Amphipathic Polymers: Tools To Fold Integral Membrane Proteins to Their Active Form

Pocanschi, Cosmin L.; Dahmane, Tassadite; Gohon, Yann; Rappaport, Fabrice; Apell, Hans-Jürgen; Kleinschmidt, Jörg H.; Popot, Jean‐Luc

doi:10.1021/bi0616706

Cited by 94 publications

(122 citation statements)

References 37 publications

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“…The resulting preparations therefore comprised a mixture of MP/ BA8-35 complexes and free BA8-35 particles. We have shown before that none of these proteins is denatured upon being trapped with APol A8-35 (17)(18)(19)(20)25). Upon flushing SA-coated chips with these preparations, an SPR signal developed, a large fraction of which resisted washing with buffer (Fig.…”

Section: Synthesis Of Biotinylatedmentioning

confidence: 85%

“…Finally, it is worth recalling that APols have proven to be an excellent medium for folding denatured MPs to their native state (ref. 25; T. Dahmane, M. Damian, S. Mary, J.-L.P., and J.-L. Banères, unpublished results), which ought to facilitate functional and structural studies of MPs overexpressed as inclusion bodies: folding can be achieved by using a functionalized APol, so that the folded protein can be immobilized thanks to the very polymer that has assisted its folding.…”

Section: Discussionmentioning

confidence: 99%

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

Charvolin

Perez

Rouvière

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Because of the importance of their physiological functions, cell membranes represent critical targets in biological research. Membrane proteins, which make up Ϸ1/3 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).diagnostics ͉ drug discovery ͉ immobilization ͉ chips bioreactors

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Section: Synthesis Of Biotinylatedmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

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

Charvolin

Perez

Rouvière

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…La dernière classe de surfactants que nous mentionnerons ici est celle des amphipols [23,40]. Les amphipols sont des polymères amphiphiles, de longueur et de composition bien définies, qui forment un complexe stable avec la protéine membranaire et la maintient ainsi en solution sans la dénaturer (Figure 1).…”

Section: Stabilisation Par Les Amphipolsunclassified

Manipulation des récepteurs couplés aux protéines G

Banères

Mouillac

2012

Med Sci (Paris)

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> Parmi les différentes classes de protéines membranaires, les récepteurs couplés aux protéines G (RCPG) représentent une famille majeure. Ils sont impliqués dans la plupart des grandes fonctions physiologiques et jouent un rôle primordial dans la communication intercellulaire et la réception des signaux sensoriels. Ils constituent la cible de 30 % des médicaments actuellement sur le marché pharmaceutique. Pour comprendre le fonctionnement de ces récepteurs, aborder leur structure tridimensionnelle et déve-lopper des médicaments sélectifs et efficaces, il est nécessaire de purifier ces protéines afin de bien les caractériser. Cependant, cette étape n'est pas triviale, car les RCPG sont présents en très petite quantité dans la membrane plasmique, et leur environnement lipidique naturel suppose une extraction et une manipulation à l'aide de détergents. Il n'existe pas d'approche universelle aujourd'hui qui permette la production, la purification et la stabilisation des RCPG. Chacun de ces récepteurs possède des caractéristiques physicochimiques uniques, une préférence particulière pour certains détergents lors de leur solubilisation et des conditions spécifiques pour leur purification. Ces dernières années, de grandes avancées en termes de surexpression, de purification et surtout de stabilisation des RCPG, en particulier grâce aux amphipols et aux nanodisques, ont ouvert des perspectives très encourageantes pour l'étude structurale et l'analyse dynamique de ces protéines membranaires. Ces différents aspects de la manipulation des RCPG sont développés dans cette revue. < avec des analyses biochimiques et structurales. Leur surexpression est donc une condition préalable obligatoire si l'on veut aborder leur structure moléculaire, leur dynamique au cours de la liaison de ligands (drogues, médicaments) ou l'étude de leurs interactions avec des partenaires de signalisation tels que les protéines G ou les arrestines. Le développement de systèmes d'expression hétérologues capables de produire des protéines recombinantes fonctionnelles avec des rendements élevés constitue donc une étape essentielle. Une large palette de systèmes cellulaires adaptés à la production des RCPG est aujourd'hui disponible (voir quelques exemples représentatifs dans le Tableau I). Expression et production bactérienne chez E. coliLa bactérie Escherichia coli est le système de production de protéines membranaires le plus commun. Il est simple, peu coûteux, le temps de génération du bacille est court, il peut être manipulé sans danger, et les volumes de culture peuvent être aisément augmentés. De plus, les rendements d'expression sont élevés et la protéine recombinante est structuralement homogène (pas de modifications post-traductionnelles chez E. coli). Les RCPG sont ciblés soit à la membrane interne, soit en corps d'inclusion cytoplasmiques, dans les deux cas sous forme de protéines de fusion par un couplage à un partenaire ou un épitope court. Les corps d'inclusion représentent une alternative intéressante, mais la protéine recombinante doit ê...

