Amphiphilic copolymers that directly extract membrane proteins and lipids from cellular membranes to form nanodiscs combine the advantages of harsher membrane mimics with those of a native-like membrane environment. Among...

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We report herein the design and synthesis of a novel series of alkyl glycoside detergents consisting of a nonionic polar headgroup that comprises two glucose moieties in a branched arrangement (DG), onto which octane-, decane-, and dodecanethiols were grafted leading to ODG, DDG, and DDDG detergents, respectively. Micellization in aqueous solution was studied by isothermal titration calorimetry, 1H NMR spectroscopy, and surface tensiometry. Critical micellar concentration values were found to decrease by a factor of ∼10 for each pair of methylene groups added to the alkyl chain, ranging from ∼0.05 to 9 mM for DDDG and ODG, respectively. Dynamic light scattering and analytical ultracentrifugation sedimentation velocity experiments were used to investigate the size and composition of the micellar aggregates, showing that the aggregation number significantly increased from ∼40 for ODG to ∼80 for DDDG. All new compounds were able to solubilize membrane proteins (MPs) from bacterial membranes, insect cells, as well as the Madin–Darby canine kidney cells. In particular, native human adenosine receptor (A2AR) and bacterial transporter (BmrA) were solubilized efficiently. Striking thermostability improvements of +13 and +8 °C were observed when ODG and DDG were, respectively, applied to wild-type and full-length A2AR. Taken together, this novel detergent series shows promising detergent potency for solubilization and stabilization of membrane proteins (MPs) and thus makes a valuable addition to the chemical toolbox available for extracting and handling these important but challenging MP targets.

Two new surfactants, F 5 OM and F 5 DM, were designed as partially fluorinated analogs of ndodecyl-β-D-maltoside (DDM). The micellization properties and the morphologies of the aggregates formed by the two surfactants in water and phosphate buffer were evaluated by NMR spectroscopy, surface tension measurement (SFT), isothermal titration calorimetry (ITC), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), and analytical ultracentrifugation (AUC). As expected, the critical micellar concentration (CMC) was found to decrease with chain length of the fluorinated tail from 2.1-2.5 mM for F 5 OM to 0.3-0.5 mM for F 5 DM, and micellization was mainly entropy-driven at 25°C. Close to their respective CMC, the micelle sizes were similar for both surfactants i.e. 7 and 13 nm for F 5 OM and F 5 DM, respectively and both increased with concentration forming 4 nm diameter rods with maximum dimensions of 50 and 70 nm, respectively, at a surfactant concentration of ~30 mM. The surfactants were found to readily solubilize lipid vesicles and extract membrane proteins (MPs) directly from Escherichia coli membranes. They were found more efficient than the commercial fluorinated detergent F 6 H 2 OM over a broad range of concentrations (1-10 mM) and even better than DDM at low concentrations (1-5 mM). When transferred into the two new surfactants, the thermal stability of the proteins bacteriorhodopsin (bR) and FhuA were higher than in the presence of their solubilization detergents and similar to that in DDM; furthermore, bR was stable over several months. The membrane enzymes SpNOX and BmrA were not as active as in DDM micelles but similarly active as in F 6 OM. Together, these findings indicate both extracting and stabilizing properties of the new maltose-based fluorinated surfactants, making them promising tools in MPs applications.

According to their lifestyle, plant pathogens are divided into biotrophic and necrotrophic organisms. Biotrophic pathogens exclusively nourish on living host cells, whereas necrotrophic pathogens rapidly kill host cells and nourish on cell walls and cell contents. To this end, the necrotrophic fungus Botrytis cinerea secretes large amounts of phytotoxic proteins and cell wall degrading enzymes. However, the precise role of these proteins during infection is unknown. Here, we report on the identification and characterization of the previously unknown toxic protein hypersensitive response inducing protein 1 (Hip1), which induces plant cell death. We found the adoption of a structurally conserved folded Alternaria alternata Alt a 1 protein structure to be a prerequisite for Hip1 to exert its necrosis-inducing activity in a host-specific manner. Localization and the induction of typical plant defense responses by Hip1 indicate recognition as a pathogen-associated molecular pattern at the plant plasma membrane. In contrast to other secreted toxic Botrytis proteins, activity of Hip1 does not depend on the presence of the receptor-associated kinases BRI1-ASSOCIATED KINASE 1 (BAK1) and SUPPRESSOR OF BIR1-1 (SOBIR1) Our results demonstrate that recognition of Hip1, even in the absence of obvious enzymatic or pore-forming activity, induces strong plant defense reactions eventually leading to plant cell death. Botrytis hip1 overexpression strains generated by CRISPR/Cas9 displayed enhanced infection, indicating the virulence-promoting potential of Hip1. Taken together, Hip1 induces a non-canonical defence response which might be a common feature of structurally conserved fungal proteins from the Alt a 1 family.