Feedback mechanisms that dissipate excess photoexcitations in light-harvesting complexes (LHCs) are necessary to avoid detrimental oxidative stress in most photosynthetic eukaryotes. Here we demonstrate the unique ability of LHCSR, a stress-related LHC from the model organism Chlamydomonas reinhardtii, to sense pH variations, reversibly tuning its conformation from a light-harvesting state to a dissipative one. This conformational change is induced exclusively by the acidification of the environment, and the magnitude of quenching is correlated to the degree of acidification of the environment. We show that this ability to respond to different pH values is missing in the related major LHCII, despite high structural homology. Via mutagenesis and spectroscopic characterization, we show that LHCSR's uniqueness relies on its peculiar C-terminus subdomain, which acts as a sensor of the lumenal pH, able to tune the quenching level of the complex.
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.
The use of classical antioxidants is limited by their low bioavailabilities, and therefore, high doses are usually required to display significant protective activity. In a recent article (J. Med. Chem. 2003, 46, 5230) we showed that the ability of the alpha-phenyl-N-tert-butylnitrone (PBN) to restore the viability of ATPase-deficient human skin fibroblasts was greatly enhanced by grafting it on a fluorinated amphiphilic carrier. With the aim of extending this concept to other antioxidants, we present here the design, the synthesis, and the physicochemical measurements of a new series of fluorinated amphiphilic antioxidant derivatives. The hydroxyl radical scavenging activity and the radical reducing potency of these newly designed compounds were respectively demonstrated in an ABTS competition and an ABTS(*+) reduction assay. We also showed that the protective effects of amphiphilic antioxidants derived from PBN, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) or lipoic acid (5-[1,2]-dithiolan-3-ylpentanoic acid) in primary cortical mixed cell cultures exposed to oxidotoxins are greatly improved compared to their parent compounds in the following rank-order: (1) PBN, (2) Trolox, and (3) lipoic acid. In contrast, the protective activity of indole-3-propionic acid was slightly decreased by grafting it on the amphiphilic carrier. Similar observations were made in in vivo experiments using aquatic invertebrate microorganisms, called rotifers, which were exposed to lethal concentrations of nonselective (H(2)O(2)) and mitochondria-selective (doxorubicin) oxidotoxins. The conclusion of these studies is that fluorinated amphiphilic PBN, Trolox, and lipoic acid derivatives exhibit very potent protective activities in in vitro and in vivo experiments. The findings demonstrated herein therefore strongly suggest that the amphiphilic character enhances the bioavailability of the antioxidants and allows for a selective targeting of mitochondria.
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.
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.
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).
Surfactants carrying fluorocarbon chains hold great promise as gentle alternatives to conventional hydrocarbon-based detergents for the solubilization and handling of integral membrane proteins. However, their inertness towards lipid bilayer membranes has limited the usefulness of fluorinated surfactants in situations where detergent-like activity is required. We demonstrate that fluorination does not necessarily preclude detergency, as exemplified by a fluorinated octyl maltoside derivative termed F6 OM. This nonionic compound readily interacts with and completely solubilizes phospholipid vesicles in a manner reminiscent of conventional detergents without, however, compromising membrane order at subsolubilizing concentrations. Owing to this mild and unusual mode of detergency, F6 OM outperforms a lipophobic fluorinated surfactant in chaperoning the functional refolding of an integral membrane enzyme by promoting bilayer insertion in the absence of micelles.
During ultrasound processing of lipid-containing food, some off-flavors can be detected, which can incite depreciation by consumers. The impacts of ultrasound treatment on sunflower oil using two different ultrasound horns (titanium and pyrex) were evaluated. An electron paramagnetic resonance study was performed to identify and quantify the formed radicals, along with the assessment of classical physicochemical parameters such as peroxide value, acid value, anisidine value, conjugated dienes, polar compounds, water content, polymer quantification, fatty acid composition, and volatiles profile. The study shows an increase of formed radicals in sonicated oils, as well as the modification of physicochemical parameters evidencing an oxidation of treated oils.
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