Emil Georg Palmkvist Stender

Mycobacterium tuberculosis ( Mtb ), the causative agent of tuberculosis (TB), is estimated to infect nearly one-quarter of the global population. A key factor in its resilience and persistence is its robust DNA repair capacity. Non-homologous end joining (NHEJ) is the primary pathway for repairing DNA double-strand breaks (DSBs) in many organisms, including Mtb , where it is mediated by the Ku protein and the multifunctional LigD enzyme. In this study, we demonstrate that Ku is essential for mycobacterial survival under DNA-damaging conditions. Using cryogenic electron microscopy (cryo-EM), we solved high-resolution structures of both the apo and DNA-bound forms of the Ku- Mtb homodimer. Our structural and biophysical analyses reveal that Ku forms an extended proteo-filament upon binding DNA. We identify critical residues involved in filament formation and DNA synapsis and show that their mutation severely impairs bacterial viability. Furthermore, we propose a model in which the C-terminus of Ku regulates DNA binding and loading and facilitates subsequent recruitment of LigD. These findings provide unique insights into bacterial DNA repair and guide future therapeutics.

Biomolecular condensates have emerged as important structures in cellular function and disease, and are thought to form through liquid‐liquid phase separation (LLPS). Thorough and efficient in vitro experiments are therefore needed to elucidate the driving forces of protein LLPS and the possibility to modulate it with drugs. Here we present Taylor dispersion‐induced phase separation (TDIPS), a method to robustly measure condensation phenomena using a commercially available microfluidic platform. It uses only nanoliters of sample, does not require extrinsic fluorescent labels, and is straightforward to implement. We demonstrate TDIPS by screening the phase behaviour of two proteins that form biomolecular condensates in vivo, PGL‐3 and Ddx4. Uniquely accessible to this method, we find an unexpected re‐entrant behaviour at very low ionic strength, where LLPS is inhibited for both proteins. TDIPS can also probe the reversibility of assemblies, which was shown for both α‐synuclein and for lysozyme, relevant for health and biotechnology, respectively. Finally, we highlight how effective inhibition concentrations and partitioning of LLPS‐modifying compounds can be screened highly efficiently.

Biomolecular condensates have emerged as important structures in cellular function and disease, and are thought to form through liquid‐liquid phase separation (LLPS). Thorough and efficient in vitro experiments are therefore needed to elucidate the driving forces of protein LLPS and the possibility to modulate it with drugs. Here we present Taylor dispersion‐induced phase separation (TDIPS), a method to robustly measure condensation phenomena using a commercially available microfluidic platform. It uses only nanoliters of sample, does not require extrinsic fluorescent labels, and is straightforward to implement. We demonstrate TDIPS by screening the phase behaviour of two proteins that form biomolecular condensates in vivo, PGL‐3 and Ddx4. Uniquely accessible to this method, we find an unexpected re‐entrant behaviour at very low ionic strength, where LLPS is inhibited for both proteins. TDIPS can also probe the reversibility of assemblies, which was shown for both α‐synuclein and for lysozyme, relevant for health and biotechnology, respectively. Finally, we highlight how effective inhibition concentrations and partitioning of LLPS‐modifying compounds can be screened highly efficiently.

The loading of the bacterial replicative helicase is an essential step for genome replication and depends on the assistance of accessory proteins. Several of these proteins have been identified across the bacterial phyla. DciA is the most common loading protein in bacteria, yet the one whose mechanism is the least understood. We have previously shown that Vc DciA from Vibrio cholerae, composed of a globular KH-like domain followed by an unfolded extension, has a strong affinity for DNA. Here, we characterized the droplets formed by Vc DciA upon interaction with a short single-stranded substrate. We demonstrate the fluidity of these droplets using light microscopy and address their network organization through electron microscopy, thereby bridging events to conclude on a liquid-liquid phase separation behavior. Additionally, we observe the recruitment of Vc DnaB inside the Vc DciA-DNA droplets. We show that DnaC from Escherichia coli is also competent to form these condensate structures in the presence of ssDNA. Our data open up possibilities for the involvement of DciA in the formation of non-membrane compartments within the bacterium, facilitating the assembly of replication players with the chromosomal DNA.

To fully utilize carbohydrates from seaweed biomass, the degradation of the family of polysaccharides known as alginates must be understood. A step in the degradation of alginate is the conversion of 4,5-unsaturated monouronates to 4-deoxy-L-erythro-5-hexoseulose catalysed by the enzyme KdgF. In this study BeKdgF from Bacteroides eggerthii from the human gut microbiota has been produced isotopically labelled in Escherichia coli. Here the ¹H, ¹³C, and ¹⁵N NMR chemical shift assignment for BeKdgF is reported.

β-lactoglobulin (BLG) is a promiscuous protein in terms of ligand interactions, having several binding sites reported for hydrophobic biomolecules such as fatty acids, lipids, and vitamins as well as detergents. BLG also interacts with neutral and anionic oligo- and polysaccharides for which the binding sites remain to be identified. The multivalency offered by these carbohydrate ligands is expected to facilitate coacervation, an electrostatically driven liquid–liquid phase separation. Using heteronuclear single quantum coherence NMR spectroscopy and monitoring chemical shift perturbations, we observed specific binding sites of modest affinity for alginate oligosaccharides (AOSs) prepared by alginate lyase degradation. Two different AOS binding sites (site 1 and site 2) centered around K75 and K101 were identified for monomeric BLG isoform A (BLGA) at pH 2.65. In contrast, only site 1 around K75 was observed for dimeric BLGA at pH 4.0. The data suggest a pH-dependent mechanism whereby both the BLGA dimer–monomer equilibrium and electrostatic interactions are exploited. This variability allows for control of coacervation and particle formation of BLGA/alginate mixtures via directed polysaccharide bridging of AOS binding sites and has implication for molecular network formation. The results are valuable for design of polyelectrolyte-based BLG particles and coacervates for carrying nutraceuticals and modulating viscosity in dairy products by use of alginates.