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.

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.

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.

Folding and stabilizing membrane proteins in amphipol A8-35.

Membrane proteins (MPs) are important pharmacological targets because of their involvement in many essential cellular processes whose dysfunction can lead to a large variety of diseases. A detailed knowledge of the structure of MPs and the molecular mechanisms of their activity is essential to the design of new therapeutic agents. However, studying MPs in vitro is challenging, because it generally implies their overexpression under a functional form, followed by their extraction from membranes and purification. Targeting an overexpressed MP to a membrane is often toxic and expression yields tend to be limited. One alternative is the formation of inclusion bodies (IBs) in the cytosol of the cell, from which MPs need then to be folded to their native conformation before structural and functional analysis can be contemplated. Folding MPs targeted to IBs is a difficult task. Specially designed amphipathic polymers called 'amphipols' (APols), which have been initially developed with the view of improving the stability of MPs in aqueous solutions compared to detergents, can be used to fold both α-helical and ß-barrel MPs. APols represent an interesting novel amphipathic medium, in which high folding yields can be achieved. In this review, the properties of APol A8-35 and of the complexes they form with MPs are summarized. An overview of the most important studies reported so far using A8-35 to fold MPs is presented. Finally, from a practical point of view, a detailed description of the folding and trapping methods is given.

Membranes Do Not Tell Proteins How To Fold.

Which properties of the membrane environment are essential for the folding and oligomerization of transmembrane proteins? Because the lipids that surround membrane proteins in situ spontaneously organize into bilayers, it may seem intuitive that interactions with the bilayer provide both hydrophobic and topological constraints that help the protein to achieve a stable and functional three-dimensional structure. However, one may wonder whether folding is actually driven by the membrane environment or whether the folded state just reflects an adaptation of integral proteins to the medium in which they function. Also, apart from the overall transmembrane orientation, might the asymmetry inherent in biosynthesis processes cause proteins to fold to out-of-equilibrium, metastable topologies? Which of the features of a bilayer are essential for membrane protein folding, and which are not? To which extent do translocons dictate transmembrane topologies? Recent data show that many membrane proteins fold and oligomerize very efficiently in media that bear little similarity to a membrane, casting doubt on the essentiality of many bilayer constraints. In the following discussion, we argue that some of the features of bilayers may contribute to protein folding, stability and regulation, but they are not required for the basic three-dimensional structure to be achieved. This idea, if correct, would imply that evolution has steered membrane proteins toward an accommodation to biosynthetic pathways and a good fit into their environment, but that their folding is not driven by the latter or dictated by insertion apparatuses. In other words, the three-dimensional structure of membrane proteins is essentially determined by intramolecular interactions and not by bilayer constraints and insertion pathways. Implications are discussed.

Synthesis of a Polyhistidine-bearing Amphipol and its Use for Immobilizing Membrane Proteins.

Amphipols (APols) are short amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions. In the present study, A8-35, a polyacrylate-based APol, was grafted with hexahistidine tags (His6-tags). The synthesis and characterization of this novel functionalized APol, named HistAPol, are described. Its ability to immobilize MPs on nickel ion-bearing surfaces was tested using two complementary methods, immobilized metal affinity chromatography (IMAC) and surface plasmon resonance (SPR). Compared to a single His6-tag fused at one extremity of a MP, the presence of several His6-tags carried by the APol belt surrounding the transmembrane domain of a MP increases remarkably the affinity of the protein/APol complex for nickel ion-bearing SPR chips, whereas it does not show such a strong effect on an IMAC resin. HistAPol-mediated immobilization, which allows reversibility of the interaction and easy regeneration of the supports and dispenses with any genetic modification of the target protein, provides a novel, promising tool for attaching MPs onto solid supports while stabilizing them.

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.

Thermal fluctuations in amphipol A8-35 particles: a neutron scattering and molecular dynamics study.

Amphipols are a class of polymeric surfactants that can stabilize membrane proteins in aqueous solutions as compared to detergents. A8-35, the best-characterized amphipol to date, is composed of a polyacrylate backbone with ~35% of the carboxylates free, ~25% grafted with octyl side-chains, and ~40% with isopropyl ones. In aqueous solutions, A8-35 self-organizes into globular particles with a molecular mass of ~40 kDa. The thermal dynamics of A8-35 particles was measured by neutron scattering in the 10-picosecond, 18-picosecond, and 1-nanosecond time-scales on natural abundance and deuterium-labeled molecules, which permitted to separate backbone and side-chain motions. A parallel analysis was performed on molecular dynamics trajectories (Perlmutter et al., Langmuir 27:10523-10537, 2011). Experimental results and simulations converge, from their respective time-scales, to show that A8-35 particles feature a more fluid hydrophobic core, predominantly containing the octyl chains, and a more rigid solvent-exposed surface, made up predominantly of the hydrophilic polymer backbone. The fluidity of the core is comparable to that of the lipid environment around proteins in the center of biological membranes, as also measured by neutron scattering. The biological activity of proteins depends sensitively on molecular dynamics, which itself is strongly dependent on the immediate macromolecular environment. In this context, the characterization of A8-35 particle dynamics constitutes a step toward understanding the effect of amphipols on membrane protein stability and function.

