Capturing structural changes of the S-1 to S-2 transition of photosystem II using time-resolved serial femtosecond crystallography

Photosystem II (PSII) catalyzes light-induced water oxidation through an S-i-state cycle, leading to the generation of di-oxygen, protons and electrons. Pumpprobe time-resolved serial femtosecond crystallography (TR-SFX) has been used to capture structural dynamics of light-sensitive proteins. In this approach, it is crucial to avoid light contamination in the samples when analyzing a particular reaction intermediate. Here, a method for determining a condition that avoids light contamination of the PSII microcrystals while minimizing sample consumption in TR-SFX is described. By swapping the pump and probe pulses with a very short delay between them, the structural changes that occur during the S-1-to-S-2 transition were examined and a boundary of the excitation region was accurately determined. With the sample flow rate and concomitant illumination conditions determined, the S-2-state structure of PSII could be analyzed at room temperature, revealing the structural changes that occur during the S-1-to-S-2 transition at ambient temperature. Though the structure of the manganese cluster was similar to previous studies, the behaviors of the water molecules in the two channels (O1 and O4 channels) were found to be different. By comparing with the previous studies performed at low temperature or with a different delay time, the possible channels for water inlet and structural changes important for the water-splitting reaction were revealed

Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL

Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previousl

Caracterización estructural y funcional de biocatalizadores termoestables para la síntesis de análogos del nucleósido 5'-monofosfato de 6-aminopurina.

The present work describes the functional and structural characterization of adenine phosphoribosyltransferase 2 from thermus thermophilus hb8 (ttaprt2). The combination of structural and substrate specificity data provided valuable information for immobilization studies. Dimeric ttaprt2 was immobilized onto glutaraldehydeactivated magresyn®amine magnetic iron oxide porous microparticles by two different strategies: a) an enzyme immobilization at ph 8.5 to encourage the immobilization process by n-termini (mttaprt2a, mttaprt2b, mttaprt2c) or b) an enzyme immobilization at ph 10.0 to encourage the immobilization process through surface exposed lysine residues (mttaprt2d, mttaprt2e, mttaprt2f). According to catalyst load experiments, mttaprt2b (activity: 480 iu g−1 biocatalyst, activity recovery: 52%) and mttaprt2f (activity: 507 iu g−1 biocatalyst, activity recovery: 44%) were chosen as optimal derivatives. The biochemical characterization studies demonstrated that immobilization process improved the thermostability of ttaprt2. Moreover, the potential reusability of mttaprt2b and mttaprt2f was also tested. Finally, mttaprt2f was employed in the synthesis of nucleoside-5′-monophosphate analogues.El presente trabajo describe la caracterización funcional y estructural de la adenina fosforribosiltransferasa 2 de thermus thermophilus hb8 (ttaprt2). La combinación de datos de especificidad estructural y de sustrato proporcionó información valiosa para los estudios de inmovilización. El ttaprt2 dimérico se inmovilizó en micropartículas porosas de óxido de hierro magnético magresyn®amine activado con glutaraldehido mediante dos estrategias diferentes: a) una inmovilización de enzima a ph 8.5 para alentar el proceso de inmovilización por n-termini (mttaprt2a, mttaprt2c) o bttaprt2c) o b) ph 10.0 para fomentar el proceso de inmovilización a través de residuos de lisina expuestos en la superficie (mttaprt2d, mttaprt2e, mttaprt2f). De acuerdo con los experimentos de carga de catalizador, mttaprt2b (actividad: 480 iu g − 1 biocatalizador, recuperación de actividad: 52%) y mttaprt2f (actividad: 507 iu g − 1 biocatalizador, recuperación de actividad: 44%) fueron elegidos como derivados óptimos. Los estudios de caracterización bioquímica demostraron que el proceso de inmovilización mejoró la termoestabilidad de ttaprt2. Además, también se probó la reutilización potencial de mttaprt2b y mttaprt2f. Finalmente, mttaprt2f se empleó en la síntesis de análogos de nucleósido-5'-monofosfato

Structural and functional characterization of thermostable biocatalysts for the synthesis of 6-aminopurine nucleoside-5′-monophospate analogues

The present work describes the functional and structural characterization of adenine phosphoribosyltransferase 2 from Thermus thermophilus HB8 (TtAPRT2). The combination of structural and substrate specificity data provided valuable information for immobilization studies. Dimeric TtAPRT2 was immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles by two different strategies: a) an enzyme immobilization at pH 8.5 to encourage the immobilization process by N-termini (MTtAPRT2A, MTtAPRT2B, MTtAPRT2C) or b) an enzyme immobilization at pH 10.0 to encourage the immobilization process through surface exposed lysine residues (MTtAPRT2D, MTtAPRT2E, MTtAPRT2F). According to catalyst load experiments, MTtAPRT2B (activity: 480 IU g−1biocatalyst, activity recovery: 52%) and MTtAPRT2F (activity: 507 IU g−1biocatalyst, activity recovery: 44%) were chosen as optimal derivatives. The biochemical characterization studies demonstrated that immobilization process improved the thermostability of TtAPRT2. Moreover, the potential reusability of MTtAPRT2B and MTtAPRT2F was also tested. Finally, MTtAPRT2F was employed in the synthesis of nucleoside-5′-monophosphate analogues.Sin financiación7.539 JCR (2019) Q1, 1/13 Agricultural Engineering2.430 SJR (2019) Q1, 15/193 BioengineeringNo data IDR 2019UE

The Amino-Terminal Region of Major Capsid Protein P3 Is Essential for Self-Assembly of Single-Shelled Core-Like Particles of Rice Dwarf Virus

The core protein P3 of Rice dwarf virus constructs asymmetric dimers, one of which is inserted by the amino-terminal region of another P3 protein. The P3 proteins with serial amino-terminal deletions, expressed in a baculovirus system, formed particles with gradually decreasing stability. The capacity for self-assembly disappeared when 52 of the amino-terminal amino acids had been deleted. These results demonstrated that insertion of the amino-terminal arm of one P3 protein into another appears to play an important role in stabilizing the core particles

Conformational alterations in unidirectional ion transport of a light-driven chloride pump revealed using X-ray free electron lasers

光でイオンを輸送する膜タンパク質の巧妙な仕組み --XFELが捉えた光駆動型イオンポンプロドプシンの構造変化--. 京都大学プレスリリース. 2022-02-28.Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3