Nikos S. Hatzakis

How curved membranes recruit amphipathic helices and protein anchoring motifs

Lipids and several specialized proteins are thought to be able to sense the curvature of membranes (MC). Here we used quantitative fluorescence microscopy to measure curvature-selective binding of amphipathic motifs on single liposomes 50-700 nm in diameter. Our results revealed that sensing is predominantly mediated by a higher density of binding sites on curved membranes instead of higher affinity. We proposed a model based on curvature-induced defects in lipid packing that related these findings to lipid sorting and accurately predicted the existence of a new ubiquitous class of curvature sensors: membrane-anchored proteins. The fact that unrelated structural motifs such as alpha-helices and alkyl chains sense MC led us to propose that MC sensing is a generic property of curved membranes rather than a property of the anchoring molecules. We therefore anticipate that MC will promote the redistribution of proteins that are anchored in membranes through other types of hydrophobic moieties.

β-Cyclodextrin-Appended Giant Amphiphile: Aggregation to Vesicle Polymersomes and Immobilisation of Enzymes

A giant amphiphile consisting of polystyrene end-capped with permethylated beta-cyclodextrin was synthesised and found to form vesicular structures when injected as a solution in THF into water. The ability of the cyclodextrins on the surface of the polymersomes to form inclusion complexes with hydrophobic compounds was tested by carrying out a competition experiment with a fluorescent probe sensitive to the polarity of the surrounding medium. It was found that 1-adamantol can displace the fluorescent probe from the cavities of the cyclodextrin moieties of the polymersomes. The recognition of molecules by cell membranes in nature is often based on interactions with specific membrane receptors. To mimic this behaviour, the enzyme horseradish peroxidase was modified with adamantane groups through a poly(ethylene glycol) spacer and its interaction with the polymersomes was investigated. It was established that the presence of adamantane moieties on each enzyme allowed a host-guest interaction with the multifunctional surface of the polymersomes.

The enzyme mechanism of nitrite reductase studied at single-molecule level

A generic method is described for the fluorescence “readout” of the activity of single redox enzyme molecules based on Förster resonance energy transfer from a fluorescent label to the enzyme cofactor. The method is applied to the study of copper-containing nitrite reductase from Alcaligenes faecalis S-6 immobilized on a glass surface. The parameters extracted from the single-molecule fluorescence time traces can be connected to and agree with the macroscopic ensemble averaged kinetic constants. The rates of the electron transfer from the type 1 to the type 2 center and back during turnover exhibit a distribution related to disorder in the catalytic site. The described approach opens the door to single-molecule mechanistic studies of a wide range of redox enzymes and the precise investigation of their internal workings.

A unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteins.

The discovery of proteins that recognize membrane curvature created a paradigm shift by suggesting that membrane shape may act as a cue for protein localization that is independent of lipid or protein composition. Here we review recent data on membrane curvature sensing by three structurally unrelated motifs: BAR domains, amphipathic helices and membrane-anchored proteins. We discuss the conclusion that the curvature of the BAR dimer is not responsible for sensing and that the sensing properties of all three motifs can be rationalized by the physicochemical properties of the curved membrane itself. We thus anticipate that membrane curvature will promote the redistribution of proteins that are anchored in membranes through any type of hydrophobic moiety, a thesis that broadens tremendously the implications of membrane curvature for protein sorting, trafficking and signaling in cell biology.

Observation of inhomogeneity in the lipid composition of individual nanoscale liposomes.

Liposomes, or vesicles, have been studied extensively both as models of biological membranes and as drug delivery vehicles. Typically it is assumed that all liposomes within the same preparation are identical. Here by employing pairs of fluorescently labeled lipids we demonstrated an up to 10-fold variation in the relative lipid composition of individual liposomes with diameters between 50 nm and 15 μm. Since the physicochemical properties of liposomes are directly linked to their composition, a direct consequence of compositional inhomogeneities is a polydispersity in the properties of the individual liposomes in an ensemble.

