Biology subjects

Meszaros, P.

Publications and source records attributed to Meszaros, P..

4 recordsLinked to original sources

bioRxiv2026

Visualizing Reaction Pathways via Reciprocal Space Kinetic Decomposition

Time-resolved serial crystallography (TR-SX) has emerged as a powerful method for capturing ultrafast structural dynamics in proteins. TR-SX continues to produce remarkable studies, revealing previously unobserved transient states and providing deeper insights into processes such as drug targeting, DNA repair, and photosynthesis. However, extracting weak structural signals from noisy time-resolved datasets remains a major challenge. Robust computational methods are therefore required to isolate the signals associated with the underlying transient states. Importantly, this should be performed in reciprocal space to preserve compatibility with established downstream structure refinement workflows. Here, we introduce a framework for kinetic decomposition directly in reciprocal space that enables separation of kinetically distinct structural states. The method decomposes crystallographic data according to a predefined kinetic model, improving the recovery of weak transient signals and enhancing mechanistic interpretation from limited time-resolved datasets. We validate the framework using simulated data based on a previously published time-resolved crystallography study and demonstrate its application to a new TR-SX dataset comprising 17 time points. We show that the method separates the reciprocal space signatures of four intermediates by incorporating kinetic information from a predefined reaction model. This establishes a workflow for extracting kinetic states directly from time-resolved X-ray diffraction data that can be seamlessly integrated into existing crystallographic structure-determination pipelines.

biophysics↗

bioRxiv2026

Single-molecule mass measurements uncover shifting RNA interactions during condensate phase transitions

RNA interactions are a key contributor to the formation and disassembly of intracellular protein condensates. Although some proteins utilize specific RNA-binding domains, these processes can also be mediated by charge interactions with intrinsically disordered regions. Due to the dynamic nature of these systems, investigating the underlying specificity and stoichiometry remains challenging. Here, we demonstrate that single-molecule mass measurements with mass photometry can capture RNA-protein interactions in phase-separated protein systems. Using the approach to investigate RNA-mediated phase shifts of tau condensates, we find that increasing the RNA concentration, which promotes phase re-entry, results in RNA-mediated tau multimerization, where each tau monomer binds a linear RNA sequence of approximately 30 nucleotides. Solution NMR and native mass spectrometry confirm the formation of stable complexes between RNA and the basic proline-rich and repeat domains of tau, which have a net charge of -29. Our findings demonstrate that mass photometry can distinguish between charge neutralization, which drives coacervation, and complex formation, which mediates phase re-entry, making it a highly complementary tool for the study of RNA-mediated phase separation.

biophysics↗

bioRxiv2025

Detection of a hybrid PrPfr state in the dark reversion of abathy phytochrome indicates inter-dimer allostery.

Phytochromes are photosensor proteins which detect light in plants, fungi, and bacteria. They photoswitch between red light absorbing (Pr) and far-red light absorbing (Pfr) states, however, thermal reversion in the dark is an equally important factor in controlling their signaling levels. Phytochromes are generally dimeric proteins, and mixed PrPfr states are therefore possible. These states have been implied in the dark reversion studies of plant phytochromes, but not in bacterial phytochromes. Here, we investigate the dark reversion kinetics of the bathy phytochrome from P. aeruginosa (PaBphP) using UV-Vis absorption spectroscopy. A single set of time-resolved spectra does not conclusively reveal the presence of a mixed PrPfr state, as both a direct Pr[->] Pfr model or a sequential Pr[->] PrPfr[->] Pfr model fit the spectral kinetics. However, a systematic analysis of dark reversion kinetics with varying Pr/Pfr ratios can only be satisfactorily fit by the sequential model, which indicates the presence of an intermediate PrPfr state. A newly designed monomeric variant of PaBphP provides strong support for this interpretation. Temperature-dependent kinetics revealed similarly low activation energies for the dark reversion processes of both proteins, consistent with a previously proposed keto-enol tautomerization preceding dark reversion. Interestingly, our results suggest allosteric regulation of dark reversion across the dimer, which we propose to be a contributing factor in phytochrome signaling.

biochemistry↗

bioRxiv2024

Cryo-EM structures of a bathy phytochrome histidine kinase reveal a unique light-dependent activation mechanism

Phytochromes are photoreceptor proteins in plants, fungi and bacteria. They can adopt two photochromic states with differential biochemical responses. The structural changes transducing the signal from the chromophore to the biochemical output modules are poorly understood due to challenges in capturing structures of the dynamic, full-length protein. Here, we present the first cryo-electron microscopy structures of the phytochrome from Pseudomonas aeruginosa (PaBphP) in its resting Pfr and photoactivated Pr state. The kinase-active Pr state has an asymmetric, dimeric structure, whereas the kinase-inactive Pfr state opens up. This behaviour is different from other known phytochromes and we explain it with the unusually short connection between the photosensory and output modules. Multiple sequence alignment of this region suggests evolutionary optimisation for different modes of signal transduction in sensor proteins. The results establish a new mechanism for light-sensing by phytochrome histidine kinases and provide input for the design of optogenetic phytochrome variants.

biochemistry↗