Amphipathic environments for determining the structure of membrane proteins by single-particle electron cryo-microscopy.

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.

Overview of Membrane Protein Sample Preparation for Single-Particle Cryo-Electron Microscopy Analysis.

Single-particle cryo-electron microscopy (cryo-EM SPA) has recently emerged as an exceptionally well-suited technique for determining the structure of membrane proteins (MPs). Indeed, in recent years, huge increase in the number of MPs solved via cryo-EM SPA at a resolution better than 3.0 Å in the Protein Data Bank (PDB) has been observed. However, sample preparation remains a significant challenge in the field. Here, we evaluated the MPs solved using cryo-EM SPA deposited in the PDB in the last two years at a resolution below 3.0 Å. The most critical parameters for sample preparation are as follows: (i) the surfactant used for protein extraction from the membrane, (ii) the surfactant, amphiphiles, nanodiscs or other molecules present in the vitrification step, (iii) the vitrification method employed, and (iv) the type of grids used. The aim is not to provide a definitive answer on the optimal sample conditions for cryo-EM SPA of MPs but rather assess the current trends in the MP structural biology community towards obtaining high-resolution cryo-EM structures.

Amphipol-facilitated elucidation of the functional tetrameric complex of full-length cytochrome P450 CYP2B4 and NADPH-cytochrome P450 oxidoreductase.

Interactions of membrane-bound mammalian cytochromes P450 (CYPs) with NADPH-cytochrome P450 oxidoreductase (POR), which are required for metabolism of xenobiotics, are facilitated by membrane lipids. A variety of membrane mimetics, such as phospholipid liposomes and nanodiscs, have been used to simulate the membrane to form catalytically active CYP:POR complexes. However, the exact mechanism(s) of these interactions are unclear because of the absence of structural information of full-length mammalian CYP:POR complexes in membranes. Herein, we report the use of amphipols (APols) to form a fully functional, soluble, homogeneous preparation of full-length CYP:POR complexes amenable to biochemical and structural study. Incorporation of CYP2B4 and POR into APols resulted in a CYP2B4:POR complex with a stoichiometry of 1:1, which was fully functional in demethylating benzphetamine at a turnover rate of 37.7 ± 2.2 min-1, with a coupling efficiency of 40%. Interestingly, the stable complex had a molecular weight (Mw) of 338 ± 22 kDa determined by multiangle light scattering, suggestive of a tetrameric complex of 2CYP2B4:2POR embedded in one APol nanoparticle. Moreover, negative stain electron microscopy (EM) validated the homogeneity of the complex and allowed us to generate a three-dimensional EM map and model consistent with the tetramer observed in solution. This first report of the full-length mammalian CYP:POR complex by transmission EM not only reveals the architecture that facilitates electron transfer but also highlights a potential use of APols in biochemical and structural studies of functional CYP complexes with redox partners.

Thermal melt circular dichroism spectroscopic studies for identifying stabilising amphipathic molecules for the voltage-gated sodium channel NavMs.

Purified integral membrane proteins require amphipathic molecules to maintain their solubility in aqueous solutions. These complexes, in turn, are used in studies to characterise the protein structures by a variety of biophysical and structural techniques, including spectroscopy, crystallography, and cryo-electron microscopy. Typically the amphilphiles used have been detergent molecules, but more recently they have included amphipols, which are polymers of different sizes and compositions designed to create smaller, more well-defined solubilised forms of the membrane proteins. In this study we used circular dichroism spectroscopy to compare the secondary structures and thermal stabilities of the NavMs voltage-gated sodium channel in different amphipols and detergents as a means of identifying amphipathic environments that maximally maintain the protein structure whilst providing a stabilising environment. These types of characterisations also have potential as means of screening for sample types that may be more suitable for crystallisation and/or cryo-electron microscopy structure determinations.

Encapsulated membrane proteins: A simplified system for molecular simulation.

