David von Stetten

European Molecular Biology Laboratory

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Antoine Royant

Institut de Biologie Structurale

P. Mehrabi

Max Planck Institute for the Structure and Dynamics of Matter

Arwen R. Pearson

Universität Hamburg

María Andrea Mroginski

Technische Universität Berlin

Peter Hildebrandt

Technische Universität Berlin

Eike C. Schulz

Max Planck Institute for the Structure and Dynamics of Matter

Guillaume Gotthard

Paul Scherrer Institute

Marjolaine Noirclerc‐Savoye

Université Grenoble Alpes

H. Schikora

Max Planck Institute for the Structure and Dynamics of Matter

Brenna Norton‐Baker

Max Planck Institute for the Structure and Dynamics of Matter

Cooperative Institutions

Universität Hamburg

115

Centre National de la Recherche Scientifique

Center for Free-Electron Laser Science

Deutsches Elektronen-Synchrotron DESY

Max Planck Institute for the Structure and Dynamics of Matter

European Synchrotron Radiation Facility

Université Grenoble Alpes

Commissariat à l'Énergie Atomique et aux Énergies Alternatives

Institut de Biologie Structurale

European X-Ray Free-Electron Laser

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An ultraviolet-driven rescue pathway for oxidative stress to eye lens protein human gamma-D crystallin

Communications Chemistry (2024)

Jake Hill Yvonne Nyathi Sam Horrell David von Stetten Danny Axford

Human gamma-D crystallin (HGD) is a major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin mutant (R36S) shows no post-translational modifications. After aging for nine months in the absence of light, a thiol-adduct (dithiothreitol) modifying surface cysteines is observed by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.

Crystallin

Ultraviolet

Eye lens

10.1038/s42004-024-01163-w

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Citations (5)

Crystal structure of a bacterial photoactivated adenylate cyclase determined at room temperature by serial femtosecond crystallography

bioRxiv (Cold Spring Harbor Laboratory) (2024)

Sofia M. Kapetanaki Nicolas Coquelle David von Stetten Martin Byrdin Ronald Rios-Santacruz

Abstract OaPAC is a recently discovered b lue-light u sing flavin adenosine dinucleotide (BLUF) photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata that uses adenosine triphosphate and translates the light signal into the production of cyclic adenosine monophosphate. Here, we report the crystal structures of the enzyme in the absence of its natural substrate determined from room temperature serial crystallography data collected at both an X-ray free electron laser and a synchrotron and we compare them with the cryo macromolecular crystallography structures obtained at a synchrotron by us and others. These results reveal slight differences in the structure of the enzyme due to data collection at different temperatures and X-ray sources. We further investigate the effect of the Y6 mutation in the blue-light using flavin adenosine dinucleotide domain, a mutation which results in a rearrangement of the hydrogen-bond network around the flavin and a notable rotation of the side-chain of the critical Q48 residue. These studies pave the way for ps - ms time-resolved serial crystallography experiments at X-ray free electron lasers and synchrotrons in order to determine the early structural intermediates and correlate them with the well-studied ps - ms spectroscopic intermediates. Synopsis Structures of the dark-adapted state of a photoactivated adenylate cyclase are determined from serial crystallography (SX) data collected at room temperature at an X-ray free electron laser (XFEL) and a synchrotron and are compared with cryo macromolecular crystallography (MX) synchrotron structures obtained by us and others. These structures of the wild-type enzyme in combination with the cryo MX synchrotron structure of a light-sensor domain mutant provide insight into the hydrogen bond network rearrangement upon blue-light illumination and pave the way for the determination of structural intermediates of the enzyme by time-resolved SX.

Crystal (programming language)

10.1101/2024.04.21.590439

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Accelerated oxidative aging in deamidated variants of the human eye lens protein γ(S)-crystallin

Biophysical Journal (2022)

Brenna Norton‐Baker P. Mehrabi Ashley O. Kwok Kyle W. Roskamp Marc A. Sprague‐Piercy

Deamidation

Crystallin

Eye lens

10.1016/j.bpj.2021.11.2606

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Pr and Pfr structures of plant phytochrome A

Research Square (Research Square) (2024)

Jon Hughes S. Nagano Kaoling Guan Peng-Yuan Chen David von Stetten

Abstract Phytochromes are biliprotein photoreceptors widespread amongst microorganisms and ubiquitous in plants where they control developmental processes as diverse as germination, stem elongation and floral induction through the photoconversion of inactive Pr to the Pfr signalling state. Here we report crystal structures of the chromophore-binding module of soybean phytochrome A, including 2.2Å XFEL structures of Pr and Pfr as well as high resolution cryogenic structures of Pr. In the Pfr structure, the chromophore is exposed to the medium, the D-ring remaining α-facial following the likely clockwise photoflip. The chromophore shifts within its pocket, while its propionate side chains, their partners as well as three neighbouring tyrosines shift radically. Helices near the chromophore show substantial shifts that might represent components of the light signal. These changes reflect those in bacteriophytochromes despite their quite different signalling mechanisms, implying that fundamental aspects of phytochrome photoactivation have been repurposed for photoregulation in the eukaryotic plant. (150 words)

Phytochrome

Chromophore

Photomorphogenesis

Phototropism

10.21203/rs.3.rs-4850459/v1

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Conformational heterogeneity of the Pfr chromophore in plant and cyanobacterial phytochromes

Frontiers in Molecular Biosciences (2015)

Francisco Velázquez Escobar David von Stetten Mina Günther-Lütkens Anke Keidel Norbert Michael