Amphipols and photosynthetic light-harvesting pigment-protein complexes.

The trimeric light-harvesting complexes II (LHCII) of plants and green algae are pigment-protein complexes involved in light harvesting and photoprotection. Different conformational states have been proposed to be responsible for their different functions. At present, detergent-solubilized LHCII is used as a model for the "light-harvesting conformation", whereas the "quenched conformation" is mimicked by LHCII aggregates. However, none of these conditions seem to perfectly reproduce the properties of LHCII in vivo. In addition, several monomeric LHC complexes are not fully stable in detergent. There is thus a need to find conditions that allow analyzing LHCs in vitro in stable and, hopefully, more native-like conformations. Here, we report a study of LHCII, the major antenna complex of plants, in complex with amphipols. We have trapped trimeric LHCII and monomeric Lhcb1 with either polyanionic or non-ionic amphipols and studied the effect of these polymers on the properties of the complexes. We show that, as compared to detergent solutions, amphipols have a stabilizing effect on LHCII. We also show that the average fluorescence lifetime of LHCII trapped in an anionic amphipol is ~30% shorter than in α-dodecylmaltoside, due to the presence of a conformation with 230-ps lifetime that is not present in detergent solutions.

Amphipol-mediated screening of molecular orthoses specific for membrane protein targets.

Specific, tight-binding protein partners are valuable helpers to facilitate membrane protein (MP) crystallization, because they can i) stabilize the protein, ii) reduce its conformational heterogeneity, and iii) increase the polar surface from which well-ordered crystals can grow. The design and production of a new family of synthetic scaffolds (dubbed αReps, for "artificial alpha repeat protein") have been recently described. The stabilization and immobilization of MPs in a functional state are an absolute prerequisite for the screening of binders that recognize specifically their native conformation. We present here a general procedure for the selection of αReps specific of any MP. It relies on the use of biotinylated amphipols, which act as a universal "Velcro" to stabilize, and immobilize MP targets onto streptavidin-coated solid supports, thus doing away with the need to tag the protein itself.

Folding membrane proteins in vitro: a table and some comments.

Thirty-three years have elapsed since the first membrane protein (MP) was brought back in vitro to its native state starting from the completely unfolded polypeptide. Folding MPs is as useful from a practical point of view as it is thought-provoking from a theoretical one. Yet, this activity is considered as a high-risk, time-consuming endeavor, full of pitfalls, its path littered with the broken careers of graduate students sacrificed on the altar of a long shot that never paid off. In fact, a surprisingly high number of MPs have actually been folded or refolded in vitro. Analysis of the literature indicates (i) that the endeavor is not as desperate as it may seem, (ii) that techniques are diversifying and improving, and (iii) that many MPs do not need the cellular biosynthetic apparatus, nor even a membrane environment, to reach a functional 3D structure. A compilation, hopefully close to complete, is presented of MPs that have been (re)folded in vitro to-date, with the conditions of their synthesis, the denaturant(s) used, if any, and the (re)folding conditions, along with a few comments. The hope is that this analysis will encourage membrane protein biochemists to consider producing their target proteins in this way, help them decide about an experimental course, and stimulate the reflection about which environments favor membrane protein folding and why.

Isolation of Escherichia coli mannitol permease, EIImtl, trapped in amphipol A8-35 and fluorescein-labeled A8-35.

Amphipols (APols) are short amphipathic polymers that keep integral membrane proteins water-soluble while stabilizing them as compared to detergent solutions. In the present work, we have carried out functional and structural studies of a membrane transporter that had not been characterized in APol-trapped form yet, namely EII(mtl), a dimeric mannitol permease from the inner membrane of Escherichia coli. A tryptophan-less and dozens of single-tryptophan (Trp) mutants of this transporter are available, making it possible to study the environment of specific locations in the protein. With few exceptions, the single-Trp mutants show a high mannitol-phosphorylation activity when in membranes, but, as variance with wild-type EII(mtl), some of them lose most of their activity upon solubilization by neutral (PEG- or maltoside-based) detergents. Here, we present a protocol to isolate these detergent-sensitive mutants in active form using APol A8-35. Trapping with A8-35 keeps EII(mtl) soluble and functional in the absence of detergent. The specific phosphorylation activity of an APol-trapped Trp-less EII(mtl) mutant was found to be ~3× higher than the activity of the same protein in dodecylmaltoside. The preparations are suitable both for functional and for fluorescence spectroscopy studies. A fluorescein-labeled version of A8-35 has been synthesized and characterized. Exploratory studies were conducted to examine the environment of specific Trp locations in the transmembrane domain of EII(mtl) using Trp fluorescence quenching by water-soluble quenchers and by the fluorescein-labeled APol. This approach has the potential to provide information on the transmembrane topology of MPs.

