Strikt anaerobe Bakterien wie Clostridium difficile und C. scatologenes verwenden GRE, um die chemisch ungünstige Decarboxylierung von 4-Hydroxyphenylacetat zu p-Cresol zu katalysieren. Das Enzymsystem besteht aus einer Decarboxylase und dem zugehörigen Aktivierungsenzym. Die 4-Hydroxyphenylacetat-Decarboxylase (4Hpad) besitzt zusätzlich zum Protein-basierten Glycinradikal eine weitere Untereinheit mit bis zu zwei [4Fe-4S] Clustern und repräsentiert hierdurch eine neue Klasse von Fe/S-Cluster-haltigen GREs, die aromatische Verbindungen umsetzen. Das Aktivierungsenzym (4Hpad-AE) weicht vom Standardtypus ab, indem es zusätzlich zum S-Adenosylmethionin(SAM)-bindenden [4Fe-4S]-Cluster (RS-Cluster) mindestens einen weiteren [4Fe-4S]-Cluster bindet. In dieser Studie wurden heterologe Expressions- und Reinigungsprotokolle für 4Hpad und 4Hpad-AE entwickelt. Kristallstrukturen von 4Hpad cokristallisiert mit den Substraten (4-Hydroxyphenylacetat, 3,4-Dihydroxyphenylacetat) und dem Inhibitor (4-Hydroxyphenylacetamid) zeigten geringe strukturelle Änderungen im aktiven Zentrum des Proteins. Die Radikalbildung am 4Hpad-AE wurde durch die Überprüfung einer klassischen reduktiven Spaltung von SAM zu den Reaktionsprodukten 5’-Deoxyadenosin und Methionin bestätigt. EPR- und Mössbauer-Spektroskopische Analysen zeigten, dass 4Hpad-AE mindestens einen zusätzlichen [4Fe-4S] Cluster neben dem einzelnen RS-Cluster enthält. Die katalytische Notwendigkeit eines zusätzlichen Clusters wurde durch eine Mutationsanalyse untersucht, wobei eine verkürzte Version des Enzyms ohne die zusätzliche Cystein-reiche Insertion konstruiert wurde. Das verkürzte Mutante ohne die Bindungsmotive für die zusätzlichen Cluster gekennzeichnet, die Konfiguration, Stöchiometrie und die Funktion der zusätzlichen Cluster diagnostizieren. / 4-hydroxyphenylacetate decarboxylase (4Hpad) is a two [4Fe-4S] cluster containing glycyl radical enzyme proposed to use a glycyl/thiyl radical dyad to catalyze the last step of tyrosine fermentation in Clostridium difficile and C. scatologenes by a Kolbe-type decarboxylation. The decarboxylation product p-cresol is a virulence factor of the human pathogen C. difficile. The small subunit of 4Hpad may have a regulatory function with the Fe/S clusters involved in complex formation and radical dissipation in the absence of substrate. The respective activating enzyme (4Hpad-AE) has one or two [4Fe-4S] cluster(s) in addition to the SAM-binding [4Fe-4S] cluster (RS cluster). The role of these auxiliary clusters is still under debate with proposed functions including structural integrity and conduit for electron transfer to the RS cluster. This study shows the optimized expression and purification protocols for the decarboxylase and the co-crystallization experiments and binding studies with 4-hydroxy-phenylacetate and 3,4-dihydroxyphenylacetate and with the inhibitor 4-hydroxy-phenylacetamide. The purification and characterization of active site mutants of decarboxylase are also done. Concerning 4-HPAD-AE, we report on the purification of code-optimized variants, and on spectroscopic and kinetic studies to characterize the respective i) SAM binding enthalpies, ii) rates for reductive cleavage of SAM and iii) putative functions of the additional Fe/S clusters. The truncated mutant lacking the binding motifs for the auxiliary clusters is characterized to diagnose the configuration, stoichiometry and function of the auxiliary clusters.