Over the past 50years there has been considerable progress in our understanding of biomolecular interactions at an atomic level. This in turn has allowed molecular simulation methods employing full atomistic modelling at ever larger scales to develop. However, some challenging areas still remain where there is either a lack of atomic resolution structures or where the simulation system is inherently complex. An area where both challenges are present is that of membranes containing membrane proteins. In this review we analyse a new practical approach to membrane protein study that offers a potential new route to high resolution structures and the possibility to simplify simulations. These new approaches collectively recognise that preservation of the interaction between the membrane protein and the lipid bilayer is often essential to maintain structure and function. The new methods preserve these interactions by producing nano-scale disc shaped particles that include bilayer and the chosen protein. Currently two approaches lead in this area: the MSP system that relies on peptides to stabilise the discs, and SMALPs where an amphipathic styrene maleic acid copolymer is used. Both methods greatly enable protein production and hence have the potential to accelerate atomic resolution structure determination as well as providing a simplified format for simulations of membrane protein dynamics. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Systematic analysis of the use of amphipathic polymers for studies of outer membrane proteins using mass spectrometry.

Membrane proteins (MPs) are essential for numerous important biological processes. Recently, mass spectrometry (MS), coupled with an array of related techniques, has been used to probe the structural properties of MPs and their complexes. Typically, detergent micelles have been employed for delivering MPs into the gas-phase, but these complexes have intrinsic properties that can limit the utility of structural studies of MPs using MS methods. Amphipols (APols) have advantages over detergent micelles and have been shown to be capable of delivering native MPs into the gas-phase. Comparing six different APols which vary in mass and charge, and the detergent n-dodecyl-ß-d-maltopyranoside, we aimed to determine which APols are most efficient for delivery of native outer membrane proteins (OMPs) into the gas-phase. We show that maintaining the solution-phase folding and global structures of three different OMPs (PagP, OmpT and tOmpA) are independent of the APol used, but differences in OMP activity can result from the different APol:OMP complexes. ESI-IMS-MS analysis of OMP:APol complexes shows that the A8-35 APol is most proficient at liberating all three OMPs into the gas-phase, without altering their gas-phase conformations.

Folding and stability of integral membrane proteins in amphipols.

Amphipols (APols) are a family of amphipathic polymers designed to keep transmembrane proteins (TMPs) soluble in aqueous solutions in the absence of detergent. APols have proven remarkably efficient at (i) stabilizing TMPs, as compared to detergent solutions, and (ii) folding them from a denatured state to a native, functional one. The underlying physical-chemical mechanisms are discussed.

Evaluation of intermolecular interactions required for thermostability of a recombinant adenovirus within a film matrix.

Thermostability of vaccines and biologic drugs are key to increasing global access to a variety of life-saving agents. In this report, we characterize interactions between a novel zwitterionic surfactant and adenovirus serotype 5 which allow the virus to remain stable at room temperature in a thin film matrix. Complexity of the adenovirus capsid and the polydispersity of the surfactant required use of a variety of techniques to achieve this goal. The CMC of the surfactant in Tris buffer (pH 6.5) was estimated to be 0.7-1.17 × 10-4 M by the pyrene 1:3 ratio method. TEM images depict micelle formation around virus capsids. An estimated Kd of the virus-surfactant interaction of 2.25 × 10-9 M was determined by isothermal titration calorimetry. Associated data suggest that this interaction may be thermodynamically favorable and entropically driven. A competitive saturation study and TEM images indicate that the surfactant also binds to hexon proteins on the virus capsid. Taken together, these data support the working hypothesis that the surfactant is capable of forming micelles in the solid and liquid state and that it forms a protective coating around the virus by binding to hexon proteins on the virus capsid during the film forming process.

Hyperosmotic Stress Allosterically Reconfigures Betaine Binding Pocket in BetP.