Long-term stability of a vaccine formulated with the amphipol-trapped major outer membrane protein from Chlamydia trachomatis.

Chlamydia trachomatis is a major bacterial pathogen throughout the world. Although antibiotic therapy can be implemented in the case of early detection, a majority of the infections are asymptomatic, requiring the development of preventive measures. Efforts have focused on the production of a vaccine using the C. trachomatis major outer membrane protein (MOMP). MOMP is purified in its native (n) trimeric form using the zwitterionic detergent Z3-14, but its stability in detergent solutions is limited. Amphipols (APols) are synthetic polymers that can stabilize membrane proteins (MPs) in detergent-free aqueous solutions. Preservation of protein structure and optimization of exposure of the most effective antigenic regions can avoid vaccination with misfolded, poorly protective protein. Previously, we showed that APols maintain nMOMP secondary structure and that nMOMP/APol vaccine formulations elicit better protection than formulations using either recombinant or nMOMP solubilized in Z3-14. To achieve a greater understanding of the structural behavior and stability of nMOMP in APols, we have used several spectroscopic techniques to characterize its secondary structure (circular dichroism), tertiary and quaternary structures (immunochemistry and gel electrophoresis) and aggregation state (light scattering) as a function of temperature and time. We have also recorded NMR spectra of (15)N-labeled nMOMP and find that the exposed loops are detectable in APols but not in detergent. Our analyses show that APols protect nMOMP much better than Z3-14 against denaturation due to continuous heating, repeated freeze/thaw cycles, or extended storage at room temperature. These results indicate that APols can help improve MP-based vaccine formulations.

Solution behavior and crystallization of cytochrome bc1 in the presence of amphipols.

Detergents classically are used to keep membrane proteins soluble in aqueous solutions, but they tend to destabilize them. This problem can be largely alleviated thanks to the use of amphipols (APols), small amphipathic polymers designed to substitute for detergents. APols adsorb at the surface of the transmembrane region of membrane proteins, keeping them water-soluble while stabilizing them bio-chemically. Membrane protein/APol complexes have proven, however, difficult to crystallize. In this study, the composition and solution properties of complexes formed between mitochondrial cytochrome bc1 and A8-35, the most extensively used APol to date, have been studied by means of size exclusion chromatography, sucrose gradient sedimentation, and small-angle neutron scattering. Stable, monodisperse preparations of bc1/A8-35 complexes can be obtained, which, depending on the medium, undergo either repulsive or attractive interactions. Under crystallization conditions, diffracting three-dimensional crystals of A8-35-stabilized cytochrome bc1 formed, but only in the concomitant presence of APol and detergent.

Amphipols for each season.

Amphipols (APols) are short amphipathic polymers that can substitute for detergents at the transmembrane surface of membrane proteins (MPs) and, thereby, keep them soluble in detergent free aqueous solutions. APol-trapped MPs are, as a rule, more stable biochemically than their detergent-solubilized counterparts. APols have proven useful to produce MPs, most noticeably by assisting their folding from the denatured state obtained after solubilizing MP inclusion bodies in either SDS or urea. They facilitate the handling in aqueous solution of fragile MPs for the purpose of proteomics, structural and functional studies, and therapeutics. Because APols can be chemically labeled or functionalized, and they form very stable complexes with MPs, they can also be used to functionalize those indirectly, which opens onto many novel applications. Following a brief recall of the properties of APols and MP/APol complexes, an update is provided of recent progress in these various fields.

Molecular dynamics simulations of a membrane protein/amphipol complex.

Amphipathic polymers known as "amphipols" provide a highly stabilizing environment for handling membrane proteins in aqueous solutions. A8-35, an amphipol with a polyacrylate backbone and hydrophobic grafts, has been extensively characterized and widely employed for structural and functional studies of membrane proteins using biochemical and biophysical approaches. Given the sensitivity of membrane proteins to their environment, it is important to examine what effects amphipols may have on the structure and dynamics of the proteins they complex. Here we present the first molecular dynamics study of an amphipol-stabilized membrane protein, using Escherichia coli OmpX as a model. We begin by describing the structure of the complexes formed by supplementing OmpX with increasing amounts of A8-35, in order to determine how the amphipol interacts with the transmembrane and extramembrane surfaces of the protein. We then compare the dynamics of the protein in either A8-35, a detergent, or a lipid bilayer. We find that protein dynamics on all accessible length scales is restrained by A8-35, which provides a basis to understanding some of the stabilizing and functional effects of amphipols that have been experimentally observed.