Les protéines [Fe-S] sont des enzymes ubiquitaires, assurant des fonctions clés au sein des organismes vivants. La biosynthèse des centres [Fe-S], à savoir les processus permettant un assemblage correct des atomes de fer et de soufre au sein des protéines cibles, requièrent la participation de machineries protéiques complexes. Parmi elles se trouve la machinerie SUF qui intervient dans des conditions de stress oxydant et de carence en fer. Elle est composée de six gènes sufABCDSE. La protéine SufA est proposée comme étant une protéine scaffold ayant pour rôle de préassembler transitoirement des centres [Fe-S] et de les transférer à des protéines cibles. Elle possède trois résidus cystéines conservés proposés comme étant les ligands des centres [Fe-S].<br />SufA est obtenue principalement sous forme apo après purification. Le centre [Fe-S] peut être reconstitué chimiquement in vitro. Dans ces conditions, SufA contient un mélange de centres [2Fe-2S] et [4Fe-4S]. Nous avons alors isolé SufA native métallée après purification à partir de tout l'opéron suf en anaérobiose, et montré qu'elle contient un centre [Fe-S], plutôt de type [2Fe-2S], transférable efficacement à la ferrédoxine. Nous avons également étudié les mécanismes moléculaires de formation du cluster dans SufA. SufA est capable de fixer à la fois du soufre, au niveau de ses trois cystéines conservées, et du fer, majoritairement au niveau d'atomes d'azote et d'oxygène. Ces éléments sont mobilisables pour la formation d'un centre [Fe-S] en milieu réducteur. Enfin, des expériences préliminaires réalisées in vitro avec des mutants dirigés n'ont pas permis d'identifier la nature exacte des ligands du centre [Fe-S] dans SufA.

Cofatores ferro-enxofre são grupos prostéticos inorgânicos ubíquos e evolutivamente ancestrais, cuja formação é dependente de complexas maquinarias protéicas. Três sistemas de formação distintos já foram determinados, denominados sistemas NIF, ISC e SUF. Apesar de bem descritos em diversos organismos, estas maquinarias são pouco caracterizadas no filo Firmicutes, o qual agrupa diversas bactérias patogênicas, e onde Enterococcus faecalis aparece como um representante clinicamente relevante. O objetivo deste estudo foi identificar a maquinaria biossintética de formação dos cofatores [Fe-S] de E. faecalis mediante análises de bioinformática, determinação das regiões promotoras do operon e de elementos cis-atuantes, padrão de expressão gênica, caracterização bioquímica dos elementos encontrados e comparação entre as maquinarias de associação do cofator [Fe-S] presente em Proteobacteria e Firmicutes através da capacidade de complementação deste sistema nos sistemas ISC e SUF de Azotobacter vinelandii e Escherichia coli, respectivamente. Metodologias de bioinformática permitiram identificar representantes da maquinaria SUF de formação dos cofatores [Fe-S], previamente identificado em Proteobacteria, apresentando os genes sufB, sufC, sufD e sufS e a presença de sufU, o único representante homólogo do sistema ISC, codificando possível proteína arcabouço, no lugar de sufA; da mesma forma, sufE e sufR não foram identificadas. A alta conservação deste sistema foi verificada em Firmicutes através de análises filogenéticas. Análise de sequências primária e estrutural de SufU verificaram um padrão estrutural similar à IscU. Modelagem molecular de SufU de E. faecalis apresentou dados de alta flexibilidade na região do sítio ativo, bem como a presença de região específica em Firmicutes, denominada região Gram-positiva (GPR), possivelmente envolvida em interações com outros fatores e/ou reguladores. SufU e o complexo SufSU são capazes de reconstituir cofactor [4Fe-4S], apresentando-se portanto como a proteína arcabouço do sistema. A enzima SufS purificada apresenta PLP ligado como cofator e atividade de cisteína desulfurase. Esta enzima apresenta um residuo catalítico essencial de cisteína na posição 365 , e necessita SufU como ativador, onde outro residuo de cisteína (128) atua como aceptor do enxofre durante a reação de transpersulfuração. SufC apresenta atividade ATPase, porém em nível reduzido em comparação ao homólogo de E. coli; SufD apresenta alta similaridade com homólogo de proteobactérias. Por outro lado, SufB não apresenta os resíduos de cisteína previamente descritos como importantes na formação dos cofatores [Fe-S] em outros organismos, assim sua função no sistema ainda deve ser determinada. Experimentos in vivo demonstraram a conservação específica de sistemas biossintéticos dos cofatores [Fe-S], onde o operon SUF de E. faecalis não foi capaz de complementar os sistemas ISC de Proteobacteria, porém complementou sistema SUF de E. coli, tornando viáveis mutantes de ambos os operons sufABCDSE e iscRSU-hscBA-fdx. / Iron-sulfur