Au cours de chimiothérapies, les cellules cancéreuses parviennent fréquemment à échapper aux effets toxiques des médicaments en développant des mécanismes de chimiorésistance qui résultent souvent de la présence d’un système d’efflux de ces médicaments. Cette chimiorésistance est corrélée à un phénomène appelé « phénotype MDR » pour (MultiDrug Resistance) et associé à la surexpression d’ATPases membranaires appartenant aux transporteurs ABC (ATP Binding Cassette). Le transporteur ABCG2 fait partie de cette grande famille de protéines. Un alignement de séquence a permis l’identification chez ABCG2 une séquence spécifique (LSGGE) très semblable à la séquence signature (VSGGE) de tous les transporteurs ABC. La mutation ponctuelle des résidus de cette séquence en alanine a produit une perte importante de fonction des mutants L352A et S353A, observée au niveau du transport et de l’activité ATPasique. Des relations structure-activité établies à partir de différents composés de la famille des flavonoïdes ont permis d’identifier MBLI 97, boéravinone G, MHT et ABI comme des composés puissants et spécifiques, capables d’abolir la résistance à de multiples drogues et chimiosensibiliser la croissance cellulaire. Le ciblage de séquences spécifiques et l'utilisation d'inhibiteurs spécifiques de ces transporteurs constituent des stratégies destinées à contrer la chimiorésistance et augmenter l’efficacité des traitements chimiothérapeutiques. / During chemotherapy, cancer cells frequently succeed to escape the toxic effects of drugs by developing mechanisms of chemoresistance which often result from the presence of an efflux system of these drugs. Such a chemoresistance is correlated to the MDR (MultiDrug Resistance) phenotype and associated to overexpression of membrane ATPases belonging to the ABC (ATP-Binding Cassette) transporters. The ABCG2 transporter belongs to this large family of proteins. Sequence alignment allowed the identification of a specific (LSGGE) sequence in ABCG2, which is quite similar to the canonical sequence signature (VSGGE) of all ABC transporters. Point mutation of these residues into alanine produced a loss of function in L352A and S353A mutants, as observed in transport and on ATPase activity. Structure-activity relationships drawn from some compounds among the family of flavonoids allowed the identification of MBLI 97, boeravinone G, MHT and ABI as potent and ABCG2-specific inhibitors, able to revert multidrug resistance and chemosensitize cell growth. The study of specific sequences and use of specific inhibitors of these transporters constitute strategies to abolish cancer cell chemoresistance and to increase the efficiency of chemotherapeutic treatments.

O sistema de captação de oligopeptideos (Opp), responsável pela captação de peptídeos com 3 ou mais resíduos de aminoácidos, representa um mecanismo importante de obtenção de nutrientes em bactérias. O operon Opp é constituído por 5 genes, sendo OppD e F responsáveis pela codificação dos componentes geradores, OppB e C codificantes para as proteínas que delimitam o poro da membrana e OppA que codifica o componente ligante. Neste trabalho identificamos uma alta identidade entre as proteínas OppA expressas por diferentes cepas de E. coli, 4 espécies do gênero Shigella ( 99 %) e diferentes sorovares de Salmonella enterica (85%) mas registramos a ocorrência de vários sítios polimórficos inter-específicos. A presença do gene OppA foi confirmada em 58 cepas diarreiogênicas de E. coli, Shigella e Salmonella. A partir da proteína OppA recombinante foi obtido soro policlonal específico que revelou a presença da proteína em todas as linhagens estudadas. Desta forma, concluímos que a proteína OppA está presente e conversada em espécies e linhagens dos três gêneros de Enterobacteriaceae estudados. / The oligopeptide uptake system (Opp), involved with the uptake of peptides formed by 3 or more amino acid residues, represent important nutrient uptake mechanism. The Opp operon is usually represented by 5 structural genes, including OppD and OppF encoding proteins involved generation of energy , OppB and OppC, encoding membrane proteins delimiting a pore and OppA encoding the protein responsible both for specificity and affinity of the transport system toward different peptide substrates. In this study, we demonstrated that the OppA proteins expressed by different E. coli strains,4 Shigella species (99%) and different serovars of Salmonella enterica (85%) were quite conserved but the occurrence of inter-species polymorphism was demonstrated. The OppA gene was detected in 58 diarrheogenic E. coli, Shigella and Salmonella strains. Using a recombinant OppA protein produced in E. coli, specific polyclonal sera were generated and successfully applied in the immunological detection of the proteins expressed by the tested strains. Thus, we conclude that the OppA protein is present and conserved among species and strains of the three test Enterobacteriaceae genera.