The transporter BetP in C. glutamicum is essential in maintaining bacterial cell viability during hyperosmotic stress and functions by co-transporting betaine and Na+ into bacterial cells. Hyperosmotic stress leads to increased intracellular K+ concentrations which in turn promotes betaine binding. While structural details of multiple end state conformations of BetP have provided high resolution snapshots, how K+ sensing by the C-terminal domain is allosterically relayed to the betaine binding site is not well understood. In this study, we describe conformational dynamics in solution of BetP using amide hydrogen/deuterium exchange mass spectrometry. These reveal how K+ alters conformation of the disordered C- and N-terminal domains to allosterically reconfigure transmembrane helices 3, 8, and 10 to enhance betaine interactions. A map of the betaine binding site, at near single amino acid resolution, reveals a critical extrahelical H-bond mediated by TM3 with betaine.

Cryo-EM structure of a monomeric yeast S. cerevisiae complex IV isolated with maltosides: Implications in supercomplex formation.

In mitochondria, complex IV (CIV) can be found as a monomer, a dimer or in association with other respiratory complexes. The atomic structure of the yeast S. cerevisiae CIV in a supercomplex (SC) with complex III (CIII) pointed to a region of significant conformational changes compared to the homologous mammalian CIV structures. These changes involved the matrix side domain of Cox5A at the CIII-CIV interface, and it was suggested that it could be required for SC formation. To investigate this, we solved the structure of the isolated monomeric CIV from S. cerevisiae stabilised in amphipol A8-35 at 3.9 Å using cryo-electron microscopy. Only a minor change in flexibility was seen in this Cox5A region, ruling out large CIV conformational shift for interaction with CIII and confirming the different fold of the yeast Cox5A subunit compared to mammalian homologues. Other differences in structure were the absence of two canonical subunits, Cox12 and Cox13, as well as Cox26, which is unique to the yeast CIV. Their absence is most likely due to the protein purification protocol used to isolate CIV from the III-IV SC.

CryoEM reconstructions of membrane proteins solved in several amphipathic solvents, nanodisc, amphipol and detergents, yield amphipathic belts of similar sizes corresponding to a common ordered solvent layer.

To maintain membrane proteins soluble in aqueous solution, amphipathic compounds are used to shield the hydrophobic patch of their membrane insertion, which forms a belt around the protein. This amphipathic belt is seldom looked at due to the difficulty to visualize it. Cryo-EM is now offering this possibility, where belts are visible in 3D reconstructions. We investigated membrane proteins solved in nanodiscs, amphipols or detergents to analyze whether the nature of the amphipathic compound influences the belt size in 3D reconstructions. We identified belt boundaries in map-density distributions and measured distances for every reconstruction. We showed that all the belts create on average similar reconstructions, whether they originate from the same protein, or from protein from different shapes and structures. There is no difference among detergents or types of nanodisc used. These observations illustrate that the belt observed in 3D reconstructions corresponds to the minimum ordered layer around membrane proteins.

Induction of Protection in Mice against a Chlamydia muridarum Respiratory Challenge by a Vaccine Formulated with the Major Outer Membrane Protein in Nanolipoprotein Particles.

Chlamydia trachomatis is a sexually transmitted bacterium that infects over 130 million individuals worldwide annually. To implement a vaccine, we developed a cell-free co-translational system to express the Chlamydia muridarum major outer membrane protein (MOMP). This approach uses a nanolipoprotein particles (tNLP) made from ApoA1 protein, amphiphilic telodendrimer and lipids that self-assemble to form 10-25 nm discs. These tNLP provide a protein-encapsulated lipid support to solubilize and fold membrane proteins. The cell-free system co-translated MOMP and ApoA1 in the presence of telodendrimer mixed with lipids. The MOMP-tNLP complex was amenable to CpG and FSL-1 adjuvant addition. To investigate the ability of MOMP-tNLP+CpG+FSL-1 to induce protection against an intranasal (i.n.) C. muridarum challenge, female mice were vaccinated intramuscularly (i.m.) or i.n. and i.m. simultaneously 4 weeks apart. Following vaccination with MOMP-tNLP+CpG+FSL-1, mice mounted significant humoral and cell-mediated immune responses. Following the i.n. challenge, mice vaccinated with MOMP-tNLP+CpG+FSL-1 i.n. + i.m. group were protected as determined by the percentage change in body weight and by the number of C. muridarum inclusion forming units (IFU) recovered from the lungs. To our knowledge, this is the first time a MOMP-based vaccine formulated in tNLP has been shown to protect against C. muridarum.

