Crystal Structures of Native and Inactivated cis-3-Chloroacrylic Acid Dehalogenase. Structural Basis for Substrate Specificity and Inactivation by (R)-Oxirane-2-Carboxylate

The bacterial degradation pathways for the nematocide 1,3-dichloropropene rely on hydrolytic dehalogenation reactions catalyzed by cis- and trans-3-chloroacrylic acid dehalogenases (cis-CaaD and CaaD, respectively). X-ray crystal structures of native cis-CaaD and cis-CaaD inactivated by (R)-oxirane-2-carboxylate were elucidated. They locate four known catalytic residues (Pro-1, Arg-70, Arg-73, and Glu-114) and two previously unknown, potential catalytic residues (His-28 and Tyr-103'). The Y103F and H28A mutants of these latter two residues displayed reductions in cis-CaaD activity confirming their importance in catalysis. The structure of the inactivated enzyme shows covalent modification of the Pro-1 nitrogen atom by (R)-2-hydroxypropanoate at the C3 position. The interactions in the complex implicate Arg-70 or a water molecule bound to Arg-70 as the proton donor for the epoxide ring-opening reaction and Arg-73 and His-28 as primary binding contacts for the carboxylate group. This proposed binding mode places the (R)-enantiomer, but not the (S)-enantiomer, in position to covalently modify Pro-1. The absence of His-28 (or an equivalent) in CaaD could account for the fact that CaaD is not inactivated by either enantiomer. The cis-CaaD structures support a mechanism in which Glu-114 and Tyr-103' activate a water molecule for addition to C3 of the substrate and His-28, Arg-70, and Arg-73 interact with the C1 carboxylate group to assist in substrate binding and polarization. Pro-1 provides a proton at C2. The involvement of His-28 and Tyr-103' distinguishes the cis-CaaD mechanism from the otherwise parallel CaaD mechanism. The two mechanisms probably evolved independently as the result of an early gene duplication of a common ancestor

Trapping and Stabilization of Integral Membrane Proteins by Hydrophobically Grafted Glucose-Based Telomers

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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)

Glucose-Based Amphiphilic Telomers Designed to Keep Membrane Proteins Soluble in Aqueous Solutions: Synthesis and Physicochemical Characterization

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

International audienceA 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, Amphiphilic molecules self-assemble in aqueous solvents because of the differential solvation properties of their hydrophilic and lipophilic groups. In water, detergents, a class of small surfactants, form micelles that can solubilize amphi-philic and lipophilic guest molecules. This property has proven highly useful in the study of membrane proteins (MPs), which are naturally water-insoluble due to the high hydrophobicity of their transmembrane surface. 1 However, the dissociating character of detergents, combined with the need to maintain an excess of them, tends to inactivate membrane proteins. This has prompted the development of milder surfactants, such as detergents with less disruptive structures, fluorinated surfac-tants, lipopeptides, or nanodiscs (for reviews, see refs 2−4). A particularly promising approach consists in generating multiple contacts between an amphiphilic polymer and the hydrophobic transmembrane surface of MPs, leading to the development of the so-called 'amphipols' (APols). 5 APols have proven very efficient at keeping MPs soluble in the absence of detergents, while stabilizing them biochemically. Their applications extend to MP folding, immobilization, structural studies by solution NMR and cryo-electron microscopy, and studies of ligand binding and the recruitment of effectors, as well as vaccination (reviewed in refs 4, 6, 7). The most extensively studied APol to date, A8−35, is an anionic polymer made of poly(acrylic acid) carrying multiple octyl chains. 5 Because its solubility depends on the carboxylate groups being charged, A8−35 suffers from certain limitations, among which are its insolubility in acidic buffers 8,9 and its sensitivity to calcium ions. 10 In addition , its charged character prevents the separation of MP/A8−3

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

International audienceNonionic 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 C₁₁ 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 <i>Halobium salinarum</i> and the outer membrane protein X of <i>Escherichia coli</i>, 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. <b>2012</b>, <i>Biochemistry</i>, DOI: 10.1021/bi201862v)