Malaria is a life threatening parasite disease caused and transmitted by infected female anopheles mosquito. However, the parasite, Plasmodium falciparum, has become resistant to most anti malarial drugs, such as chloroquine, which contributes to fever and anaemia because of its ability to digest the haemoglobin in the red blood cells. The aims of this project were to establish whether “Bac to Bac” Baculoviral Expression System is suitable for expression of pfmdr 1 gene and for purification of the pgh 1 protein. The pfmdr 1 gene encodes an ABC transporter protein, pgh 1, fixed in the cell membrane of the Plasmodium falciparuum gut, which assist in elimination of drug compounds. Furthermore, “Bac to Bac” Baculoviral Expression System uses vectors with histidine tags to clone the pfmdr 1 gene and subsequently transform these into DH10Bac cells to produce the recombinant bacmid DNA. Since pfmdr 1 gene is an AT-rich sequence, PCR was optimized, by lowering the annealing and extension temperature to 47Co and 66Co respectively. The results show that “Bac to Bac” Baculoviral Expression System can be used to express the pfmdr 1 gene, though further experiments has to be performed.

Cytoplasmic membrane proteins CvaB and CvaA and the outer membrane protein TolC form the bacteriocin colicin V (ColV) secretion system in Escherichia coli. CvaB functions as an ATP-binding cassette transporter with nucleotide-binding motifs in the C-terminal domain (CTD). To study the role of CvaB-CTD in the ColV secretion, a truncated construct of this domain was made and over-expressed. Different forms of CvaB-CTD were obtained during purification, and were identified as monomer, dimer, and oligomer on gel filtration. Nucleotide binding was shown critical for the CvaB-CTD dimerization: oligomers could be converted into dimers by nucleotide bindings; the removal of nucleotide from dimers resulted in transient monomers followed by CTD oligomerization and aggregation; no dimer form could be cross-linked from the nucleotide-binding deficient mutant D654H. The spatial proximity of the Walker A site and ABC signature motif in CTD dimer was identified through disulfide cross-linking of mixed CvaB-CTD with mutants A530C and L630C, while mutations did not dimerize individually. Those results indicated that the CvaB-CTD formed a nucleotide-dependent head-to-tail dimer. Molecular basis of differential nucleotide bindings was also studied through bioinformatics prediction and biochemical verification. Through sequence alignment and homology modeling with bound ATP or GTP, it was found that the Ser503 and Gln504 on aromatic stacking region (Y501DSQ-loop) of CvaB-CTD provided two additional hydrogen-bonds to GTP, but not to ATP. Site-directed mutations of the S503A and/or Q504L were designed based on the model. While site-directed mutagenesis studies of Walker A&B sites or the ABC signature motif affected little on the GTP-binding preference, the double mutation (S503A/Q504L) on the Y501DSQ-loop increased both ATP-binding and ATPase activity at low temperatures. The double mutant showed slight decrease of GTP-binding and about 10-fold increase of the ATP/GTP-binding ratio. Similar temperature sensitivity in nucleotide-binding and activity assays were identified in the double mutant at the same time. Mutations on the Y501DSQ-loop did not affect the ColV secretion level in vivo. Together, the Y501DSQ-loop is structurally involved in the differential binding of GTP over ATP.

