Bernard Pucci

Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.

Hemifluorinated surfactants: a non-dissociating environment for handling membrane proteins in aqueous solutions?

The instability of membrane proteins in detergent solution can generally be traced to the dissociating character of detergents and often correlates with delipidation. We examine here the possibility of substituting detergents, after membrane proteins have been solubilized, with non‐detergent surfactants whose hydrophobic moiety contains a perfluorinated region that makes it lipophobic. In order to improve its affinity for the protein surface, the fluorinated chain is terminated by an ethyl group. Test proteins included bacteriorhodopsin, the cytochrome b 6 f complex, and the transmembrane region of the bacterial outer membrane protein OmpA. All three proteins were purified using classical detergents and transferred into solutions of C2H5C6F12C2H4‐S‐poly‐Tris‐(hydroxymethyl)aminomethane (HF‐TAC). Transfer to HF‐TAC maintained the native state of the proteins and prevented their precipitation. Provided the concentration of HF‐TAC was high enough, HF‐TAC/membrane protein complexes ran as single bands upon centrifugation in sucrose gradients. Bacteriorhodopsin and the cytochrome b 6 f complex, both of which are detergent‐sensitive, exhibited increased biochemical stability upon extended storage in the presence of a high concentration of HF‐TAC as compared to detergent micelles. The stabilization of cytochrome b 6 f is at least partly due to a better retention of protein‐bound lipids.

Fluorinated and hemifluorinated surfactants as alternatives to detergents for membrane protein cell-free synthesis

Hemifluorinated and fluorinated surfactants are lipophobic and, as such, non-detergent. Although they do not solubilize biological membranes, they can, after conventional solubilization, substitute for detergents to keep membrane proteins soluble, which generally improves their stability [Breyton, Chabaud, Chaudier, Pucci and Popot (2004) FEBS Lett. 564, 312-318]. In the present study, we show that (hemi)fluorinated surfactants can be used for in vitro synthesis of membrane proteins: they do not interfere with protein synthesis, and they provide a suitable environment for MscL, a pentameric mechanosensitive channel, to fold and oligomerize to its native functional state. Following synthesis, both types of surfactants can be used to deliver MscL directly to pre-formed lipid vesicles. The electrophysiological activity of MscL synthesized in vitro in the presence of either hemi- or per-fluorinated surfactant is similar to that of the protein expressed in vivo.

Nonionic Homopolymeric Amphipols: Application to Membrane Protein Folding, Cell-Free Synthesis, and Solution Nuclear Magnetic Resonance

Nonionic amphipols (NAPols) synthesized by homotelomerization of an amphiphatic monomer are able to keep membrane proteins (MPs) stable and functional in the absence of detergent. Some of their biochemical and biophysical properties and applications have been examined, with particular attention being paid to their complementarity with the classical polyacrylate-based amphipol A8-35. Bacteriorhodopsin (BR) from Halobacterium salinarum and the cytochrome b(6)f complex from Chlamydomonas reinhardtii were found to be in their native state and highly stable following complexation with NAPols. NAPol-trapped BR was shown to undergo its complete photocycle. Because of the pH insensitivity of NAPols, solution nuclear magnetic resonance (NMR) two-dimensional (1)H-(15)N heteronuclear single-quantum coherence spectra of NAPol-trapped outer MP X from Escherichia coli (OmpX) could be recorded at pH 6.8. They present a resolution similar to that of the spectra of OmpX/A8-35 complexes recorded at pH 8.0 and give access to signals from solvent-exposed rapidy exchanging amide protons. Like A8-35, NAPols can be used to fold MPs to their native state as demonstrated here with BR and with the ghrelin G protein-coupled receptor GHS-R1a, thus extending the range of accessible folding conditions. Following NAPol-assisted folding, GHS-R1a bound four of its specific ligands, recruited arrestin-2, and activated binding of GTPγS by the G(αq) protein. Finally, cell-free synthesis of MPs, which is inhibited by A8-35 and sulfonated amphipols, was found to be very efficient in the presence of NAPols. These results open broad new perspectives on the use of amphipols for MP studies.

