Hugo Fraga

Institut de Biologie Structurale

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Paul Schanda

Institute of Science and Technology Austria

Pavel Macek

Centre National de la Recherche Scientifique

Rui Fontes

Universidade do Porto

Diego F. Gauto

Institut de Chimie des Substances Naturelles

Joaquim C. G. Esteves da Silva

Universidade do Porto

Salvador Ventura

Universitat Autònoma de Barcelona

Audrey Hessel

Institut de Pharmacologie et de Biologie Structurale

Katharina Weinhäupl

Institut de Biologie Structurale

Charles‐Adrien Arnaud

Institut de Biologie Structurale

Jia‐Ying Guan

Université Grenoble Alpes

Cooperative Institutions

Centre National de la Recherche Scientifique

Universidade do Porto

Institut de Biologie Structurale

Université Grenoble Alpes

Commissariat à l'Énergie Atomique et aux Énergies Alternatives

Universidade Federal de Santa Catarina

CEA Grenoble

Harvard University

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto

Consejo Superior de Investigaciones Científicas

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Firefly Luciferase Produces Hydrogen Peroxide as a Coproduct in Dehydroluciferyl Adenylate Formation

ChemBioChem (2006)

Hugo Fraga Diogo Fernandes J. Novotný Rui Fontes Joaquim C. G. Esteves da Silva

Abstract Firefly luciferase catalyzes the synthesis of H 2 O 2 from the same substrates as the bioluminescence reaction: ATP and luciferin ( d‐ LH 2 ). About 80 % of the enzyme‐bound intermediate d‐ luciferyl adenylate ( d‐ LH 2 ‐AMP) is oxidized into oxyluciferin, and a photon is emitted during this reaction. The enzyme pathway responsible for the generation of H 2 O 2 is a side reaction in which d‐ LH 2 ‐AMP is oxidized into dehydroluciferyl adenylate ( L‐AMP ). Like the bioluminescence reaction, the luciferase‐catalyzed synthesis of H 2 O 2 and L‐AMP is a stereospecific process, involving only the natural D enantiomer. However, the intramolecular electron transfer postulated as essential to the light emission process is not involved in this side reaction.

Luciferin

Stereospecificity

Luciferases

Enantiomeric excess

10.1002/cbic.200500443

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Citations (59)

Pyrophosphate and tripolyphosphate affect firefly luciferase luminescence because they act as substrates and not as allosteric effectors

FEBS Journal (2008)

Rui Fontes Diogo Fernandes Filipe Peralta Hugo Fraga Inês Maio

The activating and stabilizing effects of inorganic pyrophosphate, tripolyphosphate and nucleoside triphosphates on firefly luciferase bioluminescence were studied. The results obtained show that those effects are a consequence of the luciferase‐catalyzed splitting of dehydroluciferyl‐adenylate, a powerful inhibitor formed as a side product in the course of the bioluminescence reaction. Inorganic pyrophosphate, tripolyphosphate, CTP and UTP antagonize the inhibitory effect of dehydroluciferyl‐adenylate because they react with it giving rise to products that are, at least, less powerful inhibitors. Moreover, we demonstrate that the antagonizing effects depended on the rate of the splitting reactions being higher in the cases of inorganic pyrophosphate and tripolyphosphate and lower in the cases of CTP and UTP. In the case of inorganic pyrophosphate, the correlation between the rate of dehydroluciferyl‐adenylate pyrophosphorolysis and the activating effect on bioluminescence only occurs for low concentrations because inorganic pyrophosphate is, simultaneously, an inhibitor of the bioluminescence reaction. Our results demonstrate that previous reports concerning the activating effects of several nucleotides (including some that do not react with dehydroluciferyl‐adenylate) on bioluminescence were caused by the presence of inorganic pyrophosphate contamination in the preparations used.

10.1111/j.1742-4658.2008.06309.x

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Citations (39)

Deuteration of proteins boosted by cell lysates: high-resolution amide and Hα MAS NMR without re-protonation bottleneck

bioRxiv (Cold Spring Harbor Laboratory) (2024)

Federico Napoli Jia‐Ying Guan Charles‐Adrien Arnaud Pavel Macek Hugo Fraga

Amide-proton detected magic-angle spinning NMR of deuterated proteins has become a main technique in NMR-based structural biology. In standard deuteration protocols that rely on D 2 O-based culture media, non-exchangeable amide sites remain deuterated, making these sites unobservable. Here we demonstrate that proteins produced with H 2 O-based culture medium doped with deuterated cell lysate allow to overcome this “reprotonation bottleneck”, while retaining a high level of deuteration (ca. 80 %) and narrow line widths. We quantified coherence life times of several proteins prepared with this labelling pattern over a range of MAS frequencies (40-100 kHz). We demonstrate that under commonly used conditions (50-60 kHz MAS), amide 1 H line widths with our labelling approach are comparable to those of perdeuterated proteins and better than those of protonated samples at 100 kHz. For three proteins in the 33-50 kDa size range many previously unobserved amides become visible. We report how to prepare the deuterated cell lysate for our approach from fractions of perdeuterated cultures which are usually discarded, and show that such media can be used identically to commercial media. The residual protonation of Hα sites allows for well-resolved Hα-detected spectra and Hα resonance assignment, exemplified by the de novo assignment of 168 Hα sites in a 39 kDa protein. The approach based on this H 2 O/cell-lysate deuteration and MAS frequencies compatible with 1.3 or 1.9 mm rotors presents a strong sensitivity benefit over 0.7 mm/100 kHz MAS experiments.

