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).

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.

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.

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 approximately 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.

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.

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.