A 10-year meta-analysis of membrane protein structural biology: Detergents, membrane mimetics, and structure determination techniques.

Structure determination of membrane proteins is critical to the molecular understanding of many life processes, yet it has historically been a technically challenging endeavor. This past decade has given rise to a number of technological advancements, techniques, and reagents, which have facilitated membrane protein structural biology, resulting in an ever-growing number of membrane protein structures determined. To collate these advances, we have mined available literature to analyze the purification and structure determination specifics for all uniquely solved membrane protein structures from 2010 to 2019. Our analyses demonstrate the strong impact of single-particle cryo-electron microscopy on the field and illustrate how this technique has affected detergent and membrane mimetic usage. Furthermore, we detail how different structure determination methods, taxonomic domains and protein classes have unique detergent/membrane mimetic profiles, highlighting the importance of tailoring their selection. Our analyses provide a quantitative overview of where the field of membrane protein structural biology stands and how it has developed over time. We anticipate that these will serve as a useful tool to streamline future membrane protein structure determination by guiding the choice of detergent/membrane mimetic.

Isolation of intramembrane proteases in membrane-like environments.

Intramembrane proteases (IMPs) are proteolytic enzymes embedded in the lipid bilayer, where they cleave transmembrane substrates. The importance of IMPs relies on their role in a wide variety of cellular processes and diseases. In order to study the activity and function of IMPs, their purified form is often desired. The production of pure and active IMPs has proven to be a challenging task. This process unavoidably requires the use of solubilizing agents that will, to some extent, alter the native environment of these proteases. In this review we present the current solubilization and reconstitution techniques that have been applied to IMPs. In addition, we describe how these techniques had an influence on the activity and structural studies of IMPs, focusing on rhomboid proteases and γ-secretase.

Styrene maleic-acid lipid particles (SMALPs) into detergent or amphipols: An exchange protocol for membrane protein characterisation.

Membrane proteins are traditionally extracted and purified in detergent for biochemical and structural characterisation. This process is often costly and laborious, and the stripping away of potentially stabilising lipids from the membrane protein of interest can have detrimental effects on protein integrity. Recently, styrene-maleic acid (SMA) co-polymers have offered a solution to this problem by extracting membrane proteins directly from their native membrane, while retaining their naturally associated lipids in the form of stable SMA lipid particles (SMALPs). However, the inherent nature and heterogeneity of the polymer renders their use challenging for some downstream applications - particularly mass spectrometry (MS). While advances in cryo-electron microscopy (cryo-EM) have enhanced our understanding of membrane protein:lipid interactions in both SMALPs and detergent, the resolution obtained with this technique is often insufficient to accurately identify closely associated lipids within the transmembrane annulus. Native-MS has the power to fill this knowledge gap, but the SMA polymer itself remains largely incompatible with this technique. To increase sample homogeneity and allow characterisation of membrane protein:lipid complexes by native-MS, we have developed a novel SMA-exchange method; whereby the membrane protein of interest is first solubilised and purified in SMA, then transferred into amphipols or detergents. This allows the membrane protein and endogenously associated lipids extracted by SMA co-polymer to be identified and examined by MS, thereby complementing results obtained by cryo-EM and creating a better understanding of how the lipid bilayer directly affects membrane protein structure and function.

Improved protection against Chlamydia muridarum using the native major outer membrane protein trapped in Resiquimod-carrying amphipols and effects in protection with addition of a Th1 (CpG-1826) and a Th2 (Montanide ISA 720) adjuvant.

