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Abstract: We report here a simple wet chemical method to fabricate ZnO nanodiscs. Growth of the nanodiscs occurs at room temperature (35°C) in the reaction between zinc chloride and sodium hydroxide of predetermined concentration. The nanodiscs were structurally characterized using field emission scanning electron microscopy and X-ray diffraction. The diameters of the nanodiscs vary between 300nm and 500nm. X-ray diffraction data revealed that the unit cell of the nanodiscs is hexagonal. Room temperature photoluminescence was used to study the optical emission property of the nanodiscs. The nanodiscs exhibit strong PL emission peaked at ∼420nm. This singular violet emission from ZnO nanodiscs is a very interesting and rare finding. The PL emission at 420nm arises due to recombination of electron at zinc interstitials and holes in the valence band. [Copyright &y& Elsevier]

Membrane nanoobjects are very important tools to study biomembrane properties. Two types are described herein: Bicelles and Nanodiscs. Bicelles are obtained by thorough water mixing of long chain and short chain lipids and may take the form of membranous discs of 10–50 nm. Temperature-composition-hydration diagrams have been established for Phosphatidylcholines and show limited domains of existence. Bicelles can be doped with charged lipids, surfactants or with cholesterol and offer a wide variety of membranous platforms for structural biology. Internal dynamics as measured by solid-state NMR is very similar to that of liposomes in their fluid phase. Because of the magnetic susceptibility anisotropy of the lipid chains, discs may be aligned along or perpendicular to the magnetic field. They may serve as weak orienting media to provide distance information in determining the 3D structure of soluble proteins. In different conditions they show strong orienting properties which may be used to study the 3D structure, topology and dynamics of membrane proteins. Lipid Bicelles with biphenyl chains or doped with lanthanides show long lasting remnant orientation after removing the magnetic field due to smectic-like properties. An alternative to pure lipid Bicelles is provided by nanodiscs where the half torus composed by short chain lipids is replaced by proteins. This renders the nano-objects less fragile as they can be used to stabilize membrane protein assemblies to be studied by electron microscopy. Internal dynamics is again similar to liposomes except that the phase transition is abolished, possibly due to lateral constrain imposed by the toroidal proteins limiting the disc size. Advantages and drawbacks of both nanoplatforms are discussed. Unlabelled Image • Bicelles are pure synthetic lipidic discoidal particles of 20–50 nm diameter that may orient parallel or perpendicularly to magnetic fields. They are called "molecular goniometers" and mimic small pieces of biomembranes. • Bicelles are stable on narrow ranges of composition, temperature and hydration, can embed charged lipids, cholesterol, peptides and membrane proteins and are used as platforms to gain information on protein structure and dynamics by ssNMR. • Bicelle remain aligned for several hours/days outside magnetic fields and relax according to nematic or smectic properties • Nanodiscs are smaller particles (10–20 nm) that may be built with a wide variety of lipids and apolipoproteins (MSP) making the rim of the disc. • The constraints exerted by the MSP proteins forbid any temperature-driven phase transition, providing a membrane liquid-ordered state on large temperature domains. [ABSTRACT FROM AUTHOR]

Cells are surrounded by a protective lipid bilayer membrane, and membrane proteins in the bilayer control the flow of chemicals, information, and energy across this barrier. Many therapeutics target membrane proteins, and some directly target the lipid membrane itself. However, interactions within biological membranes are challenging to study due to their heterogeneity and insolubility. Mass spectrometry (MS) has become a powerful technique for studying membrane proteins, especially how membrane proteins interact with their surrounding lipid environment. Although detergent micelles are the most common membrane mimetic, nanodiscs are emerging as a promising platform for MS. Nanodiscs, nanoscale lipid bilayers encircled by two scaffold proteins, provide a controllable lipid bilayer for solubilizing membrane proteins. This Young Scientist Perspective focuses on native MS of intact nanodiscs and highlights the unique experiments enabled by making membranes fly, including studying membrane protein-lipid interactions and exploring the specificity of fragile transmembrane peptide complexes. It will also explore current challenges and future perspectives for interfacing nanodiscs with MS. Image 1 • This feature explores interfacing native MS and nanodiscs to study lipid membranes. • An overview highlights the broad uses of nanodiscs and MS. • Applications discuss protein-lipid interactions and antimicrobial peptides. • Future challenges and opportunities of complexity in nanodisc native MS. [ABSTRACT FROM AUTHOR]

