Biology subjects

Wranik, M.

Publications and source records attributed to Wranik, M..

5 recordsLinked to original sources

bioRxiv2026

Covalent bond formation caught in a LOV photoreceptor

Light-oxygen-voltage (LOV) domains are blue-light photoreceptors of plants, algae and fungi, and among the most widely used tools in optogenetics. They switch on by forming a covalent thioether bond between a conserved cysteine and their flavin chromophore, in a reaction that needs a proton to cross from the cysteine to the flavin through a pocket containing essentially no water. Its mechanism has been debated for two decades1, and because the chemistry is over within a microsecond its elementary steps have stayed hidden. Here we combine 10 time-resolved serial femtosecond crystallography snapshots and infrared spectroscopy with QM/MM calculations to resolve the entire sequence of events at 1.4 [A] resolution: from excited-state distortion of the flavin ring (10-100 ps), through hydration of a surface channel (10 ns) and a single ordered water reaching the active site as the reactive cysteine shifts between its conformations (100-500 ns), to the thioether bond itself, caught half-formed at 1 {micro}s (half the molecules reacted, half still poised) and complete at 10-100 {micro}s. That water bridges the cysteine and the flavin and shuttles the proton, lowering the barrier from [~]35 to [~]15 kcal/mol and accelerating the reaction by roughly fourteen orders of magnitude (without it, the half-life would be [~]237,000 years), then departs before the bond forms. Proteins can therefore hydrate a dehydrated active site transiently and on demand to overcome otherwise prohibitive reaction barriers, a catalytic strategy that reaches well beyond photoreceptors.

biophysics↗

bioRxiv2025

Time-resolved structures of β2-adrenergic receptor modulation by a photoswitchable beta-blocker

G protein-coupled receptors (GPCRs) regulate essential physiological responses and are important drug targets, yet their ligand-induced conformational dynamics remain poorly understood. The {beta}2-adrenergic receptor ({beta}2AR) is a prominent member of the GPCR family. It regulates bronchial and vascular function and is a significant drug target, particularly in respiratory and smooth muscle-related disorders. We employed time-resolved crystallography at X-ray free-electron lasers (XFELs) to capture the conformational dynamics of {beta}2AR bound to photoazolol-1, a beta-blocker derivative developed for photopharmacological applications. Structural snapshots of the receptor bound to trans-photoazolol-1 (pre-photoconversion), a strained intermediate, and the fully photoisomerized cis-photoazolol-1 reveal an intricate interplay between ligand chemistry and receptor plasticity. Isomerization of the azobenzene moiety induces distinct conformational changes within the orthosteric pocket, altering interactions with the extracellular loop 2 and transmembrane helices 5 and 6. Supported by functional assays, these structural shifts suggest that photoazolol-1 transitions from an inverse agonist to a neutral antagonist upon photoactivation. Our findings uncover a mechanism of GPCR modulation reminiscent of rhodopsin activation and offer a framework for designing ligands that harness light-driven transitions to achieve spatiotemporal control of receptor function.

biochemistry↗

bioRxiv2022

Molecular snapshots of drug release from tubulin over eleven orders of magnitude in time

The binding and release of ligands from their protein targets is central to fundamental biological processes as well as to drug discovery. Photopharmacology introduces chemical triggers that allow the changing of ligand affinities and thus biological activity by light. Insight into the molecular mechanisms of photopharmacology is largely missing because the relevant transitions during the light-triggered reaction cannot be resolved by conventional structural biology. Using time-resolved serial crystallography at a synchrotron and X-ray free-electron laser, we captured the release of the anti-cancer compound azo-combretastatin A4 and the resulting conformational changes in tubulin. Nine structural snapshots from 1 ns to 100 ms complemented by simulations show how cis-to-trans isomerization of the azobenzene bond leads to a switch in ligand affinity, opening of an exit channel, and collapse of the binding pocket upon ligand release. The resulting global backbone rearrangements are related to the action mechanism of microtubule-destabilizing drugs.

molecular biology↗

bioRxiv2021

In vivo photocontrol of microtubule dynamics and integrity, migration and mitosis, by the potent GFP-imaging-compatible photoswitchable reagents SBTubA4P and SBTub2M

Photoswitchable reagents to modulate microtubule stability and dynamics are an exciting tool approach towards micron- and millisecond-scale control over endogenous cytoskeleton-dependent processes. When these reagents are globally administered yet locally photoactivated in 2D cell culture, they can exert precise biological control that would have great potential for in vivo translation across a variety of research fields and for all eukaryotes. However, photopharmacologys reliance on the azobenzene photoswitch scaffold has been accompanied by a failure to translate this temporally- and cellularly-resolved control to 3D models or to in vivo applications in multi-organ animals, which we attribute substantially to the metabolic liabilities of azobenzenes. Here, we optimised the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based instead on the styrylbenzothiazole (SBT) photoswitch scaffold, that are non-responsive to the major fluorescent protein imaging channels and so enable multiplexed imaging studies. We applied these reagents to 3D systems (organoids, tissue explants) and classic model organisms (zebrafish, clawed frog) with one- and two-protein imaging experiments. We successfully used systemic treatment plus spatiotemporally-localised illuminations in vivo to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in these systems with cellular precision and second-level resolution. These nanomolar, in vivo-capable photoswitchable reagents can prove a game-changer for high-precision cytoskeleton research in cargo transport, cell motility, cell division and development. More broadly, their straightforward design can also inspire the development of similarly capable optical reagents for a range of protein targets, so bringing general in vivo photopharmacology one step closer to productive realisation.

cell biology↗

bioRxiv2019

Photoswitchable microtubule inhibitors enabling robust, GFP-orthogonal optical control over the tubulin cytoskeleton

Here we present GFP-orthogonal optically controlled reagents for reliable and repetitive in cellulo modulation of microtubule dynamics and its dependent processes. Optically controlled reagents (\"photopharmaceuticals\") have developed into powerful tools for high-spatiotemporal-precision control of endogenous biology, with numerous applications in neuroscience, embryology, and cytoskeleton research. However, the restricted chemical domain of photopharmaceutical scaffolds has constrained their properties and range of applications. Styrylbenzothiazoles are an as-yet unexplored scaffold for photopharmaceuticals, which we now rationally design to feature potent photocontrol, switching microtubule cytoskeleton function off and on according to illumination conditions. We show more broadly that this scaffold is exceptionally chemically and biochemically robust as well as substituent-tolerant, and offers particular advantages for intracellular biology through a range of desirable photopharmaceutical and drug-like properties not accessible to the current classes of photoswitches. We expect that these reagents will find powerful applications enabling robust, high precision, optically controlled cell biological experimentation in cytoskeleton research and beyond.

cell biology↗