Membrane Partitioning: “Classical” and “Nonclassical” Hydrophobic Effects

The free energy of transfer of nonpolar solutes from water to lipid bilayers is often dominated by a large negative enthalpy rather than the large positive entropy expected from the hydrophobic effect. This common observation has led to the idea that membrane partitioning is driven by the “nonclassical” hydrophobic effect. We examined this phenomenon by characterizing the partitioning of the well-studied peptide melittin using isothermal titration calorimetry (ITC) and circular dichroism (CD). We studied the temperature dependence of the entropic (−TΔS) and enthalpic (ΔH) components of free energy (ΔG) of partitioning of melittin into lipid membranes made of various mixtures of zwitterionic and anionic lipids. We found significant variations of the entropic and enthalpic components with temperature, lipid composition and vesicle size but only small changes in ΔG (entropy–enthalpy compensation). The heat capacity associated with partitioning had a large negative value of about −0.5 kcal mol−1 K−1. This hallmark of the hydrophobic effect was found to be independent of lipid composition. The measured heat capacity values were used to calculate the hydrophobic-effect free energy ΔGhΦ, which we found to dominate melittin partitioning regardless of lipid composition. In the case of anionic membranes, additional free energy comes from coulombic attraction, which is characterized by a small effective peptide charge due to the lack of additivity of hydrophobic and electrostatic interactions in membrane interfaces [Ladokhin and White J Mol Biol 309:543–552, 2001]. Our results suggest that there is no need for a special effect—the nonclassical hydrophobic effect—to describe partitioning into lipid bilayers

Concentration dependent effect of GsMTx4 on mechanosensitive channels of small conductance in E. coli spheroplasts

The spider peptide GsMTx4, at saturating concentration of 5 muM, is an effective and specific inhibitor for stretch-activated mechanosensitive (MS) channels found in a variety of eukaryotic cells. Although the structure of the peptide has been solved, the mode of action remains to be determined. Because of its amphipathic structure, the peptide is proposed to interact with lipids at the boundaries of the MS channel proteins. In addition, GsMTx4 has antimicrobial effects, inhibiting growth of several species of bacteria in the range of 5-64 microM. Previous studies on prokaryotic MS channels, which serve as model systems to explore the principle of MS channel gating, have shown that various amphipathic compounds acting at the protein-lipid interface affect MS channel gating. We have therefore analyzed the effect of different concentrations of extracellular GsMTx4 on MS channels of small conductance, MscS and MscK, in the cytoplasmic membrane of wild-type E. coli spheroplasts using the patch-clamp technique. Our study shows that the peptide GsMTx4 exhibits a biphasic response in which peptide concentration determines inhibition or potentiation of activity in prokaryotic MS channels. At low peptide concentrations of 2 and 4 microM the gating of the prokaryotic MS channels was hampered, manifested by a decrease in pressure sensitivity. In contrast, application of peptide at concentrations of 12 and 20 microM facilitated prokaryotic MS channel opening by increasing the pressure sensitivity

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

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 C12H25-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H12-TAC) among many other detergents for extracting the yeast F1F0 ATP-synthase. H12-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), H12-TAC or fluorinated surfactants such as C2H5-C6F12-C2H4-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H2F6-TAC) or C6F13-C2H4-S-poly-Tris-(hydroxymethyl)acrylamidomethane (F6-TAC), two surfactants exhibiting a comparable polar head to H12-TAC but bearing a fluorinated hydrophobic tail. Preparations from enzymes purified in the presence of H12-TAC were found to be more adapted for AFM imaging than ATP-synthase purified with DDM. Keeping H12-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 H2F6-TAC and F6-TAC were found to enhance enzyme stability and integrity as indicated by sensitivity towards inhibitors. ATPase specific activity was higher with F6-TAC than with H2F6-TAC. When enzymes were mixed with egg phosphatidylcholine, ATP-synthases purified in the presence of H2F6-TAC or F6-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

New amphiphiles to handle membrane proteins: "ménage à trois" between chemistry, physical chemistry and biochemistry.

International audienceTo 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