Molecular and dimensional profiling of highly purified extracellular vesicles by fluorescence fluctuation spectroscopy.

Cells secrete extracellular vesicles (EVs) into their microenvironment that act as mediators of intercellular communication under physiological conditions and in this context also actively participate in spreading various diseases. Large efforts are currently made to produce reliable EV samples and to develop, improve, and standardize techniques allowing their biophysical characterization. Here, we used ultrafiltration and size-exclusion chromatography for the isolation and a model-free fluorescence fluctuation analysis for the investigation of the physical and biological properties of EVs secreted by mammalian cells. Our purification strategy produced enriched samples of morphologically intact EVs free of extravesicular proteins and allowed labeling of marker molecules on the vesicle surface for single-vesicle analysis with single-molecule sensitivity. This novel approach provides information on the distribution profile of both EV size and relative expression level of a specific exosomal marker, deciphering the overall heterogeneity of EV preparations.

Structure-based engineering of a minimal porin reveals loop-independent channel closure.

Porins, like outer membrane protein G (OmpG) of Escherichia coli, are ideal templates among ion channels for protein and chemical engineering because of their robustness and simple architecture. OmpG shows fast transitions between open and closed states, which were attributed to loop 6 (L6). As flickering limits single-channel-based applications, we pruned L6 by either 8 or 12 amino acids. While the open probabilities of both L6 variants resemble that of native OmpG, their gating frequencies were reduced by 63 and 81%, respectively. Using the 3.2 Å structure of the shorter L6 variant in the open state, we engineered a minimal porin (220 amino acids), where all remaining extramembranous loops were truncated. Unexpectedly, this minimized porin still exhibited gating, but it was 5-fold less frequent than in OmpG. The residual gating of the minimal pore is hence independent of L6 rearrangements and involves narrowing of the ion conductance pathway most probably driven by global stretching-flexing deformations of the membrane-embedded ß-barrel.

Flexibility of the N-terminal mVDAC1 segment controls the channel's gating behavior.

Since the solution of the molecular structures of members of the voltage dependent anion channels (VDACs), the N-terminal α-helix has been the main focus of attention, since its strategic location, in combination with its putative conformational flexibility, could define or control the channel's gating characteristics. Through engineering of two double-cysteine mVDAC1 variants we achieved fixing of the N-terminal segment at the bottom and midpoint of the pore. Whilst cross-linking at the midpoint resulted in the channel remaining constitutively open, cross-linking at the base resulted in an "asymmetric" gating behavior, with closure only at one electric field's orientation depending on the channel's orientation in the lipid bilayer. Additionally, and while the native channel adopts several well-defined closed states (S1 and S2), the cross-linked variants showed upon closure a clear preference for the S2 state. With native-channel characteristics restored following reduction of the cysteines, it is evident that the conformational flexibility of the N-terminal segment plays indeed a major part in the control of the channel's gating behavior.

Chemical engineering of Mycobacterium tuberculosis dodecin hybrids.

The suitability for chemical engineering of the highly symmetrical Mycobacterium tuberculosis dodecin was investigated, its inner cavity providing a large compartment shields introduced compounds from bulk solvent. Hybrids were obtained by S-alkylation of cysteine mutants and characterized by spectroscopic methods, including the crystal structures of wild type and biohybrid dodecins.

Strategies and perspectives in ion-channel engineering.

Membranes form natural barriers that need to be permeable to diverse matter like ions and substrates. This permeability is controlled by ion-channel proteins, which have attracted great interest for pharmaceutical applications. Ion-channel engineering (ICE) modifies biological ion channels by chemical/biological synthetis means. The goal is to obtain ion channels with modified or novel functionality. Three functional strategies exist. The first is the manipulation of the wider pores with robust ß-barrel structures, such as those of α-hemolysin and porins. The second engineering approach focuses on the modification of narrow (mostly α-helical) pores to understand selectivity and modes of action. A third functional approach addresses channel gating by (photo)triggering the biological receptor that controls the channel. Several synthetis strategies have been developed and successfully utilized for the synthetic modification of biological ion-channels: the S-alkylation of specifically introduced Cys, protein semisynthesis by native chemical ligation, protein semisynthesis by protein trans-splicing, as well as nonsense-suppression methods. Structural studies (X-ray crystallography, NMR spectroscopy) are necessary to support the functional studies and to afford predictable engineering. The reprogramming and re-engineering of channels can be used for sensing applications, treatment of channelopathies, chemical neurobiology, and providing novel lead compounds for targeting ion channels.

