Katarzyna M. Tych

Biological function is often inferred from sequence conservation or structural similarity. However, many viral RNAs maintain regulatory functions despite extensive diversification of both sequence and structures. Using arenavirus intergenic regions (IGRs) as a model system, we investigated what level of RNA organization remains conserved when these conventional sequence-structure-function relationships break down. Combining cryogenic OrbiSIMS guided RNA structure determination with graph theory-based analysis of fifty-eight IGRs across phylogenetically diverse arenaviruses, we discovered that IGR topology, defined by the connectivity and relative integration of individual stem-loop elements, is consistently arranged around a highly connected helical core that persists across divergent IGR folds. Quantitative centrality analyses further revealed that topologically peripheral stem-loops are comparatively sparse and variable across the phylogenetic classes. Investigation of the functional consequence of the peripheral elements in Golden Gate virus (a reptile arenavirus) S-IGR revealed selective association with interferon-induced and antiviral RNA-binding proteins in human host cells infected with recombinant Lymphocytic Choriomeningitis virus (a distantly related human arenavirus). Taken together, these findings identify graph-defined topology as a recurrent organisational feature among divergent arenavirus IGRs that is not captured by sequence identity or global RNA-structure similarity. The data are consistent with a model in which a topologically central helical organisation may be associated with conserved regulatory capacity, whereas structurally variable peripheral elements may contribute to lineage- or segment-specific host-factor interaction profiles.

Biological matter consists of complex networks of fibrillar assemblies that can withstand substantial mechanical stress without brittle fracture. Replicating these properties in synthetic materials - particularly the combination of strain stiffening and structural plasticity - has remained challenging without relying on covalent polymers or permanent chemical crosslinking. Here we report a purely supramolecular hydrogel that uniquely integrates both strain stiffening and structural plasticity, and we introduce a multiscale framework for studying plasticity in supramolecular materials. The material incorporates two orthogonal interaction modes, physical crosslinks that drive strain-stiffening behaviour, and inter-fibril sliding that enables plastic deformation without loss of elastic stiffness. This work reveals structural plasticity as an emergent and designable property of supramolecular networks, opening routes toward bioinspired, adaptive soft matter beyond the elastic regime.

The doubly knotted motif is one of the least expected features in proteins, occurring in both globular and transmembrane forms. Here, we focus on globular protein members of the methyltransferase family: the TrmD–Tm1570 protein, which contains two deep 31 knots, and the single-knotted proteins TrmD and Tm1570, all from Calditerrivibrio nitroreducens. Using various biophysical experimental techniques and computer simulations with AI-based methods, we studied their thermal and thermodynamic stability, as well as their mechanical unfolding. Based on molecular dynamics (MD) simulations, with the Structure-Based Cα Model (SBM-Cα) and UNRES (coarse-grained), we show that native contacts alone are not sufficient to fold double-knotted proteins. However, native contacts are sufficient to fold the single-knotted proteins TrmD and Tm1570 into their native conformations. Using the same model, we identified four possible unfolding and untying pathways, in which each domain can self-tie independently at some stage of the process. Optical tweezers (OT) experiments show that this process is also reversible, although the stretched state remains knotted. In addition, we observed higher thermal and mechanical stability in Tm1570 compared with TrmD, which is partly attributable to the position of the knot core. Overall, our results suggest that double-knotted protein from the SPOUT family can only partially self-fold, and that full knotting may require the assistance of a chaperone.

The doubly knotted motif is one of the least expected features in proteins, occurring in both globular and transmembrane forms. Here, we focus on globular protein members of the methyltransferase family: the TrmD–Tm1570 protein, which contains two deep 3 ₁ knots, and the single-knotted proteins TrmD and Tm1570, all from Calditerrivibrio nitroreducens . Using various biophysical experimental techniques and computer simulations with AI-based methods, we studied their thermal and thermodynamic stability, as well as their mechanical unfolding. Based on molecular dynamics (MD) simulations, with the Structure-Based C α Model (SBM-C α ) and UNRES (coarse-grained), we show that native contacts alone are not sufficient to fold double-knotted proteins. However, native contacts are sufficient to fold the single-knotted proteins TrmD and Tm1570 into their native conformations. Using the same model, we identified four possible unfolding and untying pathways, in which each domain can self-tie independently at some stage of the process. Optical tweezers (OT) experiments show that this process is also reversible, although the stretched state remains knotted. In addition, we observed higher thermal and mechanical stability in Tm1570 compared with TrmD, which is partly attributable to the position of the knot core. Overall, our results suggest that double-knotted protein from the SPOUT family can only partially self-fold, and that full knotting may require the assistance of a chaperone.

