Biology subjects

Stipp, R.

Publications and source records attributed to Stipp, R..

3 recordsLinked to original sources

bioRxiv2026

Anaerobic time-resolved serial crystallography captures CO dissociation and rebinding in oxygen-sensitive -hydrogenase

Redox-active, oxygen-sensitive metalloenzymes catalyze key reactions in biological energy conversion and small-molecule activation. Understanding their mechanisms requires structural characterization of transient catalytic intermediates. Time-resolved serial crystallography (TR-SX) enables direct visualization of protein dynamics during catalysis under near-physiological conditions. Its application to oxygen-sensitive enzymes has remained challenging because strict anaerobic conditions must be maintained throughout sample preparation and data collection. Here, we establish an anaerobic room-temperature serial crystallography workflow for the oxygen-sensitive [FeFe]-hydrogenase CpI and demonstrate its applicability by determining a room-temperature structure of the CO-inhibited Hox-CO state and following inhibitory CO dissociation and rebinding on the millisecond timescale via TR-SX. The presented room-temperature Hox-CO structure closely resembles previous cryogenic models and shows no detectable evidence of oxygen-induced degradation or significant radiation damage. Time-resolved measurements reveal no detectable structural rearrangements accompanying CO dissociation and rebinding beyond displacement and return of the inhibitory ligand, indicating a rigid catalytic architecture that may facilitate rapid catalysis. The presented workflow enables time-resolved structural studies of oxygen-sensitive metalloenzymes under physiologically relevant conditions and opens the way to direct visualization of catalytic intermediates in redox enzymes.

biochemistry↗

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↗