• The new core-shell nanostructures were fabricated based on porous NiCo-P nanodiscs shelled with NiCo-LDH nanosheets. • Due to the synergistic effect between Ni-P NDs and NiCo LDH nanosheets, the hybrid nanostructure indicated good electrochemical performances. • The designed sensor using core-shell nanostructures showed excellent electrocatalytic activity for IPN oxidation with good repeatability, long-term stability, speedy response time, low detection limit, and wide linear range. • The introduced nanostructure has promising potential for the engineering of low-cost and new core-shell nanostructures with disc morphology for many fields. The development of robust and efficient earth-abundant electrocatalysts based on complex core-shell arrays with ordered two-dimensional nano-architectures for different electrochemical technologies is an ongoing challenge. In this matter, the fabrication of core-shell arrays with a core of bimetallic phosphides nanodisc-like building blocks has not been reported yet. Herein, advanced core-shell nanostructures consisting of porous nickel-cobalt phosphide nanodiscs (NiCo-P NDs) shelled with nickel-cobalt layered double hydroxide nanosheets (NiCo-LDH NSs) were rationally designed (NiCo-P@NiCo-LDH). The NiCo-P@NiCo-LDH core-shell nanodiscs/nanosheets have unique features such as high conductivity, large specific surface area, more contact area between nanomaterials and analyte, and numerous active sites for enhancing electrochemical performance. The electrocatalytic performance of fabricated core-shell arrays was evaluated toward electrochemical sensing of isoprenaline (IPN) as a templet. Some competencies of glassy carbon electrode coated with NiCo-P@NiCo-LDH core-shell arrays (NiCo-P@NiCo-LDH/GCE) to IPN determination are including speedy response time (∆t <1s), low detection limit (0.17 µM), wide linear range (0.5–2110 µM), long-term stability, good reproducibility, low cost, and portability. This report is promising for application in other electrochemical technologies and it will pave the way for the development of new core-shell nanostructures based on multi-metallic phosphide nanodiscs. Image, graphical abstract [ABSTRACT FROM AUTHOR]

Simple Summary: Located within the biological cell membranes, integral membrane proteins are responsible for a large variety of vital cellular processes. In humans, nearly a quarter of the genome codes integral membrane proteins, therefore malfunction of these proteins is associated with a variety of symptoms and diseases such as obesity, cancer and Parkinson's disease. Clearly, knowledge of membrane proteins behaviour, in both structural and functional terms, is important not only in medicine but also in the design of better drugs with improved pharmaceutical properties. Nevertheless, much still remains unknown about these proteins, mainly because of the technical challenges associated with their production and stability in vitro once removed from their native lipidic environment. Recently, several membrane mimetic systems have been developed including nanodisc lipid particles. Nanodiscs are self-assembled lipidic structures that "trap" membrane proteins into a disc shaped phospholipid bilayer that is stabilised by a belt made of a protein know as membrane scaffold protein (MSP). Membrane proteins assembled into lipidic nanodiscs can maintain their structural and functional integrity and are compatible with most biophysical methods. Here we demonstrate the use of in situ dynamic light scattering as a high-throughput screening tool to assess the best conditions for nanodisc assembly and protein incorporation. Membrane proteins play a crucial role in cell physiology by participating in a variety of essential processes such as transport, signal transduction and cell communication. Hence, understanding their structure–function relationship is vital for the improvement of therapeutic treatments. Over the last decade, based on the development of detergents, amphipoles and styrene maleic-acid lipid particles (SMALPs), remarkable accomplishments have been made in the field of membrane protein structural biology. Nevertheless, there are still many drawbacks associated with protein–detergent complexes, depending on the protein in study or experimental application. Recently, newly developed membrane mimetic systems have become very popular for allowing a structural and functional characterisation of membrane proteins in vitro. The nanodisc technology is one such valuable tool, which provides a more native-like membrane environment than detergent micelles or liposomes. In addition, it is also compatible with many biophysical and biochemical methods. Here we describe the use of in situ dynamic light scattering to accurately and rapidly probe membrane proteins' reconstitution into nanodiscs. The adenosine type 2A receptor (A2AR) was used as a case study. [ABSTRACT FROM AUTHOR]

