Biology subjects

Westenhoff, S.

Publications and source records attributed to Westenhoff, S..

11 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

XSSDense: Time-resolved X-ray Solution Scattering Density Reconstruction Using a Variational Autoencoder

Solution X-ray scattering provides unique structural information on biomolecules under biological conditions, resolving conformational heterogeneity and time-resolved structural changes. The scattering profiles contain limited information, and interpretation largely relies on fitting candidate structures guided by priors. Direct reconstruction of electron density maps is desirable, but so far has been prevented by the difficulty of incorporating such prior knowledge. Here we propose XSSDense, a framework that couples a variational autoencoder trained on electron densities from predicted or simulated protein ensembles with a genetic algorithm to refine densities against scattering data. We validate XSSDense on synthetic data for crambin, recover the conformational heterogeneity of the unfolded state of Avena sativa light-oxygen-voltage sensing domain 2, resolve a de-novo density for the pre-unfolding state of the same protein, and provide a new structural description of the signalling-state ensemble of photoactive yellow protein. XSSDense enables structurally grounded electron density reconstructions that intrinsically capture conformational heterogeneity.

biophysics↗

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

Chemical mechanism of allosteric and asymmetric dark reversion in a bacterial phytochrome uncovered by cryo-EM.

Phytochromes are light-sensitive proteins found in plants, fungi, and bacteria. They exist in two functional states, Pr and Pfr, distinguished by Z/E isomers of their bilin chromophore. The chromophore can photoswitch between these states, but also thermally converts in darkness. Despite the importance of the latter reaction, it remains unclear how it is controlled by the phytochrome. Here, we present single-particle cryo-EM measurements on the Pseudomonas aeruginosa bacteriophytochrome (PaBphP) carried out at multiple time points during dark reversion from Pr to Pfr. These experiments resolve the structure of a PrPfr hybrid state. Surprisingly, we find that only protomer B converts back to Pfr in the hybrid, while protomer A remains in Pr. We identify structural asymmetries in the precursor Pr state, which extend from the homodimer interface to a conserved histidine (H277). The hydrogen-bonding network around the chromophore is modulated, explaining how the phytochrome gains control over the activation energy of the isomerization reaction. These findings establish that dark reversion is governed by conformational selection between two substates, whereby one is "dark-reversion ready" and the other one blocks the reaction. Moreover, we explain how the equilibrium of the states is allosterically controlled across the dimer. Together, these findings provide a structural framework for tuning phytochrome signaling lifetimes in optogenetic applications. Significance statementThe dark reversion reaction of phytochromes is crucial to their signalling role in plants, bacteria, and fungi, but it is vastly understudied in terms of its chemical mechanism. It remains unsolved how the reaction can proceed at all, given that the activation energy is prohibitively high for the isomerization to occur in solution. Using time-resolved cryo-EM, we present a chemical and structural framework for understanding how the protein binding pocket regulates the reaction. Our results establish that conformational selection between two substates controls dark reversion, providing a rare example of strongly asymmetric reactivity across a dimeric protein. This opens the way for rational engineering of the lifetimes of the signaling states in phytochromes.

biochemistry↗

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

cryoSPHERE: Single-particle heterogeneous reconstruction from cryo EM

The three-dimensional structure of a protein plays a key role in determining its function. Methods like AlphaFold have revolutionized protein structure prediction based only on the amino-acid sequence. However, proteins often appear in multiple different conformations, and it is highly relevant to resolve the full conformational distribution. Single-particle cryo-electron microscopy (cryo EM) is a powerful tool for capturing a large number of images of a given protein, frequently in different conformations (referred to as particles). The images are, however, very noisy projections of the protein, and traditional methods for cryo EM reconstruction are limited to recovering a single, or a few, conformations. In this paper, we introduce cryoSPHERE, a deep learning method that takes as input a nominal protein structure, e.g. from AlphaFold, learns how to divide it into segments, and how to move these as approximately rigid bodies to fit the different conformations present in the cryo EM dataset. This formulation is shown to provide enough constraints to recover meaningful reconstructions of single protein structures. This is illustrated in three examples where we show consistent improvements over the current state-of-the-art for heterogeneous reconstruction.

biophysics↗

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↗

bioRxiv2020

High-resolution Crystal Structures of Transient Intermediates in the Phytochrome Photocycle

Phytochromes are red/far-red light photoreceptors in bacteria to plants, which elicit a variety of important physiological responses. They display a reversible photocycle between the resting (dark) Pr state and the light activated Pfr state, in which light signals are received and transduced as structural change through the entire protein to modulate the activity of the protein. It is unknown how the Pr-to-Pfr interconversion occurs as the structure of intermediates remain notoriously elusive. Here, we present short-lived crystal structures of the classical phytochrome from myxobacterium Stigmatella aurantiaca captured by an X-ray Free Electron Laser 5 ns and 33ms after light illumination of the Pr state. We observe large structural displacements of the covalently bound bilin chromophore, which trigger a bifurcated signaling pathway. The snapshots show with atomic precision how the signal progresses from the chromophore towards the output domains, explaining how plants, bacteria and fungi sense red light.

biophysics↗

bioRxiv2020

Illuminating a Phytochrome Paradigm - a Light-Activated Phosphatase in Two-Component Signaling Uncovered

Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (AgP1). Whereas AgP1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While AgP1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes inform the use of light-controllable histidine kinases and phosphatases for optogenetics.

biochemistry↗

bioRxiv2020

Signaling mechanism of phytochromes in solution

Phytochrome proteins guide the red/far-red photoresponse of plants, fungi, and bacteria. The proteins change their structure in response to light, thereby altering their biochemical activity. Crystal structures suggest that the mechanism of signal transduction from the chromophore to the output domains involves refolding of the so-called PHY tongue. It is currently not clear how the two other notable structural features of the phytochrome superfamily, the helical spine and a figure-of-eight knot, are involved in photoconversion. Here, we present solution NMR data of the complete photosensory core module from D. radiodurans (Dr BphP). Photoswitching between the resting and active states induces changes in amide chemical shifts, residual dipolar couplings, and relaxation dynamics. All observables indicate a photoinduced structural change in the knot region and lower part of the helical spine. This implies that a conformational signal is transduced from the chromophore to the helical spine through the PAS and GAF domains. The new pathway underpins functional studies of plant phytochromes and may explain photo-sensing by phytochromes under biological conditions.

biochemistry↗

bioRxiv2019

The primary structural photoresponse of phytochrome proteins captured by a femtosecond X-ray laser

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The structures reveal a twist of the D-ring, which lead to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signalling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the water dissociation is key to the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.

biophysics↗