Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.

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 tOmpA͞A8-35 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 ␤-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. membrane proteins ͉ amphipols ͉ OmpA ͉ surfactant I ntegral membrane proteins (MPs) are involved in such essential cell functions as energy transduction, import and export of nutrients and drugs, signal detection, cell-to-cell communication, etc. They comprise 20-30% of the proteins encoded in the genome of cells and a majority of the targets of currently marketed drugs (1). A detailed knowledge of their structure is essential to understanding their function and dysfunction, as well as to a wide range of biomedical and biotechnological applications. The scarcity of high-resolution MP structures (which represent Ͻ0.3% of currently available structures) can be traced to three main factors: low levels of natural abundance, difficult overexpression, and a poor stability in the presence of detergent. Detergents are generally used to handle MPs in aqueous solutions, because the highly hydrophobic character of their transmembrane surface renders MPs water-insoluble. By adsorbing onto this surface, detergents make it hydrophilic (2). However, the dissociating character of detergents, combined with the need to maintain an excess of them, frequently results in more or less rapid inactivation of solubilized MPs (3).Inactivation by detergents is a particularly serious problem in the field of solution-state NMR for the following reasons: (i) to keep highly concentrated MPs (in the mM range) from aggregating, high concentrations of detergents must generally be used (usually in the 200-to 600-mM range; see, for instance, ref. 4); (ii) high temperatures are usually resorted to, to improve the resolution of the spectra; and ...

Membrane proteins perform a host of vital cellular functions. Deciphering the molecular mechanisms whereby they fulfill these functions requires detailed biophysical and structural investigations. Detergents have proven pivotal to extract the protein from its native surroundings. Yet, they provide a milieu that departs significantly from that of the biological membrane, to the extent that the structure, the dynamics, and the interactions of membrane proteins in detergents may considerably vary, as compared to the native environment. Understanding the impact of detergents on membrane proteins is, therefore, crucial to assess the biological relevance of results obtained in detergents. Here, we review the strengths and weaknesses of alkyl phosphocholines (or foscholines), the most widely used detergent in solution-NMR studies of membrane proteins. While this class of detergents is often successful for membrane protein solubilization, a growing list of examples points to destabilizing and denaturing properties, in particular for α-helical membrane proteins. Our comprehensive analysis stresses the importance of stringent controls when working with this class of detergents and when analyzing the structure and dynamics of membrane proteins in alkyl phosphocholine detergents.

Well characterized pectin samples were incubated with cell wall-bound and -solubilized pure isoforms of pectinmethylesterase from mung bean hypocotyls (Vigna radiata). Both enzyme activity and average product structure were determined at intervals along the deesterification pathway at pH 5.6 and 7.6. The latter analyses were performed by 13 C NMR spectroscopy, and the degree of esterification was probed by both 13 C NMR and potentiometric measurements. A dichotomy was observed in the behavior of the ␣ and ␥ isoforms when compared with that of the ␤ isoenzyme. Ideal blockwise deesterification mechanisms reproduced the experimental average structures (methylester distribution) throughout the course of the reaction. In the case of the ␣ and ␥ isoforms, a single chain mechanism associated with a free carboxyl group at the second nearest neighbor position could be postulated at pH 5.6, whereas some multiple attack character was required to reproduce the data at pH 7.6. Several mechanisms that differed from the preceding ones were compatible with the data for the ␤ isoform at the two pH values. Both the nature of the polysaccharides produced in these reactions and the role of pectinmethylesterase in the cell wall-stiffening process along the growth gradient are discussed.Pectins that represent around 30% of the primary plant cell walls play a key role in plant physiology as well as in plant pathology. The general structure of pectic polymers consists of homogalacturonan linear chains (smooth regions) interspersed with highly branched galacturonic chains (hairy regions). Some of the galacturonic residues linked by ␣-1,4 glycosidic bonds are methyl-esterified at the carboxyl group. The degree of methylesterification (DE) 1 varies greatly depending on the plant organ and the degree of differentiation of the cells. Young, plastic cell walls are generally characterized by a high content of highly methylated pectin that decreases in parallel with the loss of extensibility of the walls, whereas the amount of acidic residues increases (1-3). Moreover, the balance between high and low methylated pectins varies inside the wall of a single cell (4) generating microdomains. Not only the number but also the distribution of free, unesterified galacturonate carboxyl groups within the galacturonan regions will control the gellforming capacity of the pectin and thereby the porosity and the extensibility of the apoplasm (5, 6). It is commonly accepted that the polygalacturonic backbone is polymerized in the cisGolgi cisternae, methylesterified in the medial-Golgi, substituted with side chains in the trans-Golgi, and exported to the cell walls as a highly methylated polygalacturonan (7). At a later stage, it is deesterified in muro by cell wall pectinmethylesterases (PMEs). Many proteins exhibiting PME activity have been purified, and their biochemical features such as molecular weight, optimal pH, pI, and substrate specificity have been established (8). In some cases, the corresponding genes have also been cloned and sequenced (9 -11...

Mapping the conformational landscape of G protein-coupled receptors (GPCRs), and in particular how this landscape is modulated by the membrane environment, is required to gain a clear picture of how signaling proceeds. To this end, we have developed an original strategy based on solution-state nuclear magnetic resonance combined with an efficient isotope labeling scheme. This strategy was applied to a typical GPCR, the leukotriene B4 receptor BLT2, reconstituted in a lipid bilayer. Because of this, we are able to provide direct evidence that BLT2 explores a complex landscape that includes four different conformational states for the unliganded receptor. The relative distribution of the different states is modulated by ligands and the sterol content of the membrane, in parallel with the changes in the ability of the receptor to activate its cognate G protein. This demonstrates a conformational coupling between the agonist and the membrane environment that is likely to be fundamental for GPCR signaling.

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 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).

The atomic structure of OmpX, the smallest member of the bacterial outer membrane protein family, has been previously established by X-ray crystallography and NMR spectroscopy. In apparent conflict with electrophysiological studies, the lumen of its transmembrane beta-barrel appears too tightly packed with amino acid side chains to let any solute flow through. In the present study, high-resolution solution NMR spectra were obtained of OmpX kept water-soluble by either amphipol A8-35 or the detergent dihexanoylphosphatidylcholine. Hydrogen/deuterium exchange measurements performed after prolonged equilibration show that, whatever the surfactant used, some of the amide protons of the membrane-spanning region exchange much more readily than others, which likely reflects the dynamics of the barrel.