Supramolecular assembly of pyrene-DNA conjugates: influence of pyrene substitution pattern and implications for artificial LHCs.

The supramolecular self-assembly of pyrene-DNA conjugates into nanostructures is presented. DNA functionalized with different types of pyrene isomers at the 3'-end self-assemble into nano-objects. The shape of the nanostructures is influenced by the type of pyrene isomer appended to the DNA. Multilamellar vesicles are observed with the 1,6- and 1,8-isomers, whereas conjugates of the 2,7-isomer exclusively assemble into spherical nanoparticles. Self-assembled nano-spheres obtained with the 2,7-dialkynyl pyrene isomer were used for the construction of an artificial light-harvesting complex (LHC) in combination with Cy3 as the energy acceptor

Tetraphenylethylene–DNA conjugates: influence of sticky ends and DNA sequence length on the supramolecular assembly of AIE-active vesicles

The supramolecular assembly of DNA conjugates, functionalized with tetraphenylethylene (TPE) sticky ends, into vesicular structures is described. The aggregation-induced emission (AIE) active TPE units allow to monitor the assembly process by fluorescence spectroscopy. The number of TPE modifications in the overhangs of the conjugates influences the supramolecular assembly behavior. A minimum of two TPE residues on each end are required to ensure a well-defined assembly process. The design of the presented DNA-based nanostructures offers tailored functionalization with applications in DNA nanotechnology

Supramolecular Assembly of Pyrene-DNA Conjugates into Columnar Vesicles

This poster describes the supramolecular assembly of DNA conjugates functionalized with pyrene sticky-ends. After hybridization, the 3’-end modified DNA single strands self-assembled into vesicles with diameters of 50–200 nm. Columnar packed aggregated and multilamellar vesicles were observed by cryo-EM

A small ribosome-associated ncRNA globally inhibits translation by restricting ribosome dynamics

Ribosome-associated non-coding RNAs (rancRNAs) have been recognized as an emerging class of regulatory molecules capable of fine-tuning translation in all domains of life. RancRNAs are ideally suited for allowing a swift response to changing environments and are therefore considered pivotal during the first wave of stress adaptation. Previously, we identified an mRNA-derived 18 nucleotides long rancRNA (rancRNA_18) in Saccharomyces cerevisiae that rapidly downregulates protein synthesis during hyperosmotic stress. However, the molecular mechanism of action remained enigmatic. Here, we combine biochemical, genetic, transcriptome-wide and structural evidence, thus revealing rancRNA_18 as global translation inhibitor by targeting the E-site region of the large ribosomal subunit. Ribosomes carrying rancRNA_18 possess decreased affinity for A-site tRNA and impaired structural dynamics. Cumulatively, these discoveries reveal the mode of action of a rancRNA involved in modulating protein biosynthesis at a thus far unequalled precision

Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling

AbstractMany biological processes depend on the sequential assembly of protein complexes. However, studying the kinetics of such processes by direct methods is often not feasible. As an important class of such protein complexes, pore-forming toxins start their journey as soluble monomeric proteins, and oligomerize into transmembrane complexes to eventually form pores in the target cell membrane. Here, we monitored pore formation kinetics for the well-characterized bacterial pore-forming toxin aerolysin in single cells in real time to determine the lag times leading to the formation of the first functional pores per cell. Probabilistic modeling of these lag times revealed that one slow and seven equally fast rate-limiting reactions best explain the overall pore formation kinetics. The model predicted that monomer activation is the rate-limiting step for the entire pore formation process. We hypothesized that this could be through release of a propeptide and indeed found that peptide removal abolished these steps. This study illustrates how stochasticity in the kinetics of a complex process can be exploited to identify rate-limiting mechanisms underlying multistep biomolecular assembly pathways

Dual chaperone role of the c-terminal propeptide in folding and oligomerization of the pore-forming toxin aerolysin

Throughout evolution, one of the most ancient forms of aggression between cells or organisms has been the production of proteins or peptides affecting the permeability of the target cell membrane. This class of virulence factors includes the largest family of bacterial toxins, the pore-forming toxins (PFTs). PFTs are bistable structures that can exist in a soluble and a transmembrane state. It is unclear what drives biosynthetic folding towards the soluble state, a requirement that is essential to protect the PFT-producing cell. Here we have investigated the folding of aerolysin, produced by the human pathogen Aeromonas hydrophila, and more specifically the role of the C-terminal propeptide (CTP). By combining the predictive power of computational techniques with experimental validation using both structural and functional approaches, we show that the CTP prevents aggregation during biosynthetic folding. We identified specific residues that mediate binding of the CTP to the toxin. We show that the CTP is crucial for the control of the aerolysin activity, since it protects individual subunits from aggregation within the bacterium and later controls assembly of the quaternary pore-forming complex at the surface of the target host cell. The CTP is the first example of a C-terminal chain-linked chaperone with dual function

