Structural and Spectroscopic Investigations of pH-Dependent Mo(V) Species in a Bacterial Sulfite-Oxidizing Enzyme.

Mono-pyranopterin-containing sulfite-oxidizing enzymes (SOEs), including eukaryotic sulfite oxidases and homologous prokaryotic sulfite dehydrogenases (SDHs), are molybdenum enzymes that exist in almost all forms of life, where they catalyze the direct oxidation of sulfite into sulfate, playing a key role in protecting cells and organisms against sulfite-induced damage. To decipher their catalytic mechanism, we have previously provided structural and spectroscopic evidence for direct coordination of HPO to the Mo atom at the active site of the SDH from the hyperthermophilic bacterium (SDH), mimicking the proposed sulfate-bound intermediate proposed to be formed during catalysis. In this work, by solving the X-ray crystallographic structure of the unbound enzyme, we resolve the changes in the hydrogen bonding network in the molybdenum environment that enable the stabilization of the previously characterized phosphate adduct.

Structural insights into the semiquinone form of human Cytochrome P450 reductase by DEER distance measurements between a native flavin and a spin labelled non-canonical amino acid.

The flavoprotein Cytochrome P450 reductase (CPR) is the unique electron pathway from NADPH to Cytochrome P450 (CYPs). The conformational dynamics of human CPR in solution, which involves transitions from a "locked/closed" to an "unlocked/open" state, is crucial for electron transfer. To date, however, the factors guiding these changes remain unknown.

Structural and mechanistic basis for RiPP epimerization by a radical SAM enzyme.

D-Amino acid residues, found in countless peptides and natural products including ribosomally synthesized and post-translationally modified peptides (RiPPs), are critical for the bioactivity of several antibiotics and toxins. Recently, radical S-adenosyl-L-methionine (SAM) enzymes have emerged as the only biocatalysts capable of installing direct and irreversible epimerization in RiPPs. However, the mechanism underpinning this biochemical process is ill-understood and the structural basis for this post-translational modification remains unknown.

The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains.

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle.

investigation of the conformational landscape of the GTPase UreG by SDSL-EPR.

UreG is a cytosolic GTPase involved in the maturation network of urease, an Ni-containing bacterial enzyme. Previous investigations showed that UreG features a flexible tertiary organization, making this protein the first enzyme discovered to be intrinsically disordered. To determine whether this heterogeneous behavior is maintained in the protein natural environment, UreG structural dynamics was investigated directly in intact bacteria by EPR.

Refining the Dynamic Network of T2SS Endopilus Tip Heterocomplex Combining cw-EPR and Nitroxide-Gd Distance Measurements.

The type 2 secretion system (T2SS) is a bacterial nanomachine composed of an inner membrane assembly platform, an outer membrane pore and a dynamic endopilus. T2SS endopili are organized into a homo-multimeric body formed by the major pilin capped by a heterocomplex of four minor pilins. The first model of the T2SS endopilus was recently released, even if structural dynamics insights are still required to decipher the role of each protein in the full tetrameric complex.

Investigating the efficiency of silica materials with wall-embedded nitroxide radicals for dynamic nuclear polarisation NMR.

Dynamic nuclear polarisation (DNP) can significantly enhance the sensitivity of solid-state nuclear magnetic resonance (SSNMR) experiments by transferring the electron spin polarisation of paramagnetic species to nuclei through microwave irradiation of the sample at cryogenic temperatures. Paramagnetic species required for DNP can be provided in the form of mesoporous silica materials containing nitroxide radicals either located on the porous surface or embedded in the pore walls. The present study focuses specifically on porous materials with wall-embedded radicals that were synthesised using conventional molecular imprinting protocols.

Probing the Structural Dynamics of a Bacterial Chaperone in Its Native Environment by Nitroxide-Based EPR Spectroscopy.

One of the greatest current challenges in structural biology is to study protein dynamics over a wide range of timescales in complex environments, such as the cell. Among magnetic resonances suitable for this approach, electron paramagnetic resonance spectroscopy coupled to site-directed spin labeling (SDSL-EPR) has emerged as a promising tool to study protein local dynamics and conformational ensembles. In this work, we exploit the sensitivity of nitroxide labels to report protein local dynamics at room temperature.

Iron Insertion at the Assembly Site of the ISCU Scaffold Protein Is a Conserved Process Initiating Fe-S Cluster Biosynthesis.