clusters are ubiquitous and evolutionary ancient inorganic prosthetic groups, which biosynthesis depends on complex protein machineries. Three distinct assembly systems involved in the maturation of cellular Fe-S proteins have been determined, designated the NIF, ISC and SUF systems. Although well described in several organisms, these machineries are poorly understood in the Firmicutes phylum, which groups several pathological bacteria, where Enterococcus faecalis rises as a clinical relevant representative. The aim of this study was to identify the E. faecalis [Fe-S] cluster biosynthetic machinery through bioinformatics analysis, determination of operon promoter regions and cis-acting elements, relative genetic expression pattern, biochemical characterization of putative elements, and comparison of Proteobacteria and Firmicutes machineries through the ability of complementing Azotobacter vinelandii and Escherichia coli ISC and SUF systems, respectively. Bioinformatics methods enabled us to identify representatives of the SUF machinery for [Fe-S] cluster biosynthesis, previously verified in Proteobacteria showing conserved sufB, sufC, sufD and sufS genes and the presence of sufU, the only ISC homolog representative, coding for putative scaffold protein, instead of sufA; neither sufE nor sufR are present. High conservancy of this system for Firmicutes bacteria was verified through phylogenetic analysis. Primary sequences and structural analysis of the SufU protein demonstrated its structural-like pattern to the scaffold protein IscU. E. faecalis SufU molecular modeling showed high flexibility over the active site regions, and demonstrated the existence of a specific region in Firmicutes, the Gram positive region (GPR), a possible candidate for interaction with other factors and/or regulators. SufU is able to reconstitute a [4Fe-4S] cluster, such as the complex SufSU, arising as the scaffold protein in the system. Purified SufS corresponds to a PLP containing enzyme with cysteine desulfurase activity. It encloses a catalytically essential cysteine residue at position 365, and requires SufU as activator, where another cysteine residue (128) works as a proximal sulfur acceptor site for transpersulfurization reaction. SufC presents ATPase activity, though in a reduced level, when compared to the Escherichia coli homolog; SufD also shares high similarity with proteobacterial SufD. On the other hand, SufB does not present cysteine residues previously described as important involved in the [Fe-S] cluster formation process of other organisms, therefor its function in the system still have to be determined. In vivo experiments enabled us to dfemonstrate the conservancy of specific [Fe-S] cluster biosynthetic systems, where E. faecalis SUF operon was not able to complement Proteobacteria ISC systems, but complemented E. coli SUF system, turning viable mutants of both sufABCDSE and iscRSU-hscBA-fdx operons.

Cofatores ferro-enxofre são grupos prostéticos inorgânicos ubíquos e evolutivamente ancestrais, cuja formação é dependente de complexas maquinarias protéicas. Três sistemas de formação distintos já foram determinados, denominados sistemas NIF, ISC e SUF. Apesar de bem descritos em diversos organismos, estas maquinarias são pouco caracterizadas no filo Firmicutes, o qual agrupa diversas bactérias patogênicas, e onde Enterococcus faecalis aparece como um representante clinicamente relevante. O objetivo deste estudo foi identificar a maquinaria biossintética de formação dos cofatores [Fe-S] de E. faecalis mediante análises de bioinformática, determinação das regiões promotoras do operon e de elementos cis-atuantes, padrão de expressão gênica, caracterização bioquímica dos elementos encontrados e comparação entre as maquinarias de associação do cofator [Fe-S] presente em Proteobacteria e Firmicutes através da capacidade de complementação deste sistema nos sistemas ISC e SUF de Azotobacter vinelandii e Escherichia coli, respectivamente. Metodologias de bioinformática permitiram identificar representantes da maquinaria SUF de formação dos cofatores [Fe-S], previamente identificado em Proteobacteria, apresentando os genes sufB, sufC, sufD e sufS e a presença de sufU, o único representante homólogo do sistema ISC, codificando possível proteína arcabouço, no lugar de sufA; da mesma forma, sufE e sufR não foram identificadas. A alta conservação deste sistema foi verificada em Firmicutes através de análises filogenéticas. Análise de sequências primária e estrutural de SufU verificaram um padrão estrutural similar à IscU. Modelagem molecular de SufU de E. faecalis apresentou dados de alta flexibilidade na região do sítio ativo, bem como a presença de região específica em Firmicutes, denominada região Gram-positiva (GPR), possivelmente envolvida em interações com outros fatores e/ou reguladores. SufU e o complexo SufSU são capazes de reconstituir cofactor [4Fe-4S], apresentando-se portanto como a proteína arcabouço do sistema. A enzima SufS purificada apresenta PLP ligado como cofator e atividade de cisteína desulfurase. Esta enzima apresenta um residuo catalítico essencial de cisteína na posição 365 , e necessita SufU como ativador, onde outro residuo de cisteína (128) atua como aceptor do enxofre durante a reação de transpersulfuração. SufC apresenta atividade ATPase, porém em nível reduzido em comparação ao homólogo de E. coli; SufD apresenta alta similaridade com homólogo de proteobactérias. Por outro lado, SufB não apresenta os resíduos de cisteína previamente descritos como importantes na formação dos cofatores [Fe-S] em outros organismos, assim sua função no sistema ainda deve ser determinada. Experimentos in vivo demonstraram a conservação específica de sistemas biossintéticos dos cofatores [Fe-S], onde o operon SUF de E. faecalis não foi capaz de complementar os sistemas ISC de Proteobacteria, porém complementou sistema SUF de E. coli, tornando viáveis mutantes de ambos os operons sufABCDSE e iscRSU-hscBA-fdx. / Iron-sulfur clusters are ubiquitous and evolutionary ancient inorganic prosthetic groups, which biosynthesis depends on complex protein machineries. Three distinct assembly systems involved in the maturation of cellular Fe-S proteins have been determined, designated the NIF, ISC and SUF systems. Although well described in several organisms, these machineries are poorly understood in the Firmicutes phylum, which groups several pathological bacteria, where Enterococcus faecalis rises as a clinical relevant representative. The aim of this study was to identify the E. faecalis [Fe-S] cluster biosynthetic machinery through bioinformatics analysis, determination of operon promoter regions and cis-acting elements, relative genetic expression pattern, biochemical characterization of putative elements, and comparison of Proteobacteria and Firmicutes machineries through the ability of complementing Azotobacter vinelandii and Escherichia coli ISC and SUF systems, respectively. Bioinformatics methods enabled us to identify representatives of the SUF machinery for [Fe-S] cluster biosynthesis, previously verified in Proteobacteria showing conserved sufB, sufC, sufD and sufS genes and the presence of sufU, the only ISC homolog representative, coding for putative scaffold protein, instead of sufA; neither sufE nor sufR are present. High conservancy of this system for Firmicutes bacteria was verified through phylogenetic analysis. Primary sequences and structural analysis of the SufU protein demonstrated its structural-like pattern to the scaffold protein IscU. E. faecalis SufU molecular modeling showed high flexibility over the active site regions, and demonstrated the existence of a specific region in Firmicutes, the Gram positive region (GPR), a possible candidate for interaction with other factors and/or regulators. SufU is able to reconstitute a [4Fe-4S] cluster, such as the complex SufSU, arising as the scaffold protein in the system. Purified SufS corresponds to a PLP containing enzyme with cysteine desulfurase activity. It encloses a catalytically essential cysteine residue at position 365, and requires SufU as activator, where another cysteine residue (128) works as a proximal sulfur acceptor site for transpersulfurization reaction. SufC presents ATPase activity, though in a reduced level, when compared to the Escherichia coli homolog; SufD also shares high similarity with proteobacterial SufD. On the other hand, SufB does not present cysteine residues previously described as important involved in the [Fe-S] cluster formation process of other organisms, therefor its function in the system still have to be determined. In vivo experiments enabled us to dfemonstrate the conservancy of specific [Fe-S] cluster biosynthetic systems, where E. faecalis SUF operon was not able to complement Proteobacteria ISC systems, but complemented E. coli SUF system, turning viable mutants of both sufABCDSE and iscRSU-hscBA-fdx operons.

Aim of this thesis was to shed light on the function(s) of Iba57 and Ssc1 proteins in both life cycle stages of T. brucei using RNA interference. Depletion of Ssc1 resulted in severe grow phenotype, decrease in activities of iron-sulphur cluster-containing enzyme aconitase but no increase in oxidative stress sensitivity or accumulation of ROS in mitochondrion. Down regulation of Iba57, specialized maturation factor of aconitase and homoaconitase, lead to depletion of aconitase, destabilization of Isa1 and increased sensitivity to oxidative stress and accumulation of ROS in both stages.

