Biology subjects

Chevreuil, M.

Publications and source records attributed to Chevreuil, M..

5 recordsLinked to original sources

bioRxiv2026

Interlaboratory benchmarking of differential scanning fluorimetry reveals sources of variability in protein thermal stability parameters

Differential scanning fluorimetry (DSF) is widely used to assess protein thermal stability and to compare protein variants, ligands, or formulations with limited sample consumption. Yet, the reproducibility of DSF derived parameters across laboratories, instruments and analysis workflows remains poorly defined. Here, we report a two phase interlaboratory benchmark using a shared VHH sample across 26 laboratories, 28 instruments and seven DSF instrument models. In Phase 1, participants applied their routine acquisition and analysis workflows, whereas in Phase 2, acquisition conditions were harmonised. Datasets were analysed locally and then centrally reprocessed with MoltenProt. Participant reported apparent melting temperatures (Tm) showed good within instrument repeatability but moderate inter instrument dispersion. Harmonisation of acquisition conditions did not substantially reduce this dispersion. Conversely, centralised reprocessing narrowed the distribution among retained datasets, indicating contributions from analysis workflows and data acceptance criteria in addition to instrument dependent factors. Comparison with differential scanning calorimetry (DSC) further showed that apparent Tm depended strongly on the fluorescence readout. For this VHH, single wavelength fluorescence at 350 nm showed the closest agreement with the DSC transition temperature, whereas the 350/330 nm ratio and barycentric mean generally yielded higher values. Furthermore, DSF derived apparent unfolding enthalpies were more variable than apparent Tm. Together, these findings show that DSF can be highly repeatable within a defined instrument and workflow, but that quantitative comparability across instruments depends on instrument specific effects, the choice of fluorescence readout and analysis strategy. Improved comparability will require transparent reporting of acquisition and analysis conditions, explicit data quality criteria, and well characterised reference materials to monitor instrument dependent offsets.

biophysics↗

bioRxiv2025

Activation Mechanism and Structural Assembly of the Mycobacterium tuberculosis ClpP1P2 Protease and Its Associated ATPases.

Supramolecular assemblies are integral to cellular biochemical processes, relying on their dynamic nature to fulfill essential functions. The protease ClpP1P2, paired with ATPase partners ClpC1 or ClpX, is vital for the survival of Mycobacterium tuberculosis (Mtb). While the ClpP1P2 complex requires activation by specific N-blocked dipeptides (e.g., Z-Leu-Leu) to exhibit proteolytic activity in vitro, the mechanism of in vivo activation remains unclear. In this study, we identified novel activators that enabled the structural determination of the ClpC1P1P2 complex, providing insights into its assembly. Furthermore, we discovered that trehalose - a key metabolite and molecular crowding agent in Mtb, significantly enhances the activity of both ClpC1P1P2 and ClpXP1P2 complexes without the need for activating peptides. Analytical ultracentrifugation revealed that trehalose promotes the formation of these active complexes, mimicking intracellular conditions. These findings propose a new model of Clp system activation in vivo, offering promising avenues for therapeutic targeting in tuberculosis treatment. Significance StatementThe proteolytic complex formed by the essential proteins ClpP1 and ClpP2, along with their specific ATP-dependent activators ClpX and ClpC1, has emerged as a highly attractive target for anti-tuberculosis drug development. While previous studies have shown that ClpP1P2 can be activated in vitro by small peptide activators, its in vivo activation mechanism remains unclear. In this study, we identify novel activators and demonstrate that trehalose, a key metabolite in Mycobacterium tuberculosis, enhances ClpC1P1P2 and ClpXP1P2 activity without the need for activating peptides. These findings propose a new model for Clp system activation in Mycobacterium tuberculosis, advancing our understanding of its regulation and potential as a therapeutic target.

biochemistry↗

bioRxiv2024

Restriction on Ku's Inward Translocation Caps Telomere Ends

Safeguarding chromosome ends against fusions via nonhomologous end joining (NHEJ) is essential for preserving genome integrity. Paradoxically, the conserved NHEJ core factor Ku binds telomere ends. How it is prevented from promoting NHEJ remains unclear, as does the mechanism that allows Ku to coexist with telomere-protective DNA binding proteins, e.g., Rap1 in Saccharomyces cerevisiae. Here, we reveal a direct role for Rap1 in the inhibition of Kus NHEJ function at telomeres. A single Rap1 molecule bound near a DNA end inhibits NHEJ in vivo without disrupting Ku presence. Consistent with this, Rap1 and Ku form a complex on short DNA duplexes in vitro. Cryo-EM and molecular modelling analysis of this complex shows that Rap1 obstructs Kus inward translocation on DNA - an essential step for NHEJ at broken ends. Nanopore sequencing of telomere fusions confirms the importance of this pathway in protecting native telomere ends. Collectively, our findings uncover a mechanism of telomere end protection mediated by restricting Kus inward translocation, a functional switch that prevents promiscuous NHEJ repair at telomeres.

molecular biology↗

bioRxiv2023

Raynals, an online tool for the analysis of dynamic light scattering

Dynamic light scattering (DLS) is routinely employed to assess the homogeneity and size distribution profile of samples containing microscopic particles in suspension or solubilised polymers. In this work, we introduce Raynals, an user-friendly software for the analysis of single-angle DLS data that uses the Tikhonov-Phillips regularisation. Performance is evaluated on simulated and experimental data, generated by different DLS instruments, for several proteins and gold nanoparticles. DLS data can be easily misinterpreted and the simulation tools available in Raynals allow understanding of the limitations of the measurement and its resolution. It has been designed as a tool to address quality control of biological samples, during sample preparation and optimisation, and it helps in the detection of aggregates showing the influence of large particles. Last, Raynals provides flexibility in the way the data is presented, allows exporting publication-quality figures, it is free for academic use, and can be accessed online on the eSPC data analysis platform at spc.embl-hamburg.de.

biophysics↗

bioRxiv2022

DNA-binding mechanism and evolution of Replication Protein A

Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair, in both eukaryotic and archaeal cells. By using an integrative approach that combines three crystal structures, four cryo-EM structures in complex with single-stranded DNA (ssDNA) of different lengths, we extensively characterized RPA from Pyrococcus abyssi in different states. These structures show two essential features conserved in eukaryotes: a trimeric core and a module that promotes cooperative binding to ssDNA, as well as a newly identified archaeal-specific domain. These structures reveal for the first time how ssDNA is handed over from one RPA complex to the other, and uncover an unanticipated mechanism of self-association on ssDNA tracts. This work constitutes a significant step forward in the molecular understanding of the structure and DNA-binding mechanism of RPA, with far-reaching implications for the evolution of this primordial replication factor in Archaea and Eukarya.

biochemistry↗