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

Abstract

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 ...

Molecular Architecture and Subunit Organization of TRPA1 Ion Channel Revealed by Electron Microscopy

“…The amphipol A8-35 has proven to be an effective agent for maintaining membrane proteins in a soluble, stabilized state. Amphipols have a high affinity for the TM region of membrane proteins and preferentially localize to these sites, as shown by EM and NMR (39).…”

“…A8-35 is thought to provide a more nativelike environment for a membrane protein (32) and has been utilized in a variety of NMR and electron microscopy structural studies (33)(34)(35)(36)(37)(38). One advantage of amphipols is their ability to bind tightly to the transmembrane portion of the protein, which allows the protein to remain soluble in buffers that contain no detergent or excess amphipol (39). Five EM structures have been determined using A8-35, including two structures for proton ATP synthase as well as structures of bacteriorhodopsin, mitochondrial complex I, and supercomplex B (I 1 III 2 IV 1 ) (33-38).…”

The use of amphipols as universal molecular adapters to immobilize membrane proteins onto solid supports

“…1A). Its use has been validated on a large panel of MPs (18), including bacteriorhodopsin (BR) (20), the nicotinic acetylcholine receptor (nAChR) (22), the cytochrome b 6 f and bc 1 complexes (17,18) and the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) (19,23). Of particular relevance to the present work is the fact that, although it is noncovalent, its association to MPs is strictly irreversible as long as it is not displaced by another surfactant (23,24).…”

“…APols (17,18) are short soluble polymers carrying numerous hydrophobic side chains thanks to which they can associate with the transmembrane surface of MPs (19) by multiple attachment points. Thereby, they keep them water soluble in the absence of detergent and stabilize them biochemically (see ref.…”

Visiting order on membrane proteins by using nanotechnology

“…Moreover, the fact that hydrophobic molecules and amphipathic interfaces should generally have low affinity for direct interaction with the polyanionic surface presented by the DNA-based nanotubes bodes well for widespread applicability to a range of membrane proteins (provided they do not have an intrinsic affinity for DNA or for polyanions). It should be added that the nanotube-based alignment method should also be applicable to membrane proteins that are solubilized in amphipols or in small (normally isotropic) bicelles, emerging alternatives to classical micelles (18,19).…”