Partial specific volume and solvent interactions of amphipol A8-35

Gohon, Yann; Pavlov, G. G.; Timmins, Peter; Tribet, Christophe; Popot, Jean‐Luc; Ebel, Christine

doi:10.1016/j.ab.2004.07.033

Cited by 101 publications

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“…For these particular experiments, we resorted to a polyacrylate-based polymer, A8-35, because it is by far the best characterized APol with respect to its synthesis, purification, and physicochemical properties, as well as to the preparation and properties of MP͞APol complexes (23)(24)(25)34). It was known from the onset, however, that using A8-35 instead of detergents like DPC or DHPC entailed two disadvantages: a larger particle size and the impossibility of working in neutral or acidic solution.…”

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…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 (Ϸ19 kDa), a protein that has been well studied by x-ray crystallography (26-28) and NMR spectroscopy (6, 29-32). …”

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“…Protonated and deuterated forms of APol A8-35 (hereafter HAPol and DAPol, respectively; Fig. 1a) were synthesized in our laboratory by grafting either hydrogenated or deuterated (Ն99%) octylamine and isopropylamine groups onto a hydrogenated poly(acrylic acid) precursor (22,25).Expression, Refolding, and Purification of [ 2 H, 15 N]tOmpA. The plasmid containing the coding sequence of tOmpA was kindly Abbreviations: APol, amphipol; DAPol, a partially deuterated form of A8-35; DHPC, dihexanoyl phosphatidylcholine; HAPol, fully hydrogenated form of A8-35; HSQC, heteronuclear single-quantum correlation; MP, membrane protein; tOmpA, transmembrane domain of outer membrane protein A (residues 1-171); TROSY, transverse relaxation-optimized spectroscopy.…”

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confidence: 99%

“…The synthesis and physicochemical properties of one of them, A8-35 ( Fig. 1a) have been described in detail (24,25).…”

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confidence: 99%

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

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Giusti

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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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 ...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

“…The synthesis and physicochemical properties of one of them, A8-35 ( Fig. 1a) have been described in detail (24,25).…”

mentioning

confidence: 99%

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

Zoonens

Catoire

Giusti

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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107

169

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Characterizing Protein‐Protein Interactions by Sedimentation Velocity Analytical Ultracentrifugation

2008

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This unit introduces the basic principles and practice of sedimentation velocity analytical ultracentrifugation for the study of reversible protein interactions, such as the characterization of self-association, heterogeneous association, multi-protein complexes, binding stoichiometry, and the determination of association constants. The analytical tools described include sedimentation coefficient and molar mass distributions, multi-signal sedimentation coefficient distributions, Gilbert-Jenkins theory, different forms of isotherms, and global Lamm equation modeling. Concepts for the experimental design are discussed, and a detailed step-by-step protocol guiding the reader through the experiment and the data analysis is available as an Internet resource.

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Analytical Ultracentrifugation

Schuck

Balbo

Brown

et al. 2010

Encyclopedia of Analytical Chemistry

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Analytical ultracentrifugation is a powerful and very versatile technique for the study of particles in solution ranging from sub‐nanometers to micrometers in size. It allows the characterization of their size or size distribution, low‐resolution (hydrodynamic) shape, density, and solvation properties, and, in some cases, composition and attractive or repulsive interactions. For single‐ or multicomponent systems with reversible chemical reactions, the number and stoichiometry of complexes, the timescale of their interconversion, as well as binding constants from nanomolar to millimolar range can be determined. Analytical ultracentrifugation is based on first principles and has a long history of theoretical and practical development. This present article provides an introduction of the theoretical concept, the instrumentation, and considerations for conducting experiments and data analysis.

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Partial specific volume and solvent interactions of amphipol A8-35

Cited by 101 publications

References 32 publications

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

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

Characterizing Protein‐Protein Interactions by Sedimentation Velocity Analytical Ultracentrifugation

Analytical Ultracentrifugation

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