Amphipol-trapped ExbB-ExbD membrane protein complex from Escherichia coli: a biochemical and structural case study.

Nutrient import across Gram-negative bacteria's outer membrane is powered by the proton-motive force, delivered by the cytoplasmic membrane protein complex ExbB-ExbD-TonB. Having purified the ExbB4-ExbD2 complex in the detergent dodecyl maltoside, we substituted amphipol A8-35 for detergent, forming a water-soluble membrane protein/amphipol complex. Properties of the ExbB4-ExbD2 complex in detergent or in amphipols were compared by gel electrophoresis, size exclusion chromatography, asymmetric flow field-flow fractionation, thermal stability assays, and electron microscopy. Bound detergent and fluorescently labeled amphipol were assayed quantitatively by 1D NMR and analytical ultracentrifugation, respectively. The structural arrangement of ExbB4-ExbD2 was examined by EM, small-angle X-ray scattering, and small-angle neutron scattering using a deuterated amphipol. The amphipol-trapped ExbB4-ExbD2 complex is slightly larger than its detergent-solubilized counterpart. We also investigated a different oligomeric form of the two proteins, ExbB6-ExbD4, and propose a structural arrangement of its transmembrane α-helical domains.

Synthesis of an oligonucleotide-derivatized amphipol and its use to trap and immobilize membrane proteins.

Amphipols (APols) are specially designed amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions in the absence of detergent. A8-35, a polyacrylate-based APol, has been grafted with an oligodeoxynucleotide (ODN). The synthesis, purification and properties of the resulting 'OligAPol' have been investigated. Grafting was performed by reacting an ODN carrying an amine-terminated arm with the carboxylates of A8-35. The use of OligAPol for trapping MPs and immobilizing them onto solid supports was tested using bacteriorhodopsin (BR) and the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) as model proteins. BR and OligAPol form water-soluble complexes in which BR remains in its native conformation. Hybridization of the ODN arm with a complementary ODN was not hindered by the assembly of OligAPol into particles, nor by its association with BR. BR/OligAPol and tOmpA/OligAPol complexes could be immobilized onto either magnetic beads or gold nanoparticles grafted with the complementary ODN, as shown by spectroscopic measurements, fluorescence microscopy and the binding of anti-BR and anti-tOmpA antibodies. OligAPols provide a novel, highly versatile approach to tagging MPs, without modifying them chemically nor genetically, for specific, reversible and targetable immobilization, e.g. for nanoscale applications.

Increased immunoaccessibility of MOMP epitopes in a vaccine formulated with amphipols may account for the very robust protection elicited against a vaginal challenge with Chlamydia muridarum.

There is a need to implement a vaccine to protect against Chlamydia trachomatis infections. To test a new vaccine, mice were immunized with the Chlamydia muridarum native major outer membrane protein (nMOMP) solubilized with either amphipol A8-35 or the detergent Z3-14. OVA was used as a negative control, and mice were inoculated intranasally with C. muridarum as positive controls. Animals vaccinated with nMOMP mounted strong Chlamydia-specific humoral and cell-mediated immune responses. Mice vaccinated with nMOMP/A8-35 had a higher ratio of Abs to denatured elementary bodies (EB) over live EB, recognized more synthetic MOMP peptides and had higher neutralizing titers than sera from mice immunized with nMOMP/Z3-14. T cell lymphoproliferative responses and levels of IFN-γ were also higher in mice vaccinated with nMOMP/A8-35 than with nMOMP/Z3-14. Following immunization, animals were challenged intravaginally with C. muridarum. On the basis of the number of mice with positive vaginal cultures, length of vaginal shedding, total number of positive vaginal cultures, and number of Chlamydia inclusion forming units recovered, nMOMP/A8-35 elicited a more robust protection than nMOMP/Z3-14. By depleting T cells with Abs, we determined that CD4(+) and not CD8(+) T cells mediated the protection elicited by nMOMP/A8-35. Mice were subsequently mated, and based on the number of pregnant mice and number of embryos, animals that were vaccinated with nMOMP/A8-35 or nMOMP/Z3-14 had fertility rates equivalent to the positive control group immunized with live EB and the fertility controls. In conclusion, increased accessibility of epitopes in the nMOMP/A8-35 preparation may account for the very robust protection against infection and disease elicited by this vaccine.