Chronic pain is a common and often debilitating condition. Existing treatments are either inefficacious or associated with a wide range of side effects. The progress on developing safer and more effective analgesics has been slow, in large part due to our limited understanding of the physiological mechanisms underlying pain in different diseases. Generation and propagation of action potentials is a central component of pain sensation and voltage-gated sodium channels (Na V s) play a critical role in this process. In particular, the Na V subtype 1.7, has emerged as a promising universal target for the treatment of pain. Recently, a spider venom peptide, μ-TRTX-Pn3a, was found to be a highly selective inhibitor of Na V 1.7. Here, we report the first recombinant expression method for Pn3a in a bacterial host, which provides an inexpensive route to production. Furthermore, we have developed a method for bio-conjugation of our recombinantly produced Pn3a via sortase A-mediated ligation, providing avenues for further pre-clinical development. We demonstrate how heterologous expression in bacteria enables facile isotope labelling of Pn3a, which allowed us to study the membrane binding properties of the peptide by high-resolution solution-state nuclear magnetic resonance (NMR) spectroscopy using a recently developed lipid nanodisc system. The heteronuclear NMR data indicate that the C-terminal region of the peptide undergoes a conformational change upon lipid binding. The membrane binding properties of Pn3a are further validated using isothermal titration calorimetry (ITC), which revealed that Pn3a binds to zwitterionic planar lipid bilayers with thermodynamics that are largely driven by enthalpic contributions. [ABSTRACT FROM AUTHOR]

We describe the construction, expression and purification of three new membrane scaffold proteins (MSP) for use in assembling Nanodiscs. These new MSPs have a variety of luminescent properties for use in combination with several analytical methods. "Dark" MSP has no tryptophan residues, "Ultra-Dark" replaces both tryptophan and tyrosine with non-fluorescent side chains, and "Ultra-Bright" adds additional tryptophans to the parent membrane scaffold protein to provide a dramatic increase in native tryptophan fluorescence. All MSPs were used to successfully assemble Nanodiscs nominally 10 nm in diameter, and the resultant bilayer structure was characterized. An example of the usefulness of these new scaffold proteins is provided. Image 1 • Nanodiscs. • Fluorescence spectroscopy. • Membrane Scaffold Proteins. • Fluorescence lifetime. • Protein-membrane interactions. [ABSTRACT FROM AUTHOR]

Binding of integrin alphaIIbbeta3 (αiibβ3) to its ligands is a highly restricted and regulated mechanism. Any modification of the protein structure yields a dysfunctional role, especially in a redox environment. Here, we examine the effect of nitrosative stress on the αiibβ3 reconstituted into nanodiscs. Using single molecule force spectroscopy, we measured the interaction between αiibβ3 and its ligand RGD and found that in the presence of exogenous nitric oxide (NO) two force regimes are generated: a low force regime of ~100pN indicating the presence of integrin in a normal status, and a broad spectrum of high force regime (~210–450pN) suggesting the protein modification/aggregation. By high resolution atomic force microscopy imaging, we demonstrate that both NO and nitrite (a stable product formed from NO) are involved in destabilizing the transmembrane protein complex leading to release of αiibβ3 from the lipid bilayer and protein aggregation. Our experimental setup opens new ways for testing in a membrane environment the effect of radical species on integrins under clinically relevant conditions. Unlabelled Image • Transmembrane protein integrin alphaIIbbeta3 is reconstituted into nanodiscs. • Treatment with nitric oxide of integrin-RGD complex leads to force regimes corresponding to single integrin and protein aggregates. • AFM imaging showed that nitric oxide and nitrite are responsible for the destabilization of integrin and its release from the lipid system. • Nanodiscs preserve their membrane integrity upon nitric oxide treatment. [ABSTRACT FROM AUTHOR]

