Biology subjects

Cosa, G.

Publications and source records attributed to Cosa, G..

4 recordsLinked to original sources

bioRxiv2025

Single-molecule assay reveals the impact of composition, RNA duplex, and inhibitors on the binding dynamics of SARS-CoV-2 polymerase complex

The genome replication of SARS-CoV-2, the causative agent of COVID-19, involves a multi-subunit replication complex consisting of non-structural proteins (nsps) 12, 7 and 8. While the structure of this complex is known, the dynamic behavior of the subunits interacting with RNA is missing. Here we report a single-molecule protein-induced fluorescence enhancement (SM-PIFE) assay to monitor binding dynamics between the reconstituted or co-expressed replication complex and RNA. Increasing binding times were observed, in this order, with nsp7 (none) nsp8 and nsp12, in nsp8-nsp12 mixtures and in reconstituted mixtures bearing all three proteins. Unstable, transient, and stable binding modes were recorded in the latter case, indicating that complexation is dynamic, and the correct conformation must be achieved before stable RNA binding can occur. Notably, the co-expressed protein yields mostly stable binding even at low concentrations, while the reconstituted proteins exhibit unstable binding indicating inefficient complexation with reduced protein. The SM-PIFE assay distinguishes inhibitors that impact protein binding from those that prevent replication, as demonstrated with suramin and remdesivir, respectively. The data reveals a correlation between binding lifetime/affinity, and protein activity, and underscores differences between co-expressed vs reconstituted mixtures, suggesting the existence of trapped conformations that may not evolve to productive binding.

biophysics↗

bioRxiv2024

Extended Plasmonic Nanostructures Templated by Tobacco Mosaic Virus Coat Protein

Optical and magnetic metamaterials possess interesting properties that cannot be achieved with conventional materials. However, there is currently no synthetic method offering both scalability and nanometer spatial precision. Biotemplating is a promising technique that has the potential to organize nanoscale components with high precision while being scalable and low-cost. Here we demonstrate a versatile template using hexahistidine-tagged tobacco mosaic virus coat protein. The protein self-assembles into disks which further assemble into extended nanostructures under mild conditions. Large sheets with either hexagonal or square packing and core-shell nanorods were formed, and gold nanoparticles were attached to the disks within each nanostructure to form assemblies of nanoparticle rings.

biochemistry↗

bioRxiv2024

Automated Synthesis of Wireframe DNA Nanotubes

DNA nanotechnology has revolutionized our ability to position matter at the nanoscale, but the preparation of DNA-based architectures remains laborious. To facilitate the formation of custom structures, we report a fully automated method to produce sequence- and size-defined DNA nanotubes. By programming the sequential addition of desired building blocks, rigid DX-tile-based DNA nanotubes (and flexible wireframe DNA structures) are attained, where the total number of possible constructs increases as a power function of the number of different units available. Using single-molecule fluorescence imaging, the kinetics and yield of each synthetic step can be quantitatively determined, revealing differences in self-assembly dynamics as the nanotube is built up from the solid support and providing new insights into DNA self-assembly. The exploitation of automation for both assembly and analysis (tthrough an ad-hoc developed K-means clustering algorithm) facilitates a workflow wherein the synthesis parameters may be iteratively improved upon, demonstrating how a single-molecule assembly-analysis-optimization sequence can be used to generate complex, non-covalent materials in good yield. The presented synthetic strategy is generalizable, making use of equipment already available in most standard laboratories and represents the first fully automated supramolecular assembly on a solid support.

bioengineering↗

bioRxiv2023

MRP1 inhibition by lipid-derived electrophiles during ferroptosis illustrates a role for protein alkylation in ferroptotic cell death

Ferroptosis is a regulated form of cell death characterized by lipid peroxidation and lipid hydroperoxide (LOOH) generation that offers new therapeutic opportunities. However, the molecular mechanism through which LOOH accumulation leads to cell death remains poorly understood. Importantly, LOOH breakdown forms truncated phospholipids (PLs) and highly reactive lipid-derived electrophiles (LDEs) capable of altering protein function through cysteine alkylation. While truncated PLs have been shown to mediate ferroptotic membrane permeabilization, a functional role for LDEs in the ferroptotic cell death mechanism has not been established. Here, using multidrug resistance protein 1 (MRP1) activity as an example, we demonstrate that LDEs mediate altered protein function during ferroptosis. Applying live cell fluorescence imaging, we first identified that inhibition of MRP1-mediated LDE detoxification occurs across a panel of ferroptosis inducers (FINs) with differing mechanisms of ferroptosis induction (Types I-IV FINs erastin, RSL3, FIN56 and FINO2). This MRP1 inhibition was recreated by both initiation of lipid peroxidation and treatment with the LDE 4-hydroxy-2-nonenal (4-HNE). Importantly, treatment with radical-trapping antioxidants prevented impaired MRP1 activity when working with both FINs and lipid peroxidation initiators but not 4-HNE, pinpointing LDEs as the cause of inhibited MRP1 activity during ferroptosis. Our findings, when combined with reports of widespread LDE-alkylation of key proteins during ferroptosis, sets a precedent for LDEs as critical mediators of ferroptotic cell death. LOOH breakdown to truncated phospholipids and LDEs may fully explain membrane permeabilization and modified protein function during late stage ferroptosis, offering a unified explanation of the molecular ferroptotic cell death mechanism.

biochemistry↗