Characterization of a dynamic metabolon producing the defense compound dhurrin in sorghum

Metabolite channeling by a dynamic metabolon The specialized metabolite dhurrin breaks down into cyanide when plant cell walls have been chewed, deterring insect pests. Laursen et al. found that the enzymes that synthesize dhurrin in sorghum assemble as a metabolon in lipid membranes (see the Perspective by Dsatmaichi and Facchini). The dynamic nature of metabolon assembly and disassembly provides dhurrin on an as-needed basis. Membrane-anchored cytochrome P450s cooperated with a soluble glucosyltransferase to channel intermediates toward efficient dhurrin production. Science, this issue p. 890; see also p. 829 Enzymes that synthesize a specialized metabolite congregate and disperse on an as-needed basis in the lipid membrane. Metabolic highways may be orchestrated by the assembly of sequential enzymes into protein complexes, or metabolons, to facilitate efficient channeling of intermediates and to prevent undesired metabolic cross-talk while maintaining metabolic flexibility. Here we report the isolation of the dynamic metabolon that catalyzes the formation of the cyanogenic glucoside dhurrin, a defense compound produced in sorghum plants. The metabolon was reconstituted in liposomes, which demonstrated the importance of membrane surface charge and the presence of the glucosyltransferase for metabolic channeling. We used in planta fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to study functional and structural characteristics of the metabolon. Understanding the regulation of biosynthetic metabolons offers opportunities to optimize synthetic biology approaches for efficient production of high-value products in heterologous hosts.

Influence of the preparation route on the supramolecular organization of lipids in a vesicular system.

A confocal fluorescence microscopy-based assay was used for studying the influence of the preparation route on the supramolecular organization of lipids in a vesicular system. In this work, vesicles composed of cholesterol and CTAB (1/1 mol %) or cholesterol and DOPC (2/8 mol %) and incorporating two membrane dyes were prepared by either a compressed fluid (CF)-based method (DELOS-susp) or a conventional film hydration procedure. They were subsequently immobilized and imaged individually using a confocal fluorescence microscope. Two integrated fluorescence intensities, I(dye1) and I(dye2), were assigned to each tracked vesicle, and their ratio, I(dye1)/I(dye2), was used for quantifying the degree of membrane inhomogeneity between individual vesicles within each sample. A distribution of I(dye1)/I(dye2) values was obtained for all the studied vesicular systems, indicating intrasample heterogeneity. The degree of inhomogeneity (DI) was similar for Chol/DOPC vesicles prepared by both procedures. In contrast, DI was more than double for the hydration method compared to the CF-based method in the case of Chol/CTAB vesicles, which can suffer from lipid demixing during film formation. These findings reveal a more homogeneous vesicle formation path by CFs, which warranted good homogeneity of the vesicular system, independently of the lipid mixture used.

Mixing subattolitre volumes in a quantitative and highly parallel manner with soft matter nanofluidics

Handling and mixing ultrasmall volumes of reactants in parallel can increase the throughput and complexity of screening assays while simultaneously reducing reagent consumption. Microfabricated silicon and plastic can provide reliable fluidic devices, but cannot typically handle total volumes smaller than ∼1 × 10(-12) l. Self-assembled soft matter nanocontainers can in principle significantly improve miniaturization and biocompatibility, but exploiting their full potential is a challenge due to their small dimensions. Here, we show that small unilamellar lipid vesicles can be used to mix volumes as small as 1 × 10(-19) l in a reproducible and highly parallelized fashion. The self-enclosed nanoreactors are functionalized with lipids of opposite charge to achieve reliable fusion. Single vesicles encapsulating one set of reactants are immobilized on a glass surface and then fused with diffusing vesicles of opposite charge that carry a complementary set of reactants. We find that ∼85% of the ∼1 × 10(6) cm(-2) surface-tethered nanoreactors undergo non-deterministic fusion, which is leakage-free in all cases, and the system allows up to three to four consecutive mixing events per nanoreactor.

Monitoring Shifts in the Conformation Equilibrium of the Membrane Protein Cytochrome P450 Reductase (POR) in Nanodiscs

Background: Investigating the mechanism of NADPH-dependent conformational changes of POR in nanodiscs. Results: The conformational equilibrium of compact and extended POR, shifts toward the compact form (from 30 to 60%) upon reduction by NADPH. Conclusion: The NADPH-dependent conformational changes follow the “swinging model.” Significance: This is the first time that the action of a membrane protein located in a lipid bilayer environment is probed by neutron reflectivity. Nanodiscs are self-assembled ∼50-nm2 patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.

Membrane curvature enables N-Ras lipid anchor sorting to liquid-ordered membrane phases

Trafficking and sorting of membrane-anchored Ras GTPases are regulated by partitioning between distinct membrane domains. Here, in vitro experiments and microscopic molecular theory reveal membrane curvature as a new modulator of N-Ras lipid anchor and palmitoyl chain partitioning. Membrane curvature was essential for enrichment in raft-like liquid-ordered phases; enrichment was driven by relief of lateral pressure upon anchor insertion and most likely affects the localization of lipidated proteins in general.