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Title
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Hydrogenated and fluorinated surfactants derived from Tris(hydroxymethyl)-acrylamidomethane allow the purification of a highly active yeast F1-F0 ATP-synthase with an enhanced stability.
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Authors
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J.C.Talbot,
A.Dautant,
A.Polidori,
B.Pucci,
T.Cohen-Bouhacina,
A.Maali,
B.Salin,
D.Brèthes,
J.Velours,
M.F.Giraud.
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Ref.
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J Bioenerg Biomembr, 2009,
41,
349-360.
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PubMed id
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Abstract
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Loss of stability and integrity of large membrane protein complexes as well as
their aggregation in a non-lipidic environment are the major bottlenecks to
their structural studies. We have tested
C(12)H(25)-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H(12)-TAC) among many
other detergents for extracting the yeast F(1)F(0) ATP-synthase. H(12)-TAC was
found to be a very efficient detergent for removing the enzyme from
mitochondrial membranes without altering its sensitivity towards specific
ATP-synthase inhibitors. This extracted enzyme was then solubilized by either
dodecyl maltoside (DDM), H(12)-TAC or fluorinated surfactants such as
C(2)H(5)-C(6)F(12)-C(2)H(4)-S-poly-Tris-(hydroxymethyl)acrylamidomethane
(H(2)F(6)-TAC) or
C(6)F(13)-C(2)H(4)-S-poly-Tris-(hydroxymethyl)acrylamidomethane (F(6)-TAC), two
surfactants exhibiting a comparable polar head to H(12)-TAC but bearing a
fluorinated hydrophobic tail. Preparations from enzymes purified in the presence
of H(12)-TAC were found to be more adapted for AFM imaging than ATP-synthase
purified with DDM. Keeping H(12)-TAC during the Ni-NTA IMAC purification step or
replacing it by DDM at low concentrations did not however allow preserving
enzyme activity, while fluorinated surfactants H(2)F(6)-TAC and F(6)-TAC were
found to enhance enzyme stability and integrity as indicated by sensitivity
towards inhibitors. ATPase specific activity was higher with F(6)-TAC than with
H(2)F(6)-TAC. When enzymes were mixed with egg phosphatidylcholine,
ATP-synthases purified in the presence of H(2)F(6)-TAC or F(6)-TAC were more
stable upon time than the DDM purified enzyme. Furthermore, in the presence of
lipids, an activation of ATP-synthases was observed that was transitory for
enzymes purified with DDM, but lasted for weeks for ATP-synthases isolated in
the presence of molecules with Tris polyalcoholic moieties. Relipidated enzymes
prepared with fluorinated surfactants remained highly sensitive towards
inhibitors, even after 6 weeks.
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