Iron-sulfur (Fe-S) clusters are prosthetic groups of proteins biosynthesized on scaffold proteins by highly conserved multi-protein machineries. Biosynthesis of Fe-S clusters into the ISCU scaffold protein is initiated by ferrous iron insertion, followed by sulfur acquisition, via a still elusive mechanism. Notably, whether iron initially binds to the ISCU cysteine-rich assembly site or to a cysteine-less auxiliary site via N/O ligands remains unclear.

PP2A is activated by cytochrome upon formation of a diffuse encounter complex with SET/TAF-Iβ.

Intrinsic protein flexibility is of overwhelming relevance for intermolecular recognition and adaptability of highly dynamic ensemble of complexes, and the phenomenon is essential for the understanding of numerous biological processes. These conformational ensembles-encounter complexes-lack a unique organization, which prevents the determination of well-defined high resolution structures. This is the case for complexes involving the oncoprotein SET/template-activating factor-Iβ (SET/TAF-Iβ), a histone chaperone whose functions and interactions are significantly affected by its intrinsic structural plasticity.

Identification and characterization of a noncanonical menaquinone-linked formate dehydrogenase.

The molybdenum/tungsten-bis-pyranopterin guanine dinucleotide family of formate dehydrogenases (FDHs) plays roles in several metabolic pathways ranging from carbon fixation to energy harvesting because of their reaction with a wide variety of redox partners. Indeed, this metabolic plasticity results from the diverse structures, cofactor content, and substrates used by partner subunits interacting with the catalytic hub. Here, we unveiled two noncanonical FDHs in Bacillus subtilis, which are organized into two-subunit complexes with unique features, ForCE1 and ForCE2.

Structural evidence for a reaction intermediate mimic in the active site of a sulfite dehydrogenase.

By combining X-ray crystallography, electron paramagnetic resonance techniques and density functional theory-based modelling, we provide evidence for a direct coordination of the product analogue, phosphate, to the molybdenum active site of a sulfite dehydrogenase. This interaction is mimicking the still experimentally uncharacterized reaction intermediate proposed to arise during the catalytic cycle of this class of enzymes. This work opens new perspectives for further deciphering the reaction mechanism of this nearly ubiquitous class of oxidoreductases.

Nickel and GTP Modulate UreG Structural Flexibility.

UreG is a P-loop GTP hydrolase involved in the maturation of nickel-containing urease, an essential enzyme found in plants, fungi, bacteria, and archaea. This protein couples the hydrolysis of GTP to the delivery of Ni(II) into the active site of apo-urease, interacting with other urease chaperones in a multi-protein complex necessary for enzyme activation. Whereas the conformation of () UreG was solved by crystallography when it is in complex with two other chaperones, in solution the protein was found in a disordered and flexible form, defining it as an intrinsically disordered enzyme and indicating that the well-folded structure found in the crystal state does not fully reflect the behavior of the protein in solution.

The Hunt for the Closed Conformation of the Fruit-Ripening Enzyme 1-Aminocyclopropane-1-carboxylic Oxidase: A Combined Electron Paramagnetic Resonance and Molecular Dynamics Study.

1-Aminocyclopropane-1-carboxylic oxidase (ACCO) is a non-heme iron(II)-containing enzyme involved in the biosynthesis of the phytohormone ethylene, which regulates fruit ripening and flowering in plants. The active conformation of ACCO, and in particular that of the C-terminal part, remains unclear and open and closed conformations have been proposed. In this work, a combined experimental and computational study to understand the conformation and dynamics of the C-terminal part is reported.

Modulating lifetimes and relaxation times of phenoxyl radicals through their incorporation into different hybrid nanostructures.

The development of new open shell systems is essential for advances in spin science. In this work, we report the synthesis and characterization of three nanostructured materials, namely SBA-15 silicas, periodic mesoporous organosilicas (PMOs) and lamellar polysilsesquioxanes, all functionalized with the same diazene-based phenoxyl radical precursor. The impact of the nature of the material, i.

Tuning the redox properties of a [4Fe-4S] center to modulate the activity of Mo-bisPGD periplasmic nitrate reductase.

Molybdoenzymes are ubiquitous in living organisms and catalyze, for most of them, oxidation-reduction reactions using a large range of substrates. Periplasmic nitrate reductase (NapAB) from Rhodobacter sphaeroides catalyzes the 2-electron reduction of nitrate into nitrite. Its active site is a Mo bis-(pyranopterin guanine dinucleotide), or Mo-bisPGD, found in most prokaryotic molybdoenzymes.