Amide

Magic angle spinning

Proton NMR

10.1101/2024.01.08.574509

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Citations (0)

Publisher Correction: Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR

Nature Communications (2022)

Diego F. Gauto Pavel Macek Duccio Malinverni Hugo Fraga Matteo Paloni

10.1038/s41467-022-31243-1

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Chemical synthesis and firefly luciferase produced dehydroluciferyl-coenzyme A

Tetrahedron Letters (2004)

Hugo Fraga Joaquim C. G. Esteves da Silva Rui Fontes

Coenzyme A

10.1016/j.tetlet.2004.01.050

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Citations (13)

Disulfide driven folding for a conditionally disordered protein

Scientific Reports (2017)

Hugo Fraga Jordi Pujols Marcos Gil-García Alicia Roque Ganeko Bernardo‐Seisdedos

Abstract Conditionally disordered proteins are either ordered or disordered depending on the environmental context. The substrates of the mitochondrial intermembrane space (IMS) oxidoreductase Mia40 are synthesized on cytosolic ribosomes and diffuse as intrinsically disordered proteins to the IMS, where they fold into their functional conformations; behaving thus as conditionally disordered proteins. It is not clear how the sequences of these polypeptides encode at the same time for their ability to adopt a folded structure and to remain unfolded. Here we characterize the disorder-to-order transition of a Mia40 substrate, the human small copper chaperone Cox17. Using an integrated real-time approach, including chromatography, fluorescence, CD, FTIR, SAXS, NMR, and MS analysis, we demonstrate that in this mitochondrial protein, the conformational switch between disordered and folded states is controlled by the formation of a single disulfide bond, both in the presence and in the absence of Mia40. We provide molecular details on how the folding of a conditionally disordered protein is tightly regulated in time and space, in such a way that the same sequence is competent for protein translocation and activity.

Intrinsically Disordered Proteins

Mitochondrial intermembrane space

Intermembrane space

Chaperone (clinical)

Folding (DSP implementation)

10.1038/s41598-017-17259-4

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Citations (18)

Deuteration of proteins boosted by cell lysates: high-resolution amide and Hα magic-angle-spinning (MAS) NMR without the reprotonation bottleneck

Magnetic Resonance (2024)

Federico Napoli Jia‐Ying Guan Charles‐Adrien Arnaud Pavel Macek Hugo Fraga

Abstract. Amide-proton-detected magic-angle-spinning NMR of deuterated proteins has become a main technique in NMR-based structural biology. In standard deuteration protocols that rely on D2O-based culture media, non-exchangeable amide sites remain deuterated, making these sites unobservable. Here we demonstrate that proteins produced with a H2O-based culture medium doped with deuterated cell lysate allow scientists to overcome this “reprotonation bottleneck” while retaining a high level of deuteration (ca. 80 %) and narrow linewidths. We quantified coherence lifetimes of several proteins prepared with this labeling pattern over a range of magic-angle-spinning (MAS) frequencies (40–100 kHz). We demonstrate that under commonly used conditions (50–60 kHz MAS), the amide 1H linewidths with our labeling approach are comparable to those of perdeuterated proteins and better than those of protonated samples at 100 kHz. For three proteins in the 33–50 kDa size range, many previously unobserved amides become visible. We report how to prepare the deuterated cell lysate for our approach from fractions of perdeuterated cultures which are usually discarded, and we show that such media can be used identically to commercial media. The residual protonation of Hα sites allows for well-resolved Hα-detected spectra and Hα resonance assignment, exemplified by the de novo assignment of 168 Hα sites in a 39 kDa protein. The approach based on this H2O/cell-lysate deuteration and MAS frequencies compatible with 1.3 or 1.9 mm rotors presents a strong sensitivity benefit over 0.7 mm 100 kHz MAS experiments.

Magic angle spinning

Amide

10.5194/mr-5-33-2024

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Citations (1)

Understanding calcium induced conformational changes as revealed by an 8 KDa protein from Fasciola hepatica

Hugo Fraga QT Faria Agostinho Almeida Mmr Brito A.M. Damas

Hepatica

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The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

Journal of Biological Chemistry (2014)

Hugo Fraga Joan Josep Bech‐Serra Françesc Canals Gabriel Ortega Óscar Millet

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX9C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX9C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.

Intermembrane space

Mitochondrial intermembrane space

Oxidative folding

Folding (DSP implementation)

10.1074/jbc.m114.553479

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Citations (19)

Influence of Cytoplasmatic Folding on Mitochondrial Import

Current Medicinal Chemistry (2015)

Hugo Fraga Salvador Ventura

The eukaryotic cell, with its organelle organization, represents a challenge for protein traffic. Contrary to what occurs in the endoplasmic reticulum, mitochondrial protein import is proposed to occur postranslationaly, as proteins are synthesized in cytoplasmic ribosomes and only then imported to the organelle. Because the diameter of the Tom and Tim pores is too narrow for the passage of a folded protein, it is assumed that polypeptides must be already in an unfolded, import competent, state for organelle entry. However, it has been suggested that mitochondria might be able to actively unfold proteins itself at the outer membrane. Here we discuss the influence of cytoplasmatic protein folding on mitochondrial import. Despite the contribution of active mitochondrial unfolding to protein import is not excluded, this mechanism is inconsistent with a number of experimental evidences. Accordingly, other alternative models for mitochondrial import are here discussed. Understanding the molecular constraints regulating this process is of crucial importance, since its failure can lead to a number of pathological situations.

Folding (DSP implementation)

10.2174/0929867322666150311153413

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Citations (4)