Cofatores ferro-enxofre são grupos prostéticos inorgânicos ubíquos e evolutivamente ancestrais, cuja formação é dependente de complexas maquinarias protéicas. Três sistemas de formação distintos já foram determinados, denominados sistemas NIF, ISC e SUF. Apesar de bem descritos em diversos organismos, estas maquinarias são pouco caracterizadas no filo Firmicutes, o qual agrupa diversas bactérias patogênicas, e onde Enterococcus faecalis aparece como um representante clinicamente relevante. O objetivo deste estudo foi identificar a maquinaria biossintética de formação dos cofatores [Fe-S] de E. faecalis mediante análises de bioinformática, determinação das regiões promotoras do operon e de elementos cis-atuantes, padrão de expressão gênica, caracterização bioquímica dos elementos encontrados e comparação entre as maquinarias de associação do cofator [Fe-S] presente em Proteobacteria e Firmicutes através da capacidade de complementação deste sistema nos sistemas ISC e SUF de Azotobacter vinelandii e Escherichia coli, respectivamente. Metodologias de bioinformática permitiram identificar representantes da maquinaria SUF de formação dos cofatores [Fe-S], previamente identificado em Proteobacteria, apresentando os genes sufB, sufC, sufD e sufS e a presença de sufU, o único representante homólogo do sistema ISC, codificando possível proteína arcabouço, no lugar de sufA; da mesma forma, sufE e sufR não foram identificadas. A alta conservação deste sistema foi verificada em Firmicutes através de análises filogenéticas. Análise de sequências primária e estrutural de SufU verificaram um padrão estrutural similar à IscU. Modelagem molecular de SufU de E. faecalis apresentou dados de alta flexibilidade na região do sítio ativo, bem como a presença de região específica em Firmicutes, denominada região Gram-positiva (GPR), possivelmente envolvida em interações com outros fatores e/ou reguladores. SufU e o complexo SufSU são capazes de reconstituir cofactor [4Fe-4S], apresentando-se portanto como a proteína arcabouço do sistema. A enzima SufS purificada apresenta PLP ligado como cofator e atividade de cisteína desulfurase. Esta enzima apresenta um residuo catalítico essencial de cisteína na posição 365 , e necessita SufU como ativador, onde outro residuo de cisteína (128) atua como aceptor do enxofre durante a reação de transpersulfuração. SufC apresenta atividade ATPase, porém em nível reduzido em comparação ao homólogo de E. coli; SufD apresenta alta similaridade com homólogo de proteobactérias. Por outro lado, SufB não apresenta os resíduos de cisteína previamente descritos como importantes na formação dos cofatores [Fe-S] em outros organismos, assim sua função no sistema ainda deve ser determinada. Experimentos in vivo demonstraram a conservação específica de sistemas biossintéticos dos cofatores [Fe-S], onde o operon SUF de E. faecalis não foi capaz de complementar os sistemas ISC de Proteobacteria, porém complementou sistema SUF de E. coli, tornando viáveis mutantes de ambos os operons sufABCDSE e iscRSU-hscBA-fdx. / Iron-sulfur clusters are ubiquitous and evolutionary ancient inorganic prosthetic groups, which biosynthesis depends on complex protein machineries. Three distinct assembly systems involved in the maturation of cellular Fe-S proteins have been determined, designated the NIF, ISC and SUF systems. Although well described in several organisms, these machineries are poorly understood in the Firmicutes phylum, which groups several pathological bacteria, where Enterococcus faecalis rises as a clinical relevant representative. The aim of this study was to identify the E. faecalis [Fe-S] cluster biosynthetic machinery through bioinformatics analysis, determination of operon promoter regions and cis-acting elements, relative genetic expression pattern, biochemical characterization of putative elements, and comparison of Proteobacteria and Firmicutes machineries through the ability of complementing Azotobacter vinelandii and Escherichia coli ISC and SUF systems, respectively. Bioinformatics methods enabled us to identify representatives of the SUF machinery for [Fe-S] cluster biosynthesis, previously verified in Proteobacteria showing conserved sufB, sufC, sufD and sufS genes and the presence of sufU, the only ISC homolog representative, coding for putative scaffold protein, instead of sufA; neither sufE nor sufR are present. High conservancy of this system for Firmicutes bacteria was verified through phylogenetic analysis. Primary sequences and structural analysis of the SufU protein demonstrated its structural-like pattern to the scaffold protein IscU. E. faecalis SufU molecular modeling showed high flexibility over the active site regions, and demonstrated the existence of a specific region in Firmicutes, the Gram positive region (GPR), a possible candidate for interaction with other factors and/or regulators. SufU is able to reconstitute a [4Fe-4S] cluster, such as the complex SufSU, arising as the scaffold protein in the system. Purified SufS corresponds to a PLP containing enzyme with cysteine desulfurase activity. It encloses a catalytically essential cysteine residue at position 365, and requires SufU as activator, where another cysteine residue (128) works as a proximal sulfur acceptor site for transpersulfurization reaction. SufC presents ATPase activity, though in a reduced level, when compared to the Escherichia coli homolog; SufD also shares high similarity with proteobacterial SufD. On the other hand, SufB does not present cysteine residues previously described as important involved in the [Fe-S] cluster formation process of other organisms, therefor its function in the system still have to be determined. In vivo experiments enabled us to dfemonstrate the conservancy of specific [Fe-S] cluster biosynthetic systems, where E. faecalis SUF operon was not able to complement Proteobacteria ISC systems, but complemented E. coli SUF system, turning viable mutants of both sufABCDSE and iscRSU-hscBA-fdx operons.