DDDG extracts membrane proteins together with native lipids into nanodiscs. These native nanodiscs perform comparably to MSP nanodiscs, with each method suiting different structural and functional studies.

Optical tweezers are widely used in the study of biological macromolecules but are limited by their one‐directional probing capability, potentially missing critical conformational changes. Combining fluorescence microscopy with optical tweezers, employing Förster resonance energy transfer (FRET) pairs, addresses this issue. When integrating fluorescence microscopy with optical tweezers, orthogonal protein conjugation methods are needed to enable simultaneous, site‐specific attachment of fluorophores and DNA handles, commonly used to apply force to molecules of interest. In this study, we utilized commercially available reagents for dual site‐specific labeling of the homodimeric heat shock protein 90 (Hsp90) using thiol‐maleimide and inverse electron demand Diels–Alder cycloaddition (IEDDAC) bioorthogonal reactions. In a one‐pot approach, Hsp90 modified with a cysteine mutation and the non‐canonical amino acid cyclopropene‐L‐lysine (CpK) was labeled with the FRET pair maleimide‐Atto 550 and maleimide‐Atto 647N, alongside single‐stranded methyltetrazine‐modified DNA oligonucleotide. Optical tweezers experiments with this labeled Hsp90 construct revealed structural transitions consistent with previous studies, validating the approach. Fluorescence measurements confirmed the proximity of FRET pairs in the N‐terminally closed state of Hsp90 in this experimental setup. This integrative method provides a powerful tool for probing complex protein conformational dynamics beyond the limitations of traditional optical tweezers.

Biological nanopores are powerful tools for single‐molecule detection, with promising potential as next‐generation biosensors. A major bottleneck in nanopore analysis is the fragility of the supporting lipid membranes, that easily rupture after exposure to biological samples. Membranes comprising PMOXA‐PDMS‐PMOXA (poly(2‐methyloxazoline‐b‐dimethylsiloxane‐b‐2‐methyloxazoline)) or PBD‐PEO (poly(1,2‐butadiene)‐b‐poly(ethylene oxide)) polymers may form robust alternatives, but their suitability for the reconstitution of a broad range of nanopores has not yet been investigated. Here, PBD‐PEO membranes are found to be highly robust toward applied voltages and human serum, while providing a poor environment for nanopore reconstitution. However, hybrid membranes containing a similar molar ratio of PBD ₁₁ PEO ₈ polymers and diphytanoyl phosphatidylcholine (DPhPC) lipids show the best of both worlds: highly robust membranes suitable for the reconstitution of a wide variety of nanopores. Molecular dynamics simulations reveal that lipids form ≈12 nm domains interspersed by a polymer matrix. Nanopores partition into these lipid nanodomains and sequester lipids, possibly offering the same binding strength as in a native bilayer. Nanopores reconstituted in hybrid membranes yield efficient sampling of biomolecules and enable sensing of high concentrations of human serum. This work thus shows that hybrid membranes functionalized with nanopores allow single‐molecule sensing, while forming robust interfaces, resolving an important bottleneck for novel nanopore‐based biosensors.

The existence of the phenomenon of enhanced enzyme diffusion (EED) has been a topic of debate in recent literature. One proposed mechanism to explain the origin of EED is oligomeric enzyme dissociation. We used mass photometry (MP), a label-free single-molecule technique, to investigate the dependence of the oligomeric states of several enzymes on their ligands. The studied enzymes of interest are catalase, aldolase, alkaline phosphatase, and vanillyl-alcohol oxidase (VAO). We compared the ratios of oligomeric states in the presence and absence of the substrate as well as different substrate and inhibitor concentrations. Catalase and aldolase were found to dissociate into smaller oligomers in the presence of their substrates, independently of inhibition, while for alkaline phosphatase and VAO, different behaviors were observed. Thus, we have identified a possible mechanism which explains the previously observed diffusion enhancement in vitro. This enhancement may occur due to the dissociation of oligomers through ligand binding.