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“…Specifically, amphipol A8-35 (Fig. 6A) serves as a novel environment that allows both b-barrel and a-helical membrane proteins to fold to their native state, in the absence of detergents or lipids, and to protect them against trypsin digestion as well as denaturation by urea [65,66].…”

Section: New Solubilizing Agents For Membrane Proteinsmentioning

confidence: 99%

Stabilization of Membrane Proteins: the Case of G‐Protein‐Coupled Receptors

Reyes‐Alcaraz

Tzanov

Garriga

2008

Engineering in Life Sciences

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G-protein-coupled receptors are integral membrane proteins which constitute the largest family of signal transduction molecules participating in the majority of normal physiological processes. G-protein-coupled receptors are responsible for the control of enzyme activity, ion channels and vesicle transport, and they respond to a wide variety of stimuli, like signals involved in sensory systems such as vision, taste and olfaction, but also to a diverse set of chemical signals such as lipids, hormones, neurotransmitters, amino acids, nucleotides, peptides and proteins. This family of receptors is being widely studied because of its potential use as pharmacological targets in drug development, and recently also for its potential use in the development of novel biosensors. G-protein-coupled receptors are specifically designed to fold and function in a lipid bilayer environment, where these membrane proteins are remarkably stable and achieve their optimal performance. The currently used technology for the purification of G-protein-coupled receptors consists in their extraction from the cell membrane and solubilization into detergent micelles. A common drawback of this strategy is that G-proteincoupled receptors solubilized in typical detergents show rather poor conformational stability, which may result in relatively rapid inactivation. The poor stability of detergent-solubilized samples renders many membrane proteins biochemically intractable. This precludes the determination of a high-resolution structure and imposes severe limitations for the development of applications. Thus, the enhancement of the stability of G-protein-coupled receptors is a major issue in order to facilitate structural determination and to unravel their potential in biotechnological applications. This work provides a brief overview of some current advances in the experimental methods for stabilizing G-protein-coupled receptors that can also be extended to other types of membrane proteins. Structure of G-protein-Coupled Receptors (GPCRs)The distinctive structural feature of GPCRs is the bundle of seven transmembrane (7TM) a-helices that constitutes a relevant signature in structural biology. This conformational architecture, and its functional implications, is central to pharmacology and therapeutics. Nearly 2000 GPCRs have been reported since bovine opsin was cloned in 1983 and the b-adrenergic receptor in 1986. They are classified into over 100 subfamilies as a function of their sequence homology, ligand structure and receptor function. Significant differences exist among the members of the GPCRs superfamily, in spite of the high degree of amino acid homology found among the members of a particular subfamily. The visual photoreceptor rhodopsin (Rho) was the first GPCR whose crystal structure was determined at high-resolution [1][2][3][4][5][6][7]. The natural abundance and availability of Rho has made of this protein the prototypical GPCR for structural studies. As a consequence, Rho is often selected as a model protein for biochemical and b...

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Amphipathic Polymers: Tools To Fold Integral Membrane Proteins to Their Active Form

Cited by 94 publications

References 37 publications

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

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

Manipulation des récepteurs couplés aux protéines G

Stabilization of Membrane Proteins: the Case of G‐Protein‐Coupled Receptors

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