A new vaccine formulated with the Chlamydia muridarum native major outer membrane protein (nMOMP) and amphipols was assessed in an intranasal (i.n.) challenge mouse model. nMOMP was trapped either in amphipol A8-35 (nMOMP/A8-35) or in A8-35 conjugated with Resiquimod (nMOMP/Resiq-A8-35), a TLR7/8 agonist added as adjuvant. The effects of free Resiquimod and/or additional adjuvants, Montanide ISA 720 (TLR independent) and CpG-1826 (TLR9 agonist), were also evaluated. Immunization with nMOMP/A8-35 alone administered i.n. was used as negative adjuvant-control group, whereas immunizations with C. muridarum elementary bodies (EBs) and MEM buffer, administered i.n., were used as positive and negative controls, respectively. Vaccinated mice were challenged i.n. with C. muridarum and changes in body weight, lungs weight and recovery of Chlamydia from the lungs were evaluated. All the experimental groups showed protection when compared with the negative control group. Resiquimod alone produced weak humoral and cellular immune responses, but both Montanide and CpG-1826 showed significant increases in both responses. The addition of CpG-1826 alone switched immune responses to be Th1-biased. The most robust protection was elicited in mice immunized with the three adjuvants and conjugated Resiquimod. Increased protection induced by the Resiquimod covalently linked to A8-35, in the presence of Montanide and CpG-1826 was established based on a set of parameters: (1) the ability of the antibodies to neutralize C. muridarum; (2) the increased proliferation of T-cells in vitro accompanied by higher production of IFN-γ, IL-6 and IL-17; (3) the decreased body weight loss over the 10 days after challenge; and (4) the number of IFUs recovered from the lungs at day 10 post challenge. In conclusion, a vaccine formulated with the C. muridarum nMOMP bound to amphipols conjugated with Resiquimod enhances protective immune responses that can be further improved by the addition of Montanide and CpG-1826.

BAmSA: Visualising transmembrane regions in protein complexes using biotinylated amphipols and electron microscopy.

Membrane protein (MP) complexes play key roles in all living cells. Their structural characterisation is hampered by difficulties in purifying and crystallising them. Recent progress in electron microscopy (EM) have revolutionised the field, not only by providing higher-resolution structures for previously characterised MPs but also by yielding first glimpses into the structure of larger and more challenging complexes, such as bacterial secretion systems. However, the resolution of pioneering EM structures may be difficult and their interpretation requires clues regarding the overall organisation of the complexes. In this context, we present BAmSA, a new method for localising transmembrane (TM) regions in MP complexes, using a general procedure that allows tagging them without resorting to neither genetic nor chemical modification. Labels bound to TM regions can be visualised directly on raw negative-stain EM images, on class averages, or on three-dimensional reconstructions, providing a novel strategy to explore the organisation of MP complexes.

Stabilization of a Membrane-Associated Amyloid-ß Oligomer for Its Validation in Alzheimer's Disease.

We have recently reported on the preparation of a membrane-associated ß-barrel Pore-Forming Aß42 Oligomer (ßPFOAß42). It corresponds to a stable and homogeneous Aß42 oligomer that inserts into lipid bilayers as a well-defined pore and adopts a specific structure with characteristics of a ß-barrel arrangement. As a follow-up of this work, we aim to establish ßPFOAß42's relevance in Alzheimer's disease (AD). However, ßPFOAß42 is formed under dodecyl phosphocholine (DPC) micelle conditions-intended to mimic the hydrophobic environment of membranes-which are dynamic. Consequently, dilution of the ßPFOAß42/DPC complex in a detergent-free buffer leads to dispersion of the DPC molecules from the oligomer surface, leaving the oligomer without the hydrophobic micelle belt that stabilizes it. Since dilution is required for any biological test, transfer of ßPFOAß42 from DPC micelles into another hydrophobic biomimetic membrane environment, that remains associated with ßPFOAß42 even under high dilution conditions, is a requisite for the validation of ßPFOAß42 in AD. Here we describe conditions for exchanging DPC micelles with amphipols (APols), which are amphipathic polymers designed to stabilize membrane proteins in aqueous solutions. APols bind in an irreversible but non-covalent manner to the hydrophobic surface of membrane proteins preserving their structure even under extreme dilution conditions. We tested three types of APols with distinct physical-chemical properties and found that the ßPFOAß42/DPC complex can only be trapped in non-ionic APols (NAPols). The characterization of the resulting ßPFOAß42/NAPol complex by biochemical tools and structural biology techniques allowed us to establish that the oligomer structure is maintained even under high dilution. Based on these findings, this work constitutes a first step towards the in vivo validation of ßPFOAß42 in AD.

