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Fluorinated and hemifluorinated surfactants as alternatives to detergents for membrane protein cell-free synthesis.

ABSTRACT: Hemifluorinated and fluorinated surfactants are lipophobic and, as such, non-detergent. Although they do not solubilize biological membranes, they can, after conventional solubilization, substitute for detergents to keep membrane proteins soluble, which generally improves their stability [Breyton, Chabaud, Chaudier, Pucci and Popot (2004) FEBS Lett. 564, 312-318]. In the present study, we show that (hemi)fluorinated surfactants can be used for in vitro synthesis of membrane proteins: they do not interfere with protein synthesis, and they provide a suitable environment for MscL, a pentameric mechanosensitive channel, to fold and oligomerize to its native functional state. Following synthesis, both types of surfactants can be used to deliver MscL directly to pre-formed lipid vesicles. The electrophysiological activity of MscL synthesized in vitro in the presence of either hemi- or per-fluorinated surfactant is similar to that of the protein expressed in vivo.

SUBMITTER: Park KH

PROVIDER: S-EPMC1828882 | biostudies-other | 2007 Apr

REPOSITORIES: biostudies-other

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Project description:Solubilizing membrane proteins for functional, structural and thermodynamic studies is usually achieved with the help of detergents, which, however, tend to destabilize them. Several classes of non-detergent surfactants have been designed as milder substitutes for detergents, most prominently amphipathic polymers called 'amphipols' and fluorinated surfactants. Here we test the potential usefulness of these compounds for thermodynamic studies by examining their effect on conformational transitions of the diphtheria toxin T-domain. The advantage of the T-domain as a model system is that it exists as a soluble globular protein at neutral pH yet is converted into a membrane-competent form by acidification and inserts into the lipid bilayer as part of its physiological action. We have examined the effects of various surfactants on two conformational transitions of the T-domain, thermal unfolding and pH-induced transition to a membrane-competent form. All tested detergent and non-detergent surfactants lowered the cooperativity of the thermal unfolding of the T-domain. The dependence of enthalpy of unfolding on surfactant concentration was found to be least for fluorinated surfactants, thus making them useful candidates for thermodynamic studies. Circular dichroism measurements demonstrate that non-ionic homopolymeric amphipols (NAhPols), unlike any other surfactants, can actively cause a conformational change of the T-domain. NAhPol-induced structural rearrangements are different from those observed during thermal denaturation and are suggested to be related to the formation of the membrane-competent form of the T-domain. Measurements of leakage of vesicle content indicate that interaction with NAhPols not only does not prevent the T-domain from inserting into the bilayer, but it can make bilayer permeabilization even more efficient, whereas the pH-dependence of membrane permeabilization becomes more cooperative. This article is part of a Special Issue entitled: Protein Folding in Membranes.

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Fluorinated and hemifluorinated surfactants as alternatives to detergents for membrane protein cell-free synthesis.

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