Fabrice Giusti

The use of amphipols as universal molecular adapters to immobilize membrane proteins onto solid supports

Because of the importance of their physiological functions, cell membranes represent critical targets in biological research. Membrane proteins, which make up ≈1/3 of the proteome, interact with a wide range of small ligands and macromolecular partners as well as with foreign molecules such as synthetic drugs, antibodies, toxins, or surface recognition proteins of pathogenic organisms. Whether it is for the sake of basic biomedical or pharmacological research, it is of great interest to develop tools facilitating the study of these interactions. Surface-based in vitro assays are appealing because they require minimum quantities of reagents, and they are suitable for multiplexing and high-throughput screening. We introduce here a general method for immobilizing functional, unmodified integral membrane proteins onto solid supports, thanks to amphipathic polymers called “amphipols.” The key point of this approach is that functionalized amphipols can be used as universal adapters to associate any membrane protein to virtually any kind of support while stabilizing its native state. The generality and versatility of this strategy is demonstrated by using 5 different target proteins, 2 types of supports (chips and beads), 2 types of ligands (antibodies and a snake toxin), and 2 detection methods (surface plasmon resonance and fluorescence microscopy).

The ci/bH moiety in the b6f complex studied by EPR: a pair of strongly interacting hemes.

X-band EPR features in the region of 90–150 mT have previously been attributed to heme ci of the b6 complex [Zhang H, Primak A, Bowman MK, Kramer DM, Cramer WA (2004) Biochemistry 43:16329–16336] and interpreted as arising from a high-spin species. However, the complexity of the observed spectrum is rather untypical for high-spin hemes. In this work, we show that addition of the inhibitor 2-n-nonyl-4-hydroxyquinoline N-oxide largely simplifies heme cis EPR properties. The spectrum in the presence of 2-n-nonyl-4-hydroxyquinoline N-oxide is demonstrated to be caused by a simple S = 5/2, rhombic species split by magnetic dipolar interaction (Axx = 7.5 mT) with neighboring heme bH. The large spacing of lines in the uninhibited system, by contrast, cannot be rationalized solely on the basis of magnetic dipolar coupling but is likely to encompass strong contributions from exchange interactions. The role of the H2O/OH− molecule bridging heme cis Fe atom and heme bHs propionate side chain in mediating these interactions is discussed.

Nonionic amphiphilic polymers derived from Tris(hydroxymethyl)-acrylamidomethane keep membrane proteins soluble and native in the absence of detergent.

A new family of amphipols-amphiphilic polymers designed to form water-soluble complexes with membrane proteins-was synthesized by free-radical telomerization of Tris(hydroxymethyl)-acrylamidomethane (THAM) and derivatized THAM. Some of these polymers were found to prevent aggregation and denaturation of two model membrane proteins, bacteriorhodopsin and cytochrome b(6) f, in the absence of detergent micelles.

Trapping and stabilization of integral membrane proteins by hydrophobically grafted glucose-based telomers.

Amphipols (APols) are short amphipathic polymers designed to adsorb onto the transmembrane surface of membrane proteins, keeping them water-soluble in the absence of detergent. Current APols carry charged groups, which is a limitation for certain types of applications. This has prompted the development of totally nonionic amphiphols (NAPols). In a previous work, glucose-based NAPols synthesized by free-radical cotelomerization of hydrophilic and amphiphilic monomers proved to be able to keep membrane proteins soluble (Sharma et al. Langmuir 2008, 24, 13581-13590). This provided a proof of principle, but the cumbersome synthesis prevented large-scale production and any detailed biochemical studies. In the present work, we describe a new synthesis route for NAPols based on grafting alkyl chains onto a glucosylated homotelomer. The NAPols thus prepared are highly water soluble. In aqueous solutions, they assemble into small, homogeneous particles similar to those formed by ionic APols. Two model membrane proteins, bacteriorhodopsin and the transmembrane domain of OmpA, form with NAPols small, well-defined water-soluble complexes whose size is comparable to that observed with ionic APols. Complexation by NAPols strongly stabilizes bacteriorhodopsin against denaturation. Glucosylated NAPols thus appear as a promising alternative to ionic APols for such applications as ion-exchange chromatography, isoelectrofocusing, and, possibly, structural approaches such as NMR and crystallography.

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.

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.

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.

Rapid transmembrane diffusion of ceramide and dihydroceramide spin-labelled analogues in the liquid ordered phase

In order to study the basic physical phenomena underlying complex lipid transbilayer movement in biological membranes, we have measured the transmembrane diffusion of spin-labelled analogues of sphingolipids in phosphatidylcholine (PC) large unilamellar vesicles in the absence or presence of cholesterol, going from a fluid ( liquid disordered) ld, phase to a more viscous, liquid ordered (lo), phase. We have found cholesterol to reduce the transverse diffusion of glucosylceramide (GlcCer) and galactosylceramide (GalCer) in a concentration-dependent manner. However, surprisingly, we could neither detect any influence of cholesterol on the rapid flip-flop of ceramide nor on the flip-flop of dihydroceramide, for which the τ1/2 of flip-flop remains in the order of 1 minute at 20°C in the presence of cholesterol. As a consequence of rapid flip-flop of ceramide in both the lo and the ld phase, ceramide is likely to distribute between the two monolayers of a membrane, and could in principle partition into segregated domains in each side of the plasma membrane of eukaryotic cells.

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 EIImtl, 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 EIImtl, 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 EIImtl soluble and functional in the absence of detergent. The specific phosphorylation activity of an APol-trapped Trp-less EIImtl 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 EIImtl 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.

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

We have recently reported on the preparation of a membrane-associated β-barrel Pore-Forming Aβ42 Oligomer (βPFOAβ42). It corresponds to a stable and homogeneous Aβ42 oligomer that inserts into lipid bilayers as a well-defined pore and adopts a specific structure with characteristics of a β-barrel arrangement. As a follow-up of this work, we aim to establish βPFOAβ42s relevance in Alzheimers disease (AD). However, βPFOAβ42 is formed under dodecyl phosphocholine (DPC) micelle conditions—intended to mimic the hydrophobic environment of membranes—which are dynamic. Consequently, dilution of the βPFOAβ42/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 βPFOAβ42 from DPC micelles into another hydrophobic biomimetic membrane environment, that remains associated with βPFOAβ42 even under high dilution conditions, is a requisite for the validation of βPFOAβ42 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 βPFOAβ42/DPC complex can only be trapped in non-ionic APols (NAPols). The characterization of the resulting βPFOAβ42/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 βPFOAβ42 in AD.