Protective and Inhibitory Effects of Various Types of Amphipols on the Ca 2+ -ATPase from Sarcoplasmic Reticulum: A Comparative Study † Martin Picard, ‡,§ Tassadite Dahmane, ‡,| Manuel Garrigos, § Carole Gauron, § Fabrice Giusti, | Marc le Maire, § Jean-Luc Popot, | and Philippe Champeil* ,§ Section de Biophysique des Fonctions Membranaires (Commissariat a ` l’Energie Atomique), Institut Fe ´ de ´ ratif de Recherches 46 and Laboratoire de Recherche Associe ´ 17V (UniVersite ´ Paris Sud) and Unite ´ de Recherche Associe ´ e 2096 (Centre National de la Recherche Scientifique), De ´ partement de Biologie Joliot-Curie at CEA Saclay, 91191 Gif-sur-YVette cedex, France, and Unite ´ Mixte de Recherche 7099 (Centre National de la Recherche Scientifique and UniVersite ´ Paris-7) at Institut de Biologie Physico-Chimique, CNRS FRC 550, 11 rue Pierre et Marie Curie, F-75005 Paris, France ReceiVed September 27, 2005; ReVised Manuscript ReceiVed NoVember 29, 2005 ABSTRACT: Amphipols are amphipathic polymers designed to replace or supplement detergents in membrane protein solution studies. Previous work has suggested both advantages and disadvantages to the use of a polyacrylate-based amphipol, A8-35, for studying the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA1a). We investigated this issue further using a set of four amphipols with different chemical structures. Previous size exclusion chromatography experiments had shown that A8-35 and SERCA1a/A8-35 complexes aggregate under certain conditions. We show here that aggregation can be prevented by omitting calcium from buffers or by using a sulfonated version of A8-35. A8-35 had previously been shown to protect Ca 2+ -ATPase from irreversible denaturation, while inhibiting its activity in a reversible manner. We show here that the other three amphipols tested also display these properties and that all four amphipols slow down backward calcium dissociation from the nonphosphorylated solubilized enzyme, a priori an unrelated step. As this calcium dissociation involves the opening up of the bundle of transmembrane ATPase segments, the slowing of this process may indicate that multipoint attachment of the polymers to the hydrophobic transmembrane surface damps protein dynamics (“Gulliver” effect). Damping might be the reason why amphipols also simultaneously protect membrane proteins against irreversible denaturation and may inhibit the activity of those of them that display large rearrangements of their transmembrane surface during their catalytic cycle. Integral membrane proteins are the object of intensive studies, because they fulfill essential physiological functions and constitute important biomedical targets. However, in vitro studies of membrane proteins are hampered by ag- gregation in aqueous solutions, due to the high hydrophobic- ity of the surface of their transmembrane region. Detergents prevent this aggregation by adsorbing onto transmembrane surfaces, thereby providing an interface with the hydrophilic medium (1). However, detergents are dissociating surfactants, which very often destabilize membrane proteins. One of the major challenges in membrane protein biochemistry is therefore to achieve an acceptable compromise between solubility and biochemical stability (for discussions, see, e.g., refs 2 and 3). This problem has prompted the design of milder surfactants, which may not necessarily extract proteins from biological membranes efficiently but can substitute for detergents after solubilization and are more efficient at keeping membrane proteins in solution without inactivation (for reviews, see, e.g., refs 4 and 5). “Amphipols” (APols) 1 are amphipathic polymers specially designed for this purpose (6). First-generation APols have a polyacrylate backbone onto which fatty amines are grafted (Scheme 1A) (6). APols and their uses in membrane biology have been the subject of two recent reviews (5, 7). Poly- acrylate-based APols, as well as some nonionic or zwitter- ionic APols, keep membrane proteins soluble in the absence of detergent (in most cases, after extraction from the membrane with detergents). Membrane proteins are generally more stable after trapping with APols than in detergent solution. Thus, APols are potentially useful substitutes for detergents for in vitro functional or structural studies of detergent-sensitive membrane proteins (see, e.g., refs 7 and † We thank the Human Frontier Science Program Organization for financial support to M.P. and C.G. (RGP 0060/2001-M) and to J.-L.P. (RG00223/2000-M). Work in J.-L.P.’s laboratory was supported by CNRS and Universite ´ Paris-7. T.D. is the recipient of a doctoral fellowship from the MENESR. * To whom correspondence should be addressed at URA 2096, CNRS, et SBFM/DBJC, CEA Saclay. Tel: 33 1 6908 3731. Fax: 33 1 6908 8139. E-mail: champeil@dsvidf.cea.fr. ‡ These two authors contributed equally to the project. § DBJC at CEA Saclay. | UMR 7099 at IBPC Paris. 1 Abbreviations: APol, amphipol; SAPol, a sulfonated amphipol; SR, sarcoplasmic reticulum; ATPase, adenosine triphosphatase; A8- 35, a specific type of polyacrylate-based amphipol (see Scheme 1A); C12E8, octaethylene glycol monododecyl ether; HPLC, high-pressure liquid chromatography; Kd, distribution coefficient; RS, Stokes radius; SEC, size exclusion chromatography; EGTA, [ethylenebis(oxyethyl- enenitrilo)]tetraacetic acid; quin2, 2-[(2-amino-5-methylphenoxy)- methyl]-6-methoxy-8-aminoquinoline-N,N,N′,N′-tetraacetic acid; MOPS, 4-morpholinepropanesulfonic acid; Tris, tris(hydroxymethyl)amino- methane; TES, N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid; CMC, critical micelle concentration. 1861 Biochemistry 2006, 45, 1861-1869 10.1021/bi051954a CCC: $33.50 © 2006 American Chemical Society Published on Web 01/24/2006