Structural characterization of a beta-turn mimic within a protein-protein interface.

ß-Turns are secondary structure elements not only exposed on protein surfaces, but also frequently found to be buried in protein-protein interfaces. Protein engineering so far considered mainly the backbone-constraining properties of synthetic ß-turn mimics as parts of surface-exposed loops. A ß-turn mimic, Hot═Tap, that is available in gram amounts, provides two hydroxyl groups that enhance its turn-inducing properties besides being able to form side-chain-like interactions. NMR studies on cyclic hexapeptides harboring the Hot═Tap dipeptide proved its strong ß-turn-inducing capability. Crystallographic analyses of the trimeric fibritin-foldon/Hot═Tap hybrid reveal at atomic resolution how Hot═Tap replaces a ßI'-turn by a ßII'-type structure. Furthermore, Hot═Tap adapts to the complex protein environment by participating in several direct and water-bridged interactions across the foldon trimer interface. As building blocks, ß-turn mimics capable of both backbone and side-chain mimicry may simplify the design of synthetic proteins.

Structural and functional characterization of a synthetically modified OmpG.

Chemical modification of ion channels has recently attracted attention due to their potential use in stochastic sensing and neurobiology. Among the available channel templates stable ß-barrel proteins have shown their potential for large scale chemical modifications due to their wide pore lumen. Ion-channel hybrids using the outer membrane protein OmpG were generated by S-alkylation with a synthetic modulator and functionally as well as structurally characterized. The dansyl moiety of the used modulator resulted in partial blockage of current though the OmpG channel with its gating characteristics mainly unaffected. The crystal structure of an OmpG-dansyl hybrid at 2.4Å resolution correlates this finding by showing that the modulator lines the inner walling of the OmpG pore. These results underline the suitability of OmpG as a structural base for the construction of stochastic sensors.

Xenobiotics removal from polluted water by a multifunctional constructed wetland.

Removal efficiencies on xenobiotics from polluted water in a twin-shaped constructed wetland consisting of a vertical flow chamber with the crop plant Colocasia esculenta L. Schott and a reverse vertical flow one with Ischaemum aristatum var. glaucum Honda, were assessed by chemical analysis and bioassays. After a four-month period of application, removal efficiencies of the applied pesticides parathion and omethoate were 100% with no detectable parathion and omethoate in the effluent. For the applied herbicides, the decontamination was less efficient with removal efficiencies of 36% and 0% for 4-chloro-2-methyl-phenoxyacetic acid and dicamba, respectively. As shown by toxicity assay with duckweed Lemna minor L., growth retardation may occur if the water treated for herbicide removal is used in irrigation of sensitive cultivars in agriculture or horticulture. In contrast to I. aristatum var. glaucum Honda, the crop C. esculenta L. Schott has a high yield in biomass production as a valuable source of renewable energy.

Sulphonated aromatic pollutants. Limits of microbial degradability and potential of phytoremediation.

Many synthetic sulphonated aromatic compounds are used as starting material to produce dyes and pigments, or are released as by-products in the effluents of the textile and dye industry. A large number of these chemicals are poorly biodegradable and cannot be eliminated by classical wastewater treatment plants. To limit the impact of these pollutants on the environment, new processes, based on the use of higher plants (constructed wetlands or hydroponic systems), are under development. Detergents and surfactants are essential for both industrial and domestic applications, the most important family being the alkylbenzene sulphonates. Originally, the alkyl side chains were branched and thus recalcitrant to biodegradation. Therefore, they have been replaced by linear alkylbenzene sulphonates. Although more acceptable, present formulations still have adverse environmental and toxic effects. In this context, phytoremediation appears to be a promising approach to remove these compounds from contaminated soils and waters.