The organization of metabolism is an essential feature of cellular biochemistry. Metabolism does not occur as a linear assembly of freely diffusing enzymes, but as a complex web in which multiple interactions are possible. Because of the crowded environment of the cell, there must be structured and ordered mechanisms that control metabolic pathways. The following work will examine two metabolic pathways, one that is ubiquitous among living organisms and another that is entirely unique to plants, and examine the organization of each in an attempt to further define mechanisms that are fundamental features of metabolic control. One study offers some of the first characterizations of genes involved in [Fe-S] cluster assembly in Arabidopsis. The other explores the mechanisms that control localization of an enzyme that is part of the well-characterized flavonoid biosynthetic pathway. These two distinct pathways serve as unique models for genetic and biochemical studies that contribute to our overall understanding of plant metabolism. / Ph. D.

In the search for the mitochondrion of oxymonads, DNA of Monocercomonoides exilis - an oxymonad isolated from the gut of Chinchilla, was isolated and its genome was sequenced. Sequencing resulted in a fairly complete genome which was extensively searched or genes for mitochondrion related proteins, but no reliable candidate for such gene was identified. Even genes for the ISC pathway, which is responsible for Fe-S cluster assembly and considered to be the only essential function of reduced mitochondrion-like organelles (MROs), were absent. Instead, we were able to detect the presence of a SUF pathway which functionally replaced the ISC pathway. Closer examination of the SUF pathway based on heterologous localisation revealed that this pathway localised in the cytosol. In silico analysis showed that SUF genes are highly conserved at the level of secondary and tertiary structure and most catalytic residues and motifs are present in their sequences. The functionality of these proteins was further indirectly confirmed by complementation experiments in Escherichia coli where SUF proteins of M. exilis were able to restore at least partially Fe-S cluster assembly of strains deficient in the SUF and ISC pathways. We also proved by bacterial adenylate cyclase two-hybrid system that SufB and SufC can form...

Ce manuscrit résume mes activités de recherche en thèse, dans mes différents séjours post-doctoraux et mes premières années de recherche au Commissariat à l'Energie Atomique. Elles concernent la caractérisation de biomolécules par RMN, comme des sucres, des protéines, dia ou paramagnétiques. Le manuscrit aborde aussi la caractérisation des interactions protéines-eau, du métabolisme cellulaire, et la mesure de vitesses électroniques impliquant des protéines à plusieurs centres d'oxydoréduction. <br />J'aborde ensuite mon travail récent au sein d'une équipe spécialisée dans le xénon hyperpolarisé appliqué à la phase liquide, en particulier la caractérisation de cavités hydrophobes de protéines et la forte complexation du xénon par une famille de molécules-cage nommée cryptophanes.

Ce travail de thèse propose de réaliser un nanofil électrique auto-assemblé constitué de protéines. L'unité de base de ce nanofil est une protéine chimère comprenant un domaine capable de former des fibres amyloïdes (Het-s 218-289) et un domaine capable d'effectuer des transferts d'électrons (une rubrédoxine). Le premier domaine permet la réalisation d'une fibre par auto-assemblage tandis que le deuxième est exposé à la surface de cette structure. Les caractéristiques redox du domaine exposé permettent aux électrons de se déplacer d'un bout à l'autre de la fibre par sauts successifs. Un tel nanofil a été créé et caractérisé par différentes techniques biophysiques. Ensuite, la preuve de la conduction des nanofils a été apportée sur des ensembles d'objets, de manière indirecte par électrochimie, et de manière directe par des mesures tension/courant. Ces travaux ouvrent la voie à la réalisation d'objets biocompatibles, biodégradables, possédant des propriétés électroniques exploitables dans des dispositifs technologiques.