Protective and inhibitory effects of various types of amphipols on the Ca2+-ATPase from sarcoplasmic reticulum: a comparative study.
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 Ca2+-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 Ca2+-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.
Picard
Martin
M
Section de Biophysique des Fonctions Membranaires (Commissariat à l'Energie Atomique), Institut Fédératif de Recherches 46, Département de Biologie Joliot-Curie at CEA Saclay, 91191 Gif-sur-Yvette cedex, France.
Dahmane
Tassadite
T
Garrigos
Manuel
M
Gauron
Carole
C
Giusti
Fabrice
F
le Maire
Marc
M
Popot
Jean-Luc
JL
Champeil
Philippe
P
D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D003902:Detergents / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057927:Hydrophobic and Hydrophilic Interactions; D010766:Phosphorylation; D020011:Protective Agents / Q000494:pharmacology*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000201:enzymology; D012995:Solubility; D012997:Solvents / Q000737:chemistry; D013451:Sulfonic Acids / Q000737:chemistry; D013997:Time Factors
D003902:Detergents; D004791:Enzyme Inhibitors; D020011:Protective Agents; D012997:Solvents; D013451:Sulfonic Acids; D000252:Calcium-Transporting ATPases; D002118:Calcium