Apolipoproteins are critical structural and functional components of lipoproteins, which are large supramolecular assemblies composed predominantly of lipids and proteins, and other biomolecules such as nucleic acids. A signature feature of apolipoproteins is the preponderance of amphipathic α-helical motifs that dictate their ability to make extensive non-covalent inter- or intra-molecular helix–helix interactions in lipid-free states or helix–lipid interactions with hydrophobic biomolecules in lipid-associated states. This review focuses on the latter ability of apolipoproteins, which has been capitalized on to reconstitute synthetic nanoscale binary/ternary lipoprotein complexes composed of apolipoproteins/peptides and lipids that mimic native high-density lipoproteins (HDLs) with the goal to transport drugs. It traces the historical development of our understanding of these nanostructures and how the cholesterol accepting property of HDL has been reconfigured to develop them as drug-loading platforms. The review provides the structural perspective of these platforms with different types of apolipoproteins and an overview of their synthesis. It also examines the cargo that have been loaded into the core for therapeutic and imaging purposes. Finally, it lays out the merits and challenges associated with apolipoprotein-based nanostructures with a future perspective calling for a need to develop "zip-code"-based delivery for therapeutic and diagnostic applications. [ABSTRACT FROM AUTHOR]

Recent structures of full-length ATP-binding cassette (ABC) transporter MsbA in different states indicate large conformational changes during the reaction cycle that involve transient dimerization of its nucleotide-binding domains (NBDs). However, a detailed molecular understanding of the structural changes and associated kinetics of MsbA upon ATP binding and hydrolysis is still missing. Here, we employed time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing, to investigate the kinetics and accompanying structural changes of NBD dimerization (upon ATP binding) and subsequent dissociation (upon ATP hydrolysis) in the context of isolated NBDs as well as full-length MsbA in lipid nanodiscs. Our data allowed us to structurally characterize the major states involved in the process and determine time constants for NBD dimerization and dissociation. In the full-length protein, these structural transitions occur on much faster time scales, indicating close-proximity effects and structural coupling of the transmembrane domains with the NBDs. • Time-resolved small-angle X-ray scattering initiated by stopped-flow mixing • Investigation of kinetics and structural changes of the membrane protein MsbA • Time constants for dimerization and dissociation could be determined • Differences between kinetics of isolated NBDs and the full-length protein Josts et al. used time-resolved small-angle X-ray scattering initiated by stopped-flow mixing (SF-TR-SAXS) to investigate kinetics and structural changes of the ATP-binding cassette transporter MsbA. [ABSTRACT FROM AUTHOR]

• CeO 2 nanodiscs were prepared by a template-free and surfactant-free wet chemical route. • FESEM and TEM studies revealed that CeO 2 nanodiscs consist of CeO 2 nanoparticles. • XRD and Raman studies showed crystallite size dependent variation in lattice strain and disorder. • PL studies revealed high concentration of Ce3+ and oxygen vacancies in CeO 2 nanodiscs. • CeO 2 nanodiscs with crystallite size of 8.2 nm exhibited superior photocatalytic activities. Nanodiscs of ceria (CeO 2) were prepared through a template-free and surfactant-free simple wet chemical method and were well characterized by TEM, FESEM, XRD, XPS, Raman, photoluminescence (PL) and UV–vis spectroscopy. The effects of crystallite size on the optical response and photocatalytic performance of CeO 2 nanodiscs were investigated. FESEM and TEM studies revealed that CeO 2 nanodiscs are made up of CeO 2 nanoparticles. XRD and Raman studies revealed crystallite size dependent variation in the lattice strain and disorder in the CeO 2 nanodiscs with cubic fluorite structure. PL studies revealed higher concentration of Ce3+ defects and oxygen vacancies in CeO 2 nanodiscs with smaller crystallite size. CeO 2 nanodiscs with crystallite size of 8.2 nm exhibited superior photocatalytic activities for solar degradation of MB, MG and MO. The mechanisms underlying formation of CeO 2 nanodiscs and their enhanced photocatalytic response were tentatively proposed. [ABSTRACT FROM AUTHOR]

Although lipid nanodiscs are increasingly used in the structural studies of membrane proteins, drug delivery and other applications, the interaction between the nanodisc belt and the protein to be reconstituted is a major limitation. To overcome this limitation and to further broaden the scope of nanodiscs, a family of non-ionic amphiphilic polymers synthesized by hydrophobic functionalization of fructo-oligosaccharides/inulin is reported. We show the stability of lipid nanodiscs formed by these polymers against pH and divalent metal ions, and their magnetic-alignment properties. The reported results also demonstrate that the non-ionic polymers extract membrane proteins with unprecedented efficiency., (© 2021 Wiley-VCH GmbH.)