A Bioresistant Nitroxide Spin Label for In-Cell EPR Spectroscopy: In Vitro and In Oocytes Protein Structural Dynamics Studies.

Approaching protein structural dynamics and protein-protein interactions in the cellular environment is a fundamental challenge. Owing to its absolute sensitivity and to its selectivity to paramagnetic species, site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) has the potential to evolve into an efficient method to follow conformational changes in proteins directly inside cells. Until now, the use of nitroxide-based spin labels for in-cell studies has represented a major hurdle because of their short persistence in the cellular context.

Silica materials with wall-embedded nitroxides provide efficient polarization matrices for dynamic nuclear polarization NMR.

Hybrid mesoporous silica materials with wall-embedded nitroxides are shown to efficiently polarize impregnated substrates in high-field dynamic nuclear polarization magic-angle spinning solid-state NMR experiments.

Dimerization interface and dynamic properties of yeast IF1 revealed by Site-Directed Spin Labeling EPR spectroscopy.

The mitochondrial ATPase inhibitor, IF1, regulates the activity of the mitochondrial ATP synthase. The oligomeric state of IF1 related to pH is crucial for its inhibitory activity. Although extensive structural studies have been performed to characterize the oligomeric states of bovine IF1, only little is known concerning those of yeast IF1.

Exploring intrinsically disordered proteins using site-directed spin labeling electron paramagnetic resonance spectroscopy.

Proteins are highly variable biological systems, not only in their structures but also in their dynamics. The most extreme example of dynamics is encountered within the family of Intrinsically Disordered Proteins (IDPs), which are proteins lacking a well-defined 3D structure under physiological conditions. Among the biophysical techniques well-suited to study such highly flexible proteins, Site-Directed Spin Labeling combined with EPR spectroscopy (SDSL-EPR) is one of the most powerful, being able to reveal, at the residue level, structural transitions such as folding events.

Conformational selection underlies recognition of a molybdoenzyme by its dedicated chaperone.

Molecular recognition is central to all biological processes. Understanding the key role played by dedicated chaperones in metalloprotein folding and assembly requires the knowledge of their conformational ensembles. In this study, the NarJ chaperone dedicated to the assembly of the membrane-bound respiratory nitrate reductase complex NarGHI, a molybdenum-iron containing metalloprotein, was taken as a model of dedicated chaperone.

The influence of the molecular system on the performance of heteronuclear decoupling in solid-state NMR.

The intensity of the carbon signal in a CPMAS experiment has been measured for two CH and three CH(2) moieties in four test molecules under different phase-modulated proton decoupling conditions and as a function of the spinning rate. The proton decoupling schemes investigated were the golden standard TPPM and three of the GTn family. Aim of this analysis was to better describe experimentally the impact and limitations of phase-modulated decoupling.

Structural characterization of metal-metal bonded polymer [Ru(L)(CO)(2)](n) (L = 2,2'-bipyridine) in the solid state using high-resolution NMR and DFT chemical shift calculations.

The metal bonded ruthenium polymer [Ru(0)(bpy)(CO)(2)](n) (bpy = 2,2'-bipyridine) is known to be a very promising and efficient solid material for catalysis applications, such as carbon dioxide electroreduction in pure aqueous media and the water-gas shift reaction. It also exhibits potential application for molecular electronics as a conductive molecular wire. The insolubility and relative air-sensitivity of [Ru(0)(bpy)(CO)(2)](n) as well as the lack of monocrystals make its structural characterization very challenging.

Microscopic structure of heterogeneous lipid-based formulations revealed by 13C high-resolution solid-state and 1H PFG NMR methods.

Lipid-based formulations such as lip glosses that are very alike on the base of their components may have significant differences in their expected macroscopic properties as cosmetics. To differentiate such formulations, high-resolution (13)C NMR was performed under magic angle spinning to investigate the properties at both molecular and microscopic levels. Temperature studies were carried out and no polymorphism in the solid domains could be evidenced after the thermal treatment performed for obtaining the commercial lip glosses.

Spin-coated and PECVD low dielectric constant porous organosilicate films studied by 1D and 2D solid-state NMR.

In the research field of the sub-65 nm semiconductor industry, organosilicate SiOCH films with low dielectric constant (k < 2.4) need to be developed in order to improve the performance of integrated circuits [International Roadmap for Semiconductors (ITRS), San Jose, CA, 2004]. One way to produce SiOCH films of low dielectric constant is to introduce pores into the film.