Interaction of Amphipols with Sarcoplasmic Reticulum Ca2+-ATPase

Abstract

Amphipols are short-chain amphipathic polymers designed to keep membrane proteins soluble in aqueous solutions. We have evaluated the effects of the interaction of amphipols with sarcoplasmic reticulum Ca 2؉ -ATPase either in a membrane-bound or a soluble form. If the addition of amphipols to detergent-solubilized ATPase was followed by removal of detergent, soluble complexes formed, but these complexes retained poor ATPase activity, were not very stable upon long incubation periods, and at high concentrations they experienced aggregation. Nevertheless, adding excess detergent to diluted detergent-free ATPase-amphipol complexes incubated for short periods immediately restored full activity to these complexes, showing that amphipols had protected solubilized ATPase from the rapid and irreversible inactivation that otherwise follows detergent removal. Amphipols also protected solubilized ATPase from the rapid and irreversible inactivation observed in detergent solutions if the ATPase Ca The transmembrane surface of integral membrane proteins is highly hydrophobic. Thus, detergents are generally used to facilitate the handling of these proteins in aqueous media (1). However, detergents do not always maintain solubilized membrane proteins in an active and stable state (see Refs. 2 and 3, and references therein). The use of short amphipathic polymers ("amphipols"), comprising a hydrophilic backbone with hydrophobic chains, has been proposed as an alternative approach (4). Amphipols can interact with hydrophobic surfaces at many points along their length. They should therefore form a noncovalent but stable layer at the interface between the transmembrane region of the protein and the aqueous solution. Initial studies with four integral membrane proteins showed that complexes of these proteins with amphipols could indeed be handled in surfactant-free aqueous solutions as if they were soluble proteins (4 -6); back-exchange between amphipols and detergents was also demonstrated (5), but its kinetics was not studied. Amphipols have a wide range of potential applications in biochemistry, biophysics, and biotechnology. Little is known, however, about their effects on membrane protein function. Published data are limited to cytochrome b 6 f, which was shown to retain its ability to catalyze electron transfer following injection of cytochrome b 6 f-amphipol complexes into a detergentcontaining reaction medium (4). Various degrees of inhibition were nevertheless observed, depending on the charge of the amphipols. This inhibition was hypothesized to be due to the electrostatic repulsion of one of the substrates of the reaction, but other effects of amphipols on function, an essential determinant of their usefulness in membrane biology, remain largely unknown.In this study, we investigated the interaction of amphipols (mainly amphipol A8-35, a polyacrylate backbone derivatized with octyl and isopropyl chains) with the sarcoplasmic reticulum (SR) 1 Ca 2ϩ -ATPase from rabbit skeletal muscle. This enzyme, a P-type ATPase, is respon...

Purification of Na+,K+-ATPase Expressed in Pichia pastoris Reveals an Essential Role of Phospholipid-Protein Interactions

“…With 1.4 g/g bound detergent or lipid, the masses of the renal complex would reach 367 kDa, the Stokes radius for a compact complex would be 6.2 nm, to be compared with the apparent molecular mass value from HPLC of 468 kDa or Stokes radius of 6.5 nm. Contrary to polyoxyethylene detergents (45), DDM is not known to overestimate Stokes radii of membrane proteins (24,46). Our difference can easily be accounted for by a shape effect (due to elongated shape and/or glycosylation).…”

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

“…Previous studies have firmly established the efficiency of APols at trapping and keeping any kind of MPs soluble (17,18,20,31,32). APol-mediated immobilization of MPs presents considerable advantages over existing approaches.…”

“…It is universal, easy to implement, extremely mild, technologically very light (a single APol can be used for any number of proteins), remarkably versatile (a vast variety of tags or chemical functions can mediate noncovalent or covalent attachment to virtually any kind of support, opening the way to examining MP/ligand interactions by a very large panel of observation techniques), and it does not call for any genetic or chemical modification of the target protein: Even a mixture of MPs, or a MP whose genetics is not under control and whose biochemistry is rudimentary, are eligible to the procedure, as long as they can be obtained in a detergentsoluble form. Because APols bind to membranes (31,33), native membrane fragments or lipid vesicles could presumably be anchored to supports by the same strategy. Binding studies can be carried out in surfactant-free buffers, in the absence of any interference and methodological complications due to the presence of detergents.…”

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

“…Whether the dynamics of MPs is affected by complexation with APols remains an open question. Functional data suggest that large-scale MP movements, which are unlikely in the case of the ␤-barrel region of tOmpA, may be slowed down by association with APols (23,51). The anomalous increase of NMR linewidths observed for tOmpA ␤-barrel residues between 600 and 800 MHz is slightly more pronounced in a DHPC than in an A8-35 environment (data not shown).…”