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., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kenney.)

The bacteriophage ΦX174 causes large pore formation in Escherichia coli and related bacteria. Lysis is mediated by the small membrane-bound toxin ΦX174-E, which is composed of a transmembrane domain and a soluble domain. The toxin requires activation by the bacterial chaperone SlyD and inhibits the cell wall precursor forming enzyme MraY. Bacterial cell wall biosynthesis is an important target for antibiotics; therefore, knowledge of molecular details in the ΦX174-E lysis pathway could help to identify new mechanisms and sites of action. In this study, cell-free expression and nanoparticle technology were combined to avoid toxic effects upon ΦX174-E synthesis, resulting in the efficient production of a functional full-length toxin and engineered derivatives. Pre-assembled nanodiscs were used to study ΦX174-E function in defined lipid environments and to analyze its membrane insertion mechanisms. The conformation of the soluble domain of ΦX174-E was identified as a central trigger for membrane insertion, as well as for the oligomeric assembly of the toxin. Stable complex formation of the soluble domain with SlyD is essential to keep nascent ΦX174-E in a conformation competent for membrane insertion. Once inserted into the membrane, ΦX174-E assembles into high-order complexes via its transmembrane domain and oligomerization depends on the presence of an essential proline residue at position 21. The data presented here support a model where an initial contact of the nascent ΦX174-E transmembrane domain with the peptidyl-prolyl isomerase domain of SlyD is essential to allow a subsequent stable interaction of SlyD with the ΦX174-E soluble domain for the generation of a membrane insertion competent toxin., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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.

1,4-Dihydropyridines (DHP), the most commonly used antihypertensives, function by inhibiting the L-type voltage-gated Ca 2+ (Ca v ) channels. DHP compounds exhibit chirality-specific antagonistic or agonistic effects. The structure of rabbit Ca v 1.1 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 Ca v 1.1 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., (© 2020 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

New software, called Marbles, is introduced that employs SAXS intensities to predict the shape of membrane proteins embedded into membrane nanodiscs. To gain computational speed and efficient convergence, the strategy is based on a hybrid approach that allows one to account for the contribution of the nanodisc to the SAXS intensity through a semi-analytical model, while the embedded membrane protein is treated as a set of beads, similarly to as in well known ab initio methods. The reliability and flexibility of this approach is proved by benchmarking the code, implemented in C++ with a Python interface, on a toy model and two proteins with very different geometry and size.

The properties of natural lipid bilayers are vital to the regulation of many membrane proteins. Scaffolded nanodiscs provide an in vitro lipid bilayer platform to host membrane proteins in an environment that approximates native lipid bilayers. However, the properties of scaffold-enclosed bilayers may depart significantly from those of bulk cellular membranes. Therefore, to improve the usefulness of nanodiscs it is essential to understand the properties of lipids restricted by scaffolds. We used computational molecular dynamics and modeling approaches to understand the effects of nanodisc size, scaffold type (DNA or protein), and hydrophobic modification of DNA scaffolds on bilayer stability and degree to which the properties of enclosed bilayers approximate bulk bilayers. With respect to achieving bulk bilayer behavior, we found that charge neutralization of DNA scaffolds was more important than the total hydrophobic content of their modifications: bilayer properties were better for scaffolds having a large number of short alkyl chains than those having fewer long alkyl chains. Further, complete charge neutralization of DNA scaffolds enabled better lipid binding, and more stable bilayers, as shown by steered molecular dynamics simulations that measured the force required to dislodge scaffolds from lipid bilayer patches. Considered together, our simulations provide a guide to the design of DNA-scaffolded nanodiscs suitable for studying membrane proteins.

