Biology subjects

Loewenthal, D.

Publications and source records attributed to Loewenthal, D..

4 recordsLinked to original sources

bioRxiv2026

Quinone reductase 2 dimerization is dynamically driven by ligand binding

Human quinone reductase 2 (QR2, NQO2) is a cytosolic flavoprotein involved in cell physiology and metabolism, and implicated in several diseases. However, the mechanisms that govern its oligomeric assembly and diverse functional outcomes remain incompletely understood. Here, we employ native mass spectrometry to directly resolve the dynamic oligomeric landscape of recombinant human QR2 expressed in Escherichia coli, preserving non-covalent interactions and enabling analysis of assembly behavior under native conditions. QR2 is predominantly observed as a dimer stabilized by multiple non-covalently bound ligands, giving rise to discrete species. Top-down native mass spectrometry reveals a single intact proteoform, excluding covalent modification or covalently bound flavins as drivers of oligomerization. Binding of flavin adenine dinucleotide (FAD) robustly stabilizes the dimer, while unexpectedly, flavin mononucleotide (FMN) also promotes dimer formation. As FMN and FAD differ structurally by the presence of an adenine dinucleotide moiety, we hypothesized that purine nucleotide binding itself may modulate QR2 assembly. Consistent with this, we identify a new concentration-dependent effect of guanosine-triphosphate (GTP) on QR2 dimerization. Functional reductase assays show that flavin-stabilized dimers exhibit the highest catalytic activity, whereas GTP-induced dimers retain reduced activity. Binding of the inhibitor YB537 abolishes activity despite promoting dimer formation. Together, these findings reveal a ligand-dependent structural plasticity in QR2 oligomerization that is decoupled from reductase function, suggesting that QR2 dimerization serves a wider regulatory role beyond simply supporting reductase catalysis.

molecular biology↗

bioRxiv2026

Continuous mass photometry by single molecule trapping

The dynamic choreography of biomolecular interactions underpins the processes of life, but its direct observation remains challenging. Here, we introduce confined diffusion mass photometry, enabling hour-long, mass-resolved observation of individual biomolecules, their complexes and interactions with up to sub-kDa mass precision and ms temporal resolution. Our approach represents a quantitative time-resolved single-molecule measurement modality for studying complex biomolecular mechanisms in action.

biophysics↗

bioRxiv2026

Molecular-level observation of the self-assembly of a virus-like particle

Biomolecular assembly is a cornerstone of cellular organisation and function. Revealing its underlying principles is essential for understanding biological processes, and their malfunction in disease. Viral capsid assembly is the archetypal self-assembly system that has been central in establishing the fundamental principles of biological self-assembly, providing a conceptual and geometric framework that underpins the current understanding of supramolecular biomolecular systems, the development of new biomaterials, and advancing therapeutic design. Yet, despite decades of experimental efforts, observation and quantification of virus self-assembly pathways and dynamics have remained elusive. Here, we combine mass photometry with a non-perturbative single molecule trapping method, enabling direct, real-time monitoring of the self-assembly of individual virus-like particles (VLPs) with molecular resolution. We show that weak, diffusion-limited, and reversible multivalent interactions control the assembly process by facilitating stochastic selection of a limited set of on-path, topologically closed intermediates. Assembly is finely tuned by the transition rates between these topologically closed configurations and proceeds through a sequence of effectively irreversible first-passage events. The intrinsic first-passage times are a consequence of VLP symmetry, creating a separation of timescales between the formation of the first closed intermediate and subsequent elongation. This separation results in a nucleation and growth mechanism that yields an equilibrium distribution consistent with the law of mass action, despite the overall irreversibility of assembly. Our approach enables direct and complete characterisation of both the thermodynamics and the kinetics governing VLP assembly and reveals how the system achieves specific assembly of one final structure with high fidelity despite the availability of thousands of assembly intermediates. More broadly, our approach provides a general framework for visualising and quantifying the dynamics of multimeric biological machines at the molecular level.

biophysics↗

bioRxiv2024

Best practice mass photometry: A guide to optimal single molecule mass measurement

Mass photometry (MP) has emerged as a powerful approach to study biomolecular structure, dynamics and interactions. The capabilities of the method ultimately hinge on the ability to accurately measure the tiny optical contrast generated by individual molecules landing at a glass-water interface, which enables mass-resolved quantification of biomolecular mixtures. Ideally, this capability is only limited by shot noise inherent to photon detection, but in practice depends on additional parameters and details of the assay. Here, we focus on the key parameters affecting MP performance, and present simple steps that can be taken to achieve optimal MP performance in terms of mass resolution, quantitative detection limit and analyte concentration range without compromising the ease and simplicity of the technique.

biophysics↗