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

NMR study of a membrane protein in detergent-free aqueous solution
May 6, 2005 (received for review February 16, 2005)
Published online before print June 14, 2005
Manuela Zoonens *, Laurent J. Catoire *, Fabrice Giusti, and Jean-Luc Popot
PNAS | June 21, 2005
Unit? Mixte de Recherche 7099, Centre National de la Recherche Scientifique (CNRS)/Universit? Paris-7, Institut de Biologie Physico-Chimique (CNRS FRC 550), 11 Rue Pierre et Marie Curie, F-75005 Paris, France
Communicated by Donald M. Engelman, Yale University, New Haven, CT
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. 2H,15N-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 -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
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Integral 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 <0.3% 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 (iii) those detergents that tend to be less destabilizing, such as digitonin or surfactants of the Tween series, are unsuitable for solution NMR, where a primary requirement is that the MP/detergent complex be as small as possible. As a result, the only MPs whose native structure has been studied by solution NMR to date are exceptionally robust ones, such as the transmembrane domains of glycophorin A (5) and OmpA (6), OmpX (7), and PagP (8), all of which are sturdy enough to resist denaturation by SDS at room temperature (9?12). It appears likely that a more general extension of solution NMR to MP studies will depend primarily on three major types of technical progress: (i) higher magnetic fields and improved pulse sequences (13?15), (ii) more efficient approaches to producing micromolar amounts of isotopically labeled MPs (16?19), and (iii) novel surfactants, milder than classical detergents but nevertheless allowing the acquisition of NMR spectra of a quality sufficient for structure determination. The latter issue is the focus of the present work.
The frequent instability of MPs in detergent solutions has prompted the development of alternative media based on the use of nondetergent surfactants and/or nonmicellar phases (for reviews, see, e.g., refs. 20 and 21). Over the past few years, we have endeavored to develop a novel family of surfactants dubbed "amphipols" (APols) (22). APols are amphiphilic polymers designed to bind to the transmembrane surface of MPs in a noncovalent but quasi-irreversible manner. APols are not (or are extremely weak) detergents and, as a rule, they are unable to extract MPs from biological membranes (reviewed in ref. 23). Nevertheless, they can maintain in solution MPs extracted by classical detergents after being substituted to the latter (22). MPs complexed by APols are in their native state, generally much more stable than in detergent solution, and they remain water-soluble in the absence of detergent or free APols (23). Although several families of APols have now been described (21, 23), the best-characterized ones feature a polyacrylate backbone derived with fatty amines (22). The synthesis and physicochemical properties of one of them, A8-35 (Fig. 1a) have been described in detail (24, 25).
As a model MP, we chose the transmembrane domain of outer MP A from Escherichia coli, tOmpA (19 kDa), a protein that has been well studied by x-ray crystallography (26?28) and NMR spectroscopy (6, 29?32).
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