Quantitative real-time analysis of the efflux by the MacAB-TolC tripartite efflux pump clarifies the role of ATP hydrolysis within mechanotransmission mechanism

Tripartite efflux pumps built around ATP-binding cassette (ABC) transporters are membrane protein machineries that perform vectorial export of a large variety of drugs and virulence factors from Gram negative bacteria, using ATP-hydrolysis as energy source. Determining the number of ATP molecules consumed per transport cycle is essential to understanding the efficiency of substrate transport. Using a reconstituted pump in a membrane mimic environment, we show that MacAB-TolC from Escherichia coli couples substrate transport to ATP-hydrolysis with high efficiency. Contrary to the predictions of the currently prevailing “molecular bellows” model of MacB-operation, which assigns the power stroke to the ATP-binding by the nucleotide binding domains of the transporter, by utilizing a novel assay, we report clear synchronization of the substrate transfer with ATP-hydrolysis, suggesting that at least some of the power stroke for the substrate efflux is provided by ATP-hydrolysis. Our findings narrow down the window for energy consumption step that results in substrate transition into the TolC-channel, expanding the current understanding of the efflux cycle of the MacB-based tripartite assemblies. Based on that we propose a modified model of the MacB cycle within the context of tripartite complex assembly

NMR study of a membrane protein in detergent-free aqueous solution

One of the major obstacles to membrane protein (MP) structural studies is the destabilizing effect of detergents. Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep MPs water-soluble under mild conditions. In the present work, we have explored the feasibility of studying the structure of APol-complexed MPs by NMR. As a test MP, we chose the 171-residue transmembrane domain of outer MP A from Escherichia coli (tOmpA), whose x-ray and NMR structures in detergent are known. (2)H,(15)N-labeled tOmpA was produced as inclusion bodies, refolded in detergent solution, trapped with APol A8-35, and the detergent removed by adsorption onto polystyrene beads. The resolution of transverse relaxation-optimized spectroscopy–heteronuclear single-quantum correlation spectra of tOmpA/A8-35 complexes was found to be close to that of the best spectra obtained in detergent solutions. The dispersion of chemical shifts indicated that the protein had regained its native fold and retained it during the exchange of surfactants. MP–APol interactions were mapped by substituting hydrogenated for deuterated A8-35. The resulting dipolar broadening of amide proton linewidths was found to be limited to the β-barrel region of tOmpA, indicating that A8-35 binds specifically to the hydrophobic transmembrane surface of the protein. The potential of this approach to MP studies by solution NMR is discussed

How amphipols embed membrane proteins : global solvent accessibility and interaction with a flexible protein terminus

Amphipathic polymers called amphipols provide a valuable alternative to detergents for keeping integral membrane proteins soluble in aqueous buffers. Here, we characterize spatial contacts of amphipol A8-35 with membrane proteins from two architectural classes: The 8-stranded β-barrel outer membrane protein OmpX and the α-helical protein bacteriorhodopsin. OmpX is well structured in A8-35, with its barrel adopting a fold closely similar to that in dihexanoylphosphocholine micelles. The accessibility of A8-35-trapped OmpX by a water-soluble paramagnetic molecule is highly similar to that in detergent micelles and resembles the accessibility in the natural membrane. For the α-helical protein bacteriorhodopsin, previously shown to keep its fold and function in amphipols, NMR data show that the imidazole protons of a polyhistidine tag at the N-terminus of the protein are exchange protected in the presence of detergent and lipid bilayer nanodiscs, but not in amphipols, indicating the absence of an interaction in the latter case. Overall, A8-35 exhibits protein interaction properties somewhat different from detergents and lipid bilayer nanodiscs, while maintaining the structure of solubilized integral membrane proteins

Electrostatically-driven fast association and perdeuteration allow detection of transferred cross-relaxation for G protein-coupled receptor ligands with equilibrium dissociation constants in the high-to-low nanomolar range.

International audienceThe mechanism of signal transduction mediated by G protein-coupled receptors is a subject of intense research in pharmacological and structural biology. Ligand association to the receptor constitutes a critical event in the activation process. Solution-state NMR can be amenable to high-resolution structure determination of agonist molecules in their receptor-bound state by detecting dipolar interactions in a transferred mode, even with equilibrium dissociation constants below the micromolar range. This is possible in the case of an inherent ultra-fast diffusive association of charged ligands onto a highly charged extracellular surface, and by slowing down the (1)H-(1)H cross-relaxation by perdeuterating the receptor. Here, we demonstrate this for two fatty acid molecules in interaction with the leukotriene BLT2 receptor, for which both ligands display a submicromolar affinity

Inter- and intramolecular contacts in a membrane protein/surfactant complex observed by heteronuclear dipole-to-dipole cross-relaxation.

International audienceHeteronuclear dipole-to-dipole cross-relaxation has been applied to exploring intermolecular interactions and intramolecular spatial proximities in a large supramolecular structure comprised of a beta-barrel membrane protein, OmpX, in complex with a polymeric surfactant, amphipol A8-35. The experiments, performed in either the laboratory or the rotating frame, reveal the existence of intermolecular contacts between aromatic amino acids and specific groups of the polymer, in addition to intra-protein dipolar interactions, some of them involving carbonyl carbons. This study opens the perspective of collecting by NMR spectroscopy a new kind of through-space structural information involving aromatic and carbonyl (13)C atoms of large proteins

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