Phytochromes are biological photoreceptors that can be reversibly photoconverted between a dark and photoactivated state. The underlying reaction sequences are initiated by the photoisomerization of the tetrapyrrole cofactor, which in plant and cyanobacterial phytochromes are a phytochromobilin (PΦB) and a phycocyanobilin (PCB), respectively. The transition between the two states represents an on/off-switch of the output module activating or deactivating downstream physiological processes. In addition, the photoactivated state, i.e., Pfr in canonical phytochromes, can be thermally reverted to the dark state (Pr). The present study aimed to improve our understanding of the specific reactivity of various PΦB- and PCB-binding phytochromes in the Pfr state by analysing the cofactor structure by vibrational spectroscopic techniques. Resonance Raman (RR) spectroscopy revealed two Pfr conformers (Pfr-I and Pfr-II) forming a temperature-dependent conformational equilibrium. The two sub-states-found in all phytochromes studied, albeit with different relative contributions-differ in structural details of the C-D and A-B methine bridges. In the Pfr-I sub-state the torsion between the rings C and D is larger by ca. 10° compared to Pfr-II. This structural difference is presumably related to different hydrogen bonding interactions of ring D as revealed by time-resolved IR spectroscopic studies of the cyanobacterial phytochrome Cph1. The transitions between the two sub-states are evidently too fast (i.e., nanosecond time scale) to be resolved by NMR spectroscopy which could not detect a structural heterogeneity of the chromophore in Pfr. The implications of the present findings for the dark reversion of the Pfr state are discussed.

Phytochrome

Tetrapyrrole

Chromophore

Phycocyanobilin

Conformational isomerism

Molecular switch

Dark state

10.3389/fmolb.2015.00037

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Citations (30)

Experimental Determination of Transition Dipole Moment Directions in Representative Fluorescent Proteins

Biophysical Journal (2015)

Alina Kevorkova David von Stetten Antoine Royant Josef Lazar

Fluorescent protein

10.1016/j.bpj.2014.11.1780

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X-ray structure of the cyan fluorescent protein SCFP3A (K206A mutant)

David von Stetten Joachim Goedhart Marjolaine Noirclerc‐Savoye Mickaël Lelimousin Linda Joosen

Cyan

Yellow fluorescent protein

10.2210/pdb2ydz/pdb

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Tracking the route of molecular oxygen in O ₂ -tolerant membrane-bound [NiFe] hydrogenase

Proceedings of the National Academy of Sciences (2018)

Jacqueline Kalms Andrea Schmidt Stefan Frielingsdorf Tillmann Utesch Guillaume Gotthard

Significance Tracking the route of substrates, intermediates, and inhibitors in proteins is fundamental in understanding their specific function. However, following the route of gases like molecular oxygen within enzymes has always been challenging. In protein X-ray crystallography, gases can be mimicked using krypton or xenon (with a higher electron count); however, these have a different physical behavior compared to true substrates/inhibitors. In our crystal structure of the O 2 -tolerant membrane-bound [NiFe] hydrogenase (MBH) from Ralstonia eutropha , we were able to show the direct path of molecular oxygen between the enzyme exterior and the active site with the “soak-and-freeze” derivatization method. This technique might be useful to detect O 2 traveling routes in many other enzymes.

Hydrogenase

Ralstonia

Cupriavidus necator

Molecular oxygen

Krypton

10.1073/pnas.1712267115

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Citations (44)

Mutational Analysis of Deinococcus radiodurans Bacteriophytochrome Reveals Key Amino Acids Necessary for the Photochromicity and Proton Exchange Cycle of Phytochromes

Journal of Biological Chemistry (2008)

Jeremiah R. Wagner Junrui Zhang David von Stetten Mina Günther Daniel H. Murgida

The ability of phytochromes (Phy) to act as photointerconvertible light switches in plants and microorganisms depends on key interactions between the bilin chromophore and the apoprotein that promote bilin attachment and photointerconversion between the spectrally distinct red light-absorbing Pr conformer and far red light-absorbing Pfr conformer. Using structurally guided site-directed mutagenesis combined with several spectroscopic methods, we examined the roles of conserved amino acids within the bilin-binding domain of Deinococcus radiodurans bacteriophytochrome with respect to chromophore ligation and Pr/Pfr photoconversion. Incorporation of biliverdin IXalpha (BV), its structure in the Pr state, and its ability to photoisomerize to the first photocycle intermediate are insensitive to most single mutations, implying that these properties are robust with respect to small structural/electrostatic alterations in the binding pocket. In contrast, photoconversion to Pfr is highly sensitive to the chromophore environment. Many of the variants form spectrally bleached Meta-type intermediates in red light that do not relax to Pfr. Particularly important are Asp-207 and His-260, which are invariant within the Phy superfamily and participate in a unique hydrogen bond matrix involving the A, B, and C pyrrole ring nitrogens of BV and their associated pyrrole water. Resonance Raman spectroscopy demonstrates that substitutions of these residues disrupt the Pr to Pfr protonation cycle of BV with the chromophore locked in a deprotonated Meta-R(c)-like photoconversion intermediate after red light irradiation. Collectively, the data show that a number of contacts contribute to the unique photochromicity of Phy-type photoreceptors. These include residues that fix the bilin in the pocket, coordinate the pyrrole water, and possibly promote the proton exchange cycle during photoconversion.

10.1074/jbc.m709355200

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Citations (201)

Examples of fits of the structure factor from Dynamics of a family of cyan fluorescent proteins probed by incoherent neutron scattering

Maksym Golub Virginia Guillon Guillaume Gotthard Dominik Zeller Nicolás Martínez

Cyan

Dynamics

Structure factor

10.6084/m9.figshare.7738976.v1

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