The transient receptor potential (TRP) vanilloid 2 (TRPV2) and TRP vanilloid 5 (TRPV5) cation channels play an important role in various physiological and pathophysiological processes. The heterologous expression and purification of these channels is critical for functional and structural characterization of these important proteins. Full-length rat TRPV2 and rabbit TRPV5 can both be expressed in Saccharomyces cerevisiae and affinity purified using the 1D4 epitope and antibody to yield pure, functional channels. Further, these channels can be reconstituted into lipid nanodiscs for a more functionally relevant environment. Presented here are protocols for the expression of full-length rat TRPV2 and rabbit TRPV5 in Saccharomyces cerevisiae, their affinity purification, and their reconstitution into nanodiscs for structural and functional studies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Reconstitution of detergent-solubilized membrane proteins into phospholipid bilayers allows for functional and structural studies under close-to-native conditions that greatly support protein stability and function. Here we outline the detailed steps for membrane protein reconstitution to result in proteoliposomes and nanodiscs. Reconstitution can be achieved via a number of different strategies. The protocols for preparation of proteoliposomes use detergent removal via dialysis or via nonpolar polystyrene beads, or a mixture of the two methods. In this chapter, the protocols for nanodiscs apply polystyrene beads only. Proteoliposome preparation methods allow for substantial control of the lipid-to-protein ratio, from minimal amounts of phospholipid to high concentrations, type of phospholipid, and mixtures of phospholipids. In addition, dialysis affords a fairly large degree of control and variation of parameters such as rate of reconstitution, temperature, buffer conditions, and proteoliposome size. For the nanodisc approach, which is highly advantageous for ensuring equal access to both membrane sides of the protein as well as fast reconstitution of only a single membrane protein into a well-defined bilayer environment in each nanodisc, the protocols outline how a number of these parameters are more restricted in comparison to the proteoliposome protocols.

Recombinant mammalian proteins are routinely produced in E. coli and thus lack post-translational modifications. KRAS4b is processed at both the N- and C-terminus, resulting in an acetylation of the N-terminus (at Thr, after aminopeptidase removal of the original N-term Met) and farnesylation/carboxymethylation of the C-terminal Cys (after proteolytic cleavage of the original C-terminal three amino acids, Val-Iso-Met). Processing of KRAS enables it to associate with the plasma membrane and fulfill its function in cell signaling. We describe here the production of recombinant KRAS4b from our modified baculovirus/insect cell expression system that accurately incorporates these in vivo modifications to allow experiments that anchor KRAS4b to membrane mimetics (e.g., nanodiscs and liposomes).

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., Competing Interests: Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Cell-free protein expression systems and lipid nanoparticle technologies are core platforms for membrane protein synthesis. The implementation of preassembled nanodiscs allows the co-translational insertion of membrane proteins into tailored lipid bilayers in the absence of any artificial hydrophobic compounds. This strategy is particularly interesting for detergent sensitive or otherwise critical membrane proteins such as G-protein-coupled receptors (GPCRs). Cell-free expression reactions are completed within a day and the formed GPCR/nanodisc particles can be purified directly out of the reaction mixture by affinity tags and without any further manipulation. The streamlined procedure reduces risk of GPCR denaturation and the sample quality can further be supported by supplying chaperones or other beneficial compounds directly into the expression reactions.GPCRs inserted into nanoparticle membranes are excellent tools for a variety of applications such as ligand screening, engineering or even structural characterization. In this chapter, we provide protocols for the reaction set-up and efficient cell-free production of functionally folded GPCRs reaching μM concentrations in the final expression reactions. We further exemplify the tuning of GPCR sample quality and discuss their application for throughput ligand screening and for the analysis of ligand-binding characteristics.

For a successful characterization of channelrhodopsins with biophysical methods like FTIR, Raman, EPR and NMR spectroscopy and X-ray crystallography, large amounts of purified protein are requested. For proteins of eukaryotic origin, which are poorly expressing in bacterial systems or not at all, the yeast Pichia pastoris represents a promising alternative for overexpression. Here we describe the methods for cloning, overexpression and mutagenesis as well as the purification procedures for channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) and the scaffold protein MSP1D1 for reconstitution of the membrane proteins into nanodiscs. Finally, protocols are provided to study CaChR1 by FTIR difference spectroscopy and by time-resolved UV/Vis spectroscopy.