The Structure and Symmetry of the Radial Spoke Protein Complex in \u3cem\u3eChlamydomonas\u3c/em\u3e Flagella

The radial spoke is a key element in a transducer apparatus controlling the motility of eukaryotic cilia. The transduction biomechanics is a long-standing question in cilia biology. The radial spoke has three regions – a spoke head, a bifurcated neck and a stalk. Although the neck and the stalk are asymmetric, twofold symmetry of the head has remained controversial. In this work we used single particle cryo-electron microscopy (cryo-EM) analysis to generate a 3D structure of the whole radial spoke at unprecedented resolution. We show the head region at 15 Å (1.5 nm) resolution and confirm twofold symmetry. Using distance constraints generated by cross-linking mass spectrometry, we locate two components, RSP2 and RSP4, at the head and neck regions. Our biophysical analysis of isolated RSP4, RSP9, and RSP10 affirmed their oligomeric state. Our results enable us to redefine the boundaries of the regions and propose a model of organization of the radial spoke component proteins

Ultrasensitive Label-Free Detection of Protein-Membrane Interaction Exemplified by Toxin-Liposome Insertion.

Measuring the high-affinity binding of proteins to liposome membranes remains a challenge. Here, we show an ultrasensitive and direct detection of protein binding to liposome membranes using high throughput second harmonic scattering (SHS). Perfringolysin O (PFO), a pore-forming toxin, with a highly membrane selective insertion into cholesterol-rich membranes is used. PFO inserts only into liposomes with a cholesterol concentration >30%. Twenty mole-percent cholesterol results in neither SHS-signal deviation nor pore formation as seen by cryo-electron microscopy of PFO and liposomes. PFO inserts into cholesterol-rich membranes of large unilamellar vesicles in an aqueous solution with Kd = (1.5 ± 0.2) × 10-12 M. Our results demonstrate a promising approach to probe protein-membrane interactions below sub-picomolar concentrations in a label-free and noninvasive manner on 3D systems. More importantly, the volume of protein sample is ultrasmall (<10 μL). These findings enable the detection of low-abundance proteins and their interaction with membranes

Dissecting Out the Molecular Mechanism of Insecticidal Activity of Ostreolysin A6/Pleurotolysin B Complexes on Western Corn Rootworm

Ostreolysin A6 (OlyA6) is a protein produced by the oyster mushroom (Pleurotus ostreatus). It binds to membrane sphingomyelin/cholesterol domains, and together with its protein partner, pleurotolysin B (PlyB), it forms 13-meric transmembrane pore complexes. Further, OlyA6 binds 1000 times more strongly to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with PlyB, OlyA6 has potent and selective insecticidal activity against the western corn rootworm. We analysed the histological alterations of the midgut wall columnar epithelium of western corn rootworm larvae fed with OlyA6/PlyB, which showed vacuolisation of the cell cytoplasm, swelling of the apical cell surface into the gut lumen, and delamination of the basal lamina underlying the epithelium. Additionally, cryo-electron microscopy was used to explore the membrane interactions of the OlyA6/PlyB complex using lipid vesicles composed of artificial lipids containing CPE, and western corn rootworm brush border membrane vesicles. Multimeric transmembrane pores were formed in both vesicle preparations, similar to those described for sphingomyelin/cholesterol membranes. These results strongly suggest that the molecular mechanism of insecticidal action of OlyA6/PlyB arises from specific interactions of OlyA6 with CPE, and the consequent formation of transmembrane pores in the insect midgut

Cryo-EM structure of lysenin pore elucidates membrane insertion by an aerolysin family protein

Lysenin from the coelomic fluid of the earthworm Eisenia fetida belongs to the aerolysin family of small β-pore-forming toxins (β-PFTs), some members of which are pathogenic to humans and animals. Despite efforts, a high-resolution structure of a channel for this family of proteins has been elusive and therefore the mechanism of activation and membrane insertion remains unclear. Here we determine the pore structure of lysenin by single particle cryo-EM, to 3.1 Å resolution. The nonameric assembly reveals a long β-barrel channel spanning the length of the complex that, unexpectedly, includes the two pre-insertion strands flanking the hypothetical membrane-insertion loop. Examination of other members of the aerolysin family reveals high structural preservation in this region, indicating that the membrane-insertion pathway in this family is conserved. For some toxins, proteolytic activation and pro-peptide removal will facilitate unfolding of the pre-insertion strands, allowing them to form the β-barrel of the channel