<p>Malaria is a life threatening parasite disease caused and transmitted by infected female anopheles mosquito. However, the parasite, Plasmodium falciparum, has become resistant to most anti malarial drugs, such as chloroquine, which contributes to fever and anaemia because of its ability to digest the haemoglobin in the red blood cells. The aims of this project were to establish whether “Bac to Bac” Baculoviral Expression System is suitable for expression of pfmdr 1 gene and for purification of the pgh 1 protein. The pfmdr 1 gene encodes an ABC transporter protein, pgh 1, fixed in the cell membrane of the Plasmodium falciparuum gut, which assist in elimination of drug compounds. Furthermore, “Bac to Bac” Baculoviral Expression System uses vectors with histidine tags to clone the pfmdr 1 gene and subsequently transform these into DH10Bac cells to produce the recombinant bacmid DNA. Since pfmdr 1 gene is an AT-rich sequence, PCR was optimized, by lowering the annealing and extension temperature to 47Co and 66Co respectively. The results show that “Bac to Bac” Baculoviral Expression System can be used to express the pfmdr 1 gene, though further experiments has to be performed.</p>

Made available in DSpace on 2015-03-26T13:51:45Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1393738 bytes, checksum: a972ec00e49dcfa2c5d44a8c6f9480a0 (MD5) Previous issue date: 2009-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The plants produce antifungous proteins and specialized antibiotics (phytoanticipins and phytoalexins) in order to defend from phytopathogenic fungus. Only the pathogens able to avoid those defensive plant responses at the initial stages of the infection are able to cause disease. The fungus Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara is the etiological agent of the anthracnose in the common bean plant (Phaseolus vulgaris L.), that is an important disease contributing to reduce the productivity of the bean plant crop in Brazil. The genes abcCl1 and cypCl1 were isolated from this phytopathogen and their deduced aminoacid sequences have high similarity with ABC transporters and cytochromes P450, respectively. Those genes are found in unique copy and clustered in genome of the fungus, as revealed by either the sequencing of one DNA 8,4 kb fragment from the genomic library of C. lindemuthianum and the hybridization profile. Those genes also show an increased expression in response to different toxic compounds, mainly when the fungus is grown at the presence of eugenol, hygromycin and pisatin phytoalexin. Therefore, besides their genes to be physically linked, the proteins ABCCl1 and CYPCl1 can also be involved in the same functional process: the fungus resistance to the toxic compounds produced by plants or antagonistic microorganisms. In the functional analysis of the gene abcCl1, the mutants abcCl1&#8722; showed reduction in their virulence to the leaf of the common bean plant, compared with the wild line of C. lindemuthianum. Those results indicate the gene abcCl1 to codify an ABC transporter that is necessary to pathogenicity of C. lindemuthianum, probably because providing the fungus with the ability to overcome the defense mechanism of the plant. The cytochrome P450, that is codified by the gene cypCl1, can represent a complementary detoxification mechanism used by this fungus during the establishment of the disease. / Para se defenderem de fungos fitopatogênicos, as plantas produzem proteínas antifúngicas e antibióticos especializados (fitoanticipinas e fitoalexinas). Somente patógenos que conseguem evitar estas respostas defensivas da planta nos estágios iniciais da infecção são capazes de causar doença. O fungo Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara é o agente etiológico da antracnose do feijoeiro comum (Phaseolus vulgaris L.), uma importante doença que contribui para redução da produtividade da cultura do feijoeiro no Brasil. Neste fitopatógeno foram isolados dois genes, abcCl1 e cypCl1, cujas sequências de aminoácidos deduzidas possuem alta similaridade com transportadores ABC e citocromos P450, respectivamente. Estes genes estão presentes em cópia única e agrupados no genoma do fungo, conforme revelado no sequenciamento de um fragmento de DNA de 8,4 kb da biblioteca genômica de C. lindemuthianum, bem como no perfil de hibridização. Estes genes também apresentam aumento de expressão em resposta a diferentes compostos tóxicos, principalmente quando o fungo é crescido na presença de eugenol, higromicina ou fitoalexina pisatina. Assim, além de seus genes estarem ligados fisicamente, as proteínas ABCCl1 e CYPCl1 podem, também, estarem envolvidas em um mesmo processo funcional: a resistência do fungo a compostos tóxicos produzidos por plantas ou microrganismos antagonistas. Na análise funcional do gene abcCl1, foi observado que mutantes abcCl1&#8722; apresentam redução na virulência a folhas de feijoeiro comum, em comparação com a linhagem selvagem de C. lindemuthianum. Estes resultados indicam que o gene abcCl1 codifica um transportador ABC, que é necessário para a patogenicidade de C. lindemuthianum, provavelmente por conferir ao fungo a capacidade de superar algum mecanismo de defesa da planta. O citocromo P450, codificado pelo gene cypCl1, pode representar um mecanismo complementar de destoxificação empregado por este fungo durante o estabelecimento da doença.