The prototypic multidrug (Mdr) transporter MdfA from Escherichia coli efflux chemically- dissimilar substrates in exchange for protons. Similar to other transporters, MdfA purportedly functions by alternating access of a central substrate binding pocket to either side of the membrane. Accordingly, MdfA should open at the cytoplasmic side and/or laterally toward the membrane to enable access of drugs into its pocket. At the end of the cycle, the periplasmic side is expected to open to release drugs. Two distinct conformations of MdfA have been captured by X-ray crystallography: An outward open (O o ) conformation, stabilized by a Fab fragment, and a ligand-bound inward-facing (I f ) conformation, possibly stabilized by a mutation (Q131R). Here, we investigated how these structures relate to ligand-dependent conformational dynamics of MdfA in lipid bilayers. For this purpose, we combined distances measured by double electron-electron resonance (DEER) between pairs of spin labels in MdfA, reconstituted in nanodiscs, with cysteine cross-linking of natively expressed membrane-embedded MdfA variants. Our results suggest that in a membrane environment, MdfA assumes a relatively flexible, outward-closed/inward-closed (O c /I c ) conformation. Unexpectedly, our data show that neither the substrate TPP nor protonation induces large-scale conformational changes. Rather, we identified a substrate-responsive lateral gate, which is open toward the inner leaflet of the membrane but closes upon drug binding. Together, our results suggest a modified model for the functional conformational cycle of MdfA that does not invoke canonical elements of alternating access., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The Ca 2+ sensor synaptotagmin-1 and the SNARE complex cooperate to trigger neurotransmitter release. Structural studies elucidated three distinct synaptotagmin-1-SNARE complex binding modes involving 'polybasic', 'primary' and 'tripartite' interfaces of synaptotagmin-1. We investigated these interactions using NMR and fluorescence spectroscopy. Synaptotagmin-1 binds to the SNARE complex through the polybasic and primary interfaces in solution. Ca 2+ -free synaptotagmin-1 binds to SNARE complexes anchored on PIP 2 -containing nanodiscs. R398Q/R399Q and E295A/Y338W mutations at the primary interface, which strongly impair neurotransmitter release, disrupt and enhance synaptotagmin-1-SNARE complex binding, respectively. Ca 2+ induces tight binding of synaptotagmin-1 to PIP 2 -containing nanodiscs, disrupting synaptotagmin-1-SNARE interactions. Specific effects of mutations in the polybasic region on Ca 2+ -dependent synaptotagmin-1-PIP 2 -membrane interactions correlate with their effects on release. Our data suggest that synaptotagmin-1 binds to the SNARE complex through the primary interface and that Ca 2+ releases this interaction, inducing PIP 2 /membrane binding and allowing cooperation between synaptotagmin-1 and the SNAREs in membrane fusion to trigger release., Competing Interests: RV, KJ, JR No competing interests declared, (© 2020, Voleti et al.)

Membrane proteins exist in lipid bilayers and mediate solute transport, signal transduction, cell-cell communication and energy conversion. Their activities are fundamental for life, which make them prominent subjects of study, but access to only a limited number of high-resolution structures complicates their mechanistic understanding. The absence of such structures relates mainly to difficulties in expressing and purifying high quality membrane protein samples in large quantities. An additional layer of complexity stems from the presence of intra- and/or extra-cellular domains constituted by unstructured intrinsically disordered regions (IDR), which can be hundreds of residues long. Although IDRs form key interaction hubs that facilitate biological processes, these are regularly removed to enable structural studies. To advance mechanistic insight into intact intrinsically disordered membrane proteins, we have developed a protocol for their purification. Using engineered yeast cells for optimized expression and purification, we have purified to homogeneity two very different human membrane proteins each with >300 residues long IDRs; the sodium proton exchanger 1 and the growth hormone receptor. Subsequent to their purification we have further explored their incorporation into membrane scaffolding protein nanodiscs, which will enable future structural studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)