Cécile Breyton

Hemifluorinated surfactants: a non-dissociating environment for handling membrane proteins in aqueous solutions?

The instability of membrane proteins in detergent solution can generally be traced to the dissociating character of detergents and often correlates with delipidation. We examine here the possibility of substituting detergents, after membrane proteins have been solubilized, with non‐detergent surfactants whose hydrophobic moiety contains a perfluorinated region that makes it lipophobic. In order to improve its affinity for the protein surface, the fluorinated chain is terminated by an ethyl group. Test proteins included bacteriorhodopsin, the cytochrome b 6 f complex, and the transmembrane region of the bacterial outer membrane protein OmpA. All three proteins were purified using classical detergents and transferred into solutions of C2H5C6F12C2H4‐S‐poly‐Tris‐(hydroxymethyl)aminomethane (HF‐TAC). Transfer to HF‐TAC maintained the native state of the proteins and prevented their precipitation. Provided the concentration of HF‐TAC was high enough, HF‐TAC/membrane protein complexes ran as single bands upon centrifugation in sucrose gradients. Bacteriorhodopsin and the cytochrome b 6 f complex, both of which are detergent‐sensitive, exhibited increased biochemical stability upon extended storage in the presence of a high concentration of HF‐TAC as compared to detergent micelles. The stabilization of cytochrome b 6 f is at least partly due to a better retention of protein‐bound lipids.

Amphipols and fluorinated surfactants: Two alternatives to detergents for studying membrane proteins in vitro.

Handling integral membrane proteins in aqueous solutions traditionally relies on the use of detergents, which are surfactants capable of dispersing the components of biological membranes into mixed micelles. The dissociating character of detergents, however, most often causes solubilized membrane proteins to be unstable. This has prompted the development of alternative, less-aggressive surfactants designed to keep membrane proteins soluble, after they have been solubilized, under milder conditions. A short overview is presented of the structure, properties, and uses of two families of such surfactants: amphiphilic polymers (amphipols) and fluorinated surfactants.

Micellar and Biochemical Properties of (Hemi)Fluorinated Surfactants Are Controlled by the Size of the Polar Head

Surfactants with fluorinated and hemifluorinated alkyl chains have yielded encouraging results in terms of membrane protein stability; however, the molecules used hitherto have either been chemically heterogeneous or formed heterogeneous micelles. A new series of surfactants whose polar head size is modulated by the presence of one, two, or three glucose moieties has been synthesized. Analytical ultracentrifugation and small-angle neutron scattering show that fluorinated surfactants whose polar head bears a single glucosyl group form very large cylindrical micelles, whereas those with two or three glucose moieties form small, homogeneous, globular micelles. We studied the homogeneity and stability of the complexes formed between membrane proteins and these surfactants by using bacteriorhodopsin and cytochrome b(6)f as models. Homogeneous complexes were obtained only with surfactants that form homogeneous micelles. Surfactants bearing one or two glucose moieties were found to be stabilizing, whereas those with three moieties were destabilizing. Fluorinated and hemifluorinated surfactants with a two-glucose polar head thus appear to be very promising molecules for biochemical applications and structural studies. They were successfully used for cell-free synthesis of the ion channel MscL.

New Amphiphiles to Handle Membrane Proteins: "Ménage à Trois" Between Chemistry, Physical Chemistry, and Biochemistry

To perform biochemical and structural studies of membrane proteins (MPs), amphiphilic molecules that mimic the hydrophobic environment of lipids are required. Over the past decades, detergents, a particular class of amphiphiles, have been the most widely used for MP study. However, detergents tend to be inactivating for MPs, which has prompted the recent design of alternative strategies. The present review focuses on fluorinated amphiphiles, also called fluorinated surfactants, whose hydrophobic tail is partially fluorinated. Fluorinated chains are lipophobic, bulkier, and more rigid than the hydrogenated ones. In consequence, fluorinated surfactants (F-surfactants) would poorly interfere with protein–protein, protein–lipid, and protein–cofactor interactions, thus contributing to the stability of solubilized MPs. Here, we first introduce the concepts motivating the exploration of different F-surfactant families. We then focus on the design and the surface and self-aggregation properties of two recent series—including some original compounds—of F-surfactants bearing branched polar heads. The promising biochemical applications of F-surfactants, from the literature from 2010, are reviewed. Finally, we provide an overview of other recently developed nonconventional amphiphiles with sugar-based head groups.

Analytical Ultracentrifugation and Size-Exclusion Chromatography Coupled with Light Scattering for the Characterization of Membrane Proteins in Solution

Analytical ultracentrifugation (AUC) sedimentation velocity (SV) with absorbance and interference detection and size-exclusion chromatography coupled with static and dynamic light scattering, absorbance, and refractive index detections (SEC/MALS) are two techniques that combine separation and analysis, in an absolute manner, of the mass and size of macromolecules in solution. We present here how they can be applied to the study of membrane proteins. We describe briefly the principles of the species separation, what the detection systems used measures, some theoretical background, the steps for data analysis with the example of the outer membrane protein FhuA, and emphasize the complementarity between these two techniques.