryo-electron microscopy to elucidate the structure of an agonist-bound activated DRD2-G(i)complex reconstituted into a phospholipid membrane. The extracellular ligand-binding site of DRD2 is remodelled in response to agonist binding, with conformational changes in extracellular loop 2, transmembrane domain 5 (TM5), TM6 and TM7, propagating to opening of the intracellular G(i)-binding site. The DRD2-G(i)structure represents, to our knowledge, the first experimental model of a G-protein-coupled receptor-G-protein complex embedded in a phospholipid bilayer, which serves as a benchmark to validate the interactions seen in previous detergent-bound structures. The structure also reveals interactions that are unique to the membrane-embedded complex, including helix 8 burial in the inner leaflet, ordered lysine and arginine side chains in the membrane interfacial regions, and lipid anchoring of the G protein in the membrane. Our model of the activated DRD2 will help to inform the design of subtype-selective DRD2 ligands for multiple human central nervous system disorders., The structure of the D2 dopamine receptor in complex with its G protein reveals how dopamine receptors are activated and, importantly, how a G-protein-coupled receptor can interact with its G protein in a phospholipid membrane.