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.

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.

Self-aggregation of tetradecyltrimethylammonium bromide (TTAB, [CH3(CH2)13N+(CH3)3Br-]) and polyoxyethylene 23 lauryl ether (Brij-35, [CH3 (CH2)11(OCH2CH2)23OH]) binary surfactant mixture in aqueous medium was studied using tensiometric, conductometric, density, quasielastic light scattering, potentiometric, and fluorometric measurements. The binary surfactant mixture was studied well above the Krafft temperature, which was evaluated by conductance measurements. Rubingh's nonideal solution theory predicted nonideal mixing and attractive interaction between the constituent surfactants in the mixed micelle. Moreover, attractive interaction between the two surfactants in the mixed micelle is explained by assuming that water acts as a bridge between the hydrophilic polar groups of the surfactant molecules. The chain-chain interaction among the surfactant does not seem to be high in this case. The partial specific volume of pure as well as binary surfactant mixtures was also evaluated, and it was inferred that the mixed micelles are more hydrated compared to individual components. The excess Gibbs free energy of mixing was evaluated, and it indicated relatively more stable mixed micelles for this binary combination. Surface tension measurements indicate an existence of a second state of aggregation for the mixed surfactant system, which is supported by the break in conductance−concentration of surfactant profile. The Krafft temperature of TTAB decreases as the nonionic surfactant content increases in the mixed system. Quasielastic light scattering studies suggest an increase in the hydrodynamic radius of the micelle in the mixed surfactant system.

A novel class of nonionic amphipols (NAPols) designed to handle membrane proteins in aqueous solutions has been synthesized, and its solution properties have been examined. These were synthesized through free radical cotelomerization of glucose-based hydrophilic and amphiphilic monomers derived from tris(hydroxymethyl)acrylamidomethane using azobisisobutyronitrile as the initiator and thiol as the transfer agent. The molecular weight and the hydrophilic/lipophilic balance of the cotelomers were modulated by varying the thiol/monomers and the hydrophilic monomer/amphiphilic monomer ratios, respectively, and were characterized by 'H NMR, UV, gel permeation chromatography, and Fourier transform infrared spectroscopy. Their physicochemical properties in aqueous solution were studied by dynamic light scattering, aqueous size-exclusion chromatography, analytical ultracentrifugation, and surface-tension measurements. NAPols are highly soluble in water and form, within a large concentration range, well-defined supramolecular assemblies with a diameter of approximately 6-7 nm, a narrow particle size distribution, and an average molecular weight close to 50 x 10(3) g x mol(-1). Varying the hydrophilic/amphiphilic monomer ratio of NAPols in the range of 3.0-4.9, the degree of polymerization in the range of 51-78, and the resulting average molar mass in the range of 20-29 x 10(3) g x mol(-1) has little incidence on their solution properties. Glucose-based NAPols efficiently kept soluble in aqueous solutions two test membrane proteins: bacteriorhodopsin and the transmembrane domain of Escherichia coli's outer membrane protein A.