<i>In crystallo</i>optical spectroscopy (<i>ic</i>OS) as a complementary tool on the macromolecular crystallography beamlines of the ESRF

Stetten, David von; Giraud, Thierry; Carpentier, Philippe; Sever, F.; Terrien, M.; Dobias, Fabien; Juers, D.H.; Flot, David; Mueller-Dieckmann, Christoph; Leonard, Gordon A.; Sanctis, Daniele de; Royant, Antoine

doi:10.1107/s139900471401517x

Cited by 70 publications

(65 citation statements)

References 103 publications

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“…More than 1,000 crystals were tested to optimize the protocol used to provide both high-resolution diffraction and homogeneous accumulation of Pdxl in the I 320 state. Online UV-Vis (visible) microspectrophotometry 23 was performed to ensure that crystals used in diffraction experiments contained Pdxl predominantly in the I 320 state ( Fig. 3a ).…”

Section: Resultsmentioning

confidence: 99%

Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis

et al. 2017

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Substrate channeling has emerged as a common mechanism for enzymatic intermediate transfer. A conspicuous gap in knowledge concerns the use of covalent lysine imines in the transfer of carbonyl-group-containing intermediates, despite their wide use in enzymatic catalysis. Here we show how imine chemistry operates in the transfer of covalent intermediates in pyridoxal 5΄-phosphate biosynthesis by the Arabidopsis thaliana enzyme Pdxl. An initial ribose 5-phosphate lysine imine is converted to the chromophoric l320 intermediate, simultaneously bound to two lysine residues and partially vacating the active site, which creates space for glyceraldehyde 3-phosphate to bind. Crystal structures show how substrate binding, catalysis and shuttling are coupled to conformational changes around strand β6 of the Pdxl (βα)8-barrel. The dual-specificity active site and imine relay mechanism for migration of carbonyl intermediates provide elegant solutions to the challenge of coordinating a complex sequence of reactions that follow a path of over 20 Å between substrate-and product-binding sites.

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Section: Resultsmentioning

confidence: 99%

Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis

et al. 2017

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“…UV-Vis absorption spectra were recorded at the ID29S-Cryobench laboratory of the ESRF (von Stetten et al, 2015) using a high-sensitivity fixedgrating QE65Pro spectrophotometer with a back-thinned CCD detector (Ocean Optics, Dunedin, Florida, USA) and a balanced deuterium-halogen DH2000-BAL light source (Ocean Optics). Spectra were averaged from ten 400 ms acquisitions from $50 mm thick crystals flash-cooled at 100 K.…”

Section: In Crystallo Uv-visible Absorption Spectroscopymentioning

confidence: 99%

“…Optical spectroscopy analysis. We performed an initial spectroscopic characterization after transporting one irradiated crystal from the goniometer of beamline ID29 to that of the optical spectroscopy facility ID29S-Cryobench (von Stetten et al, 2015). UV-visible absorption spectra could be measured from a non-irradiated area and an area from which an X-ray data set had been recorded, corresponding to a deposited dose of 389 kGy (Fig.…”

Section: Specific Radiation Damage In Mneongreenmentioning

confidence: 99%

“…5c; von Stetten et al, 2015) shows that the protein is not affected, and also that most of the strong bands correspond to the pre-resonance Raman signal of the chromophore [even though the excitation wavelength (785 nm) is distant from the absorption maximum of the chromophore at 506 nm]. The eight strongest peaks that decrease upon irradiation (red arrows in Fig.…”

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confidence: 97%

“…Table 2 Principal Raman modes of mNeonGreen. Amide I 1667 = von Stetten et al (2015) calculated phases of a mNeonGreen structure refined against data set 1. The three maps (2 À 1), (3 À 1) and (5 À 1) are represented in Fig.…”

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Structural analysis of the bright monomeric yellow-green fluorescent protein mNeonGreen obtained by directed evolution

Clavel

Gotthard

Stetten

et al. 2016

Acta Crystallogr D Struct Biol

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Until recently, genes coding for homologues of the autofluorescent protein GFP had only been identified in marine organisms from the phyla Cnidaria and Arthropoda. New fluorescent-protein genes have now been found in the phylum Chordata, coding for particularly bright oligomeric fluorescent proteins such as the tetrameric yellow fluorescent protein lanYFP from Branchiostoma lanceolatum. A successful monomerization attempt led to the development of the bright yellow-green fluorescent protein mNeonGreen. The structures of lanYFP and mNeonGreen have been determined and compared in order to rationalize the directed evolution process leading from a bright, tetrameric to a still bright, monomeric fluorescent protein. An unusual discolouration of crystals of mNeonGreen was observed after X-ray data collection, which was investigated using a combination of X-ray crystallography and UV-visible absorption and Raman spectroscopies, revealing the effects of specific radiation damage in the chromophore cavity. It is shown that X-rays rapidly lead to the protonation of the phenolate O atom of the chromophore and to the loss of its planarity at the methylene bridge.

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An Aromatic Dyad Motif in Dye Decolourising Peroxidases Has Implications for Free Radical Formation and Catalysis

Chaplin

Chicano

Hampshire

et al. 2019

Chemistry A European J

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Dye decolouring peroxidases (DyPs) are the most recent class of heme peroxidase to be discovered. On reacting with H2O2, DyPs form a high‐valent iron(IV)‐oxo species and a porphyrin radical (Compound I) followed by stepwise oxidation of an organic substrate. In the absence of substrate, the ferryl species decays to form transient protein‐bound radicals on redox active amino acids. Identification of radical sites in DyPs has implications for their oxidative mechanism with substrate. Using a DyP from Streptomyces lividans, referred to as DtpA, which displays low reactivity towards synthetic dyes, activation with H2O2 was explored. A Compound I EPR spectrum was detected, which in the absence of substrate decays to a protein‐bound radical EPR signal. Using a newly developed version of the Tyrosyl Radical Spectra Simulation Algorithm, the radical EPR signal was shown to arise from a pristine tyrosyl radical and not a mixed Trp/Tyr radical that has been widely reported in DyP members exhibiting high activity with synthetic dyes. The radical site was identified as Tyr374, with kinetic studies inferring that although Tyr374 is not on the electron‐transfer pathway from the dye RB19, its replacement with a Phe does severely compromise activity with other organic substrates. These findings hint at the possibility that alternative electron‐transfer pathways for substrate oxidation are operative within the DyP family. In this context, a role for a highly conserved aromatic dyad motif is discussed.

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In crystallooptical spectroscopy (icOS) as a complementary tool on the macromolecular crystallography beamlines of the ESRF

Cited by 70 publications

References 103 publications

Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis

Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis

Structural analysis of the bright monomeric yellow-green fluorescent protein mNeonGreen obtained by directed evolution

An Aromatic Dyad Motif in Dye Decolourising Peroxidases Has Implications for Free Radical Formation and Catalysis

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