Co-delivery of amphipol-conjugated adjuvant with antigen, and adjuvant combinations, enhance immune protection elicited by a membrane protein-based vaccine against a mucosal challenge with Chlamydia.

INTRODUCTION: Chlamydial infections are spread worldwide and a vaccine is needed to control this pathogen. The goals of this study were to determine if the delivery of an adjuvant associated to the antigen, via a derivatized amphipol, and adjuvant combinations improve vaccine protection. METHODS: A novel approach, trapping the Chlamydia muridarum (Cm) native MOMP (nMOMP) with amphipols (A8-35), bearing a covalently conjugated peptide (EP67), was used. Adjuvants incorporated were: EP67 either conjugated to A8-35, which was used to trap nMOMP (nMOMP/EP67-A8-35), or free as a control, added to nMOMP/A8-35 complexes (nMOMP/A8-35+EP67); Montanide ISA 720 to enhance humoral responses, and CpG-1826 to elicit robust cell-mediated immunity (CMI). BALB/c mice were immunized by mucosal and systemic routes. Intranasal immunization with live Cm was used as positive control and three negative controls were included. Mice were challenged intranasally with Cm and changes in body weight, lungs weight and number of Cm-inclusion forming units (IFU) recovered from the lungs were evaluated to establish protection. To assess local responses levels of IFN- γ and Cm-specific IgA were determined in lungs' supernatants. RESULTS: Structural assays demonstrated that nMOMP secondary structure and thermal stability were maintained when A8-35 was covalently modified. Mice vaccinated with nMOMP/EP67-A8-35 were better protected than animals immunized with nMOMP/A8-35+EP67. Addition of Montanide enhanced Th2 responses and improved protection. Including CpG-1826 further broadened, intensified and switched to Th1-biased immune responses. With delivery of nMOMP and the three adjuvants, as determined by changes in body weight, lungs weight and number of IFU recovered from lungs, protection at 10 days post-challenge was equivalent to that induced by immunization with live Cm. CONCLUSIONS: Covalent association of EP67 to A8-35, used to keep nMOMP water-soluble, improves protection over that conferred by free EP67. Adjuvant combinations including EP67+Montanide+CpG-1826, by broadening and intensifying cellular and humoral immune responses, further enhanced protection.

FPOP-LC-MS/MS Suggests Differences in Interaction Sites of Amphipols and Detergents with Outer Membrane Proteins.

Amphipols are a class of novel surfactants that are capable of stabilizing the native state of membrane proteins. They have been shown to be highly effective, in some cases more so than detergent micelles, at maintaining the structural integrity of membrane proteins in solution, and have shown promise as vehicles for delivering native membrane proteins into the gas phase for structural interrogation. Here, we use fast photochemical oxidation of proteins (FPOP), which irreversibly labels the side chains of solvent-accessible residues with hydroxyl radicals generated by laser photolysis of hydrogen peroxide, to compare the solvent accessibility of the outer membrane protein OmpT when solubilized with the amphipol A8-35 or with n-dodecyl-ß-maltoside (DDM) detergent micelles. Using quantitative mass spectrometry analyses, we show that fast photochemical oxidation reveals differences in the extent of solvent accessibility of residues between the A8-35 and DDM solubilized states, providing a rationale for the increased stability of membrane proteins solubilized with amphipol compared with detergent micelles, as a result of additional intermolecular contacts. Graphical Abstract ᅟ.