Non-Ionic Amphiphilic Homopolymers: Synthesis, Solution Properties, and Biochemical Validation

A novel type of nonionic amphipols for handling membrane proteins in detergent-free aqueous solutions has been obtained through free-radical homo-telomerization of an acrylamide-based monomer comprising a C(11) alkyl chain and two glucose moieties, using a thiol as transfer reagent. By controlling the thiol/monomer ratio, the number-average molecular weight of the polymers was varied from 8 to 63 kDa. Homopolymeric nonionic amphipols were found to be highly soluble in water and to self-organize, within a large concentration range, into small, compact particles of ~6 nm diameter with a narrow size distribution, regardless of the molecular weight of the polymer. They proved able to trap and stabilize two test membrane proteins, bacteriorhodopsin from Halobium salinarum and the outer membrane protein X of Escherichia coli, under the form of small and well-defined complexes, whose size, composition, and shape were studied by aqueous size-exclusion chromatography, analytical ultracentrifugation, and small-angle neutron scattering. As shown in a companion paper, nonionic amphipols can be used for membrane protein folding, cell-free synthesis, and solution NMR studies (Bazzacco et al. 2012, Biochemistry, DOI: 10.1021/bi201862v).

Micellar and Biochemical Properties of (Hemi)Fluorinated Surfactants Are Controlled by the Size of the Polar Head

Surfactants with fluorinated and hemifluorinated alkyl chains have yielded encouraging results in terms of membrane protein stability; however, the molecules used hitherto have either been chemically heterogeneous or formed heterogeneous micelles. A new series of surfactants whose polar head size is modulated by the presence of one, two, or three glucose moieties has been synthesized. Analytical ultracentrifugation and small-angle neutron scattering show that fluorinated surfactants whose polar head bears a single glucosyl group form very large cylindrical micelles, whereas those with two or three glucose moieties form small, homogeneous, globular micelles. We studied the homogeneity and stability of the complexes formed between membrane proteins and these surfactants by using bacteriorhodopsin and cytochrome b(6)f as models. Homogeneous complexes were obtained only with surfactants that form homogeneous micelles. Surfactants bearing one or two glucose moieties were found to be stabilizing, whereas those with three moieties were destabilizing. Fluorinated and hemifluorinated surfactants with a two-glucose polar head thus appear to be very promising molecules for biochemical applications and structural studies. They were successfully used for cell-free synthesis of the ion channel MscL.

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.

A class of mild surfactants that keep integral membrane proteins water-soluble for functional studies and crystallization

Abstract Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties. Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-β-D-maltoside (‘C12-b-M’) as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors and the human Patched protein receptor by approximately 10°C compared to C12-b-M. Another surfactant yielded the highest stabilization of the human Patched protein receptor compared to C12-b-M (13°C) but was inferior for the G protein-coupled receptors. In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b6 f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M.

In the cauldron of cell-free synthesis of membrane proteins: playing with new surfactants

Cell-free protein synthesis is a well-known technique for the roles it has played in deciphering the genetic code and in the beginnings of signal sequence studies. Since then, many efforts have been made to optimise this technique and, recently, to adapt it to membrane protein production with yields compatible with structural investigations. The versatility of the method allows membrane proteins to be obtained directly stabilised in surfactant micelles or inserted in a lipidic environment (proteoliposome, bicelle, and nanodisc) at the end of synthesis. Among the surfactants used, non-detergent ones such as fluorinated surfactants proved to be a good alternative in terms of colloidal stability and preservation of the integrity of membrane proteins, as shown for Escherichia coli homo-pentameric channel, MscL (Park et al., Biochem. J., 403: 183-187). Here we report cell-free expression of Escherichia coli leader peptidase (a transmembrane protease), Halobacterium salinarium bacteriorhodopsin (a transmembrane protein binding a hydrophobic cofactor) and E. coli MscL in the presence of non-detergent surfactants, amphipols and fluorinated surfactants in comparison to their expression in classical detergents. The results confirm the potentialities of fluorinated surfactants and, although pointing to limitations in using the first generations amphipols, results are discussed in the light of membrane protein refolding, especially in the case of bacteriorhodopsin. Preliminary experiments using new generations of amphipols supports choices made in developing new molecules.

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.