O sistema de captação de oligopeptideos (Opp), responsável pela captação de peptídeos com 3 ou mais resíduos de aminoácidos, representa um mecanismo importante de obtenção de nutrientes em bactérias. O operon Opp é constituído por 5 genes, sendo OppD e F responsáveis pela codificação dos componentes geradores, OppB e C codificantes para as proteínas que delimitam o poro da membrana e OppA que codifica o componente ligante. Neste trabalho identificamos uma alta identidade entre as proteínas OppA expressas por diferentes cepas de E. coli, 4 espécies do gênero Shigella ( 99 %) e diferentes sorovares de Salmonella enterica (85%) mas registramos a ocorrência de vários sítios polimórficos inter-específicos. A presença do gene OppA foi confirmada em 58 cepas diarreiogênicas de E. coli, Shigella e Salmonella. A partir da proteína OppA recombinante foi obtido soro policlonal específico que revelou a presença da proteína em todas as linhagens estudadas. Desta forma, concluímos que a proteína OppA está presente e conversada em espécies e linhagens dos três gêneros de Enterobacteriaceae estudados. / The oligopeptide uptake system (Opp), involved with the uptake of peptides formed by 3 or more amino acid residues, represent important nutrient uptake mechanism. The Opp operon is usually represented by 5 structural genes, including OppD and OppF encoding proteins involved generation of energy , OppB and OppC, encoding membrane proteins delimiting a pore and OppA encoding the protein responsible both for specificity and affinity of the transport system toward different peptide substrates. In this study, we demonstrated that the OppA proteins expressed by different E. coli strains,4 Shigella species (99%) and different serovars of Salmonella enterica (85%) were quite conserved but the occurrence of inter-species polymorphism was demonstrated. The OppA gene was detected in 58 diarrheogenic E. coli, Shigella and Salmonella strains. Using a recombinant OppA protein produced in E. coli, specific polyclonal sera were generated and successfully applied in the immunological detection of the proteins expressed by the tested strains. Thus, we conclude that the OppA protein is present and conserved among species and strains of the three test Enterobacteriaceae genera.

Les transporteurs à ATP binding cassette (ABC) peuvent transporter une grande variété de substrats utilisant l'ATP-Mg2+ comme source d'énergie. Ces transporteurs sont présents dans toutes les formes de vie et sont impliqués dans la résistance aux médicaments, comprenant les anticancéreux et les antibiotiques. Mes travaux de thèse se concentrent sur le transporteur BmrA (130 kDa) de Bacillus subtilis utilisé en tant que système modèle et homologue de la P-glycoprotéine humaine impliquée dans la multirésistance aux anticancéreux. Dans ces travaux nous montrons que la reconstitution de cette protéine dans les lipides de Bacillus subtilis répond aux deux exigences centrales pour RMN: haut rapport signal sur bruit et la stabilité de l'échantillon sur une période de plusieurs années. Les spectres obtenus indiquent une protéine bien repliée et une préparation très homogène, comme en témoignent les lignes de résonance étroites et la dispersion du signal typique de la distribution de structure secondaire attendue de la protéine membranaire. Nous avons adapté la méthode GRecon utilisée dans les études de microscopie électronique pour la reconstitution des protéines membranaires pour la RMN à l'état solide. Nous avons suivi en détail la reconstitution du transporteur ABC BmrA par dialyse comme référence, et établi des conditions optimales de reconstitution en utilisant un gradient combiné de saccharose / cyclodextrine / lipide caractérisant GRecon. Les spectres RMN de l‘échantillon reconstitué par GRecon sont très similaire à ceux obtenus précédemment sur des échantillons reconstitués par dialyse. La préparation d'échantillons par GRecon présente un gain de temps de près d'un ordre de grandeur. Afin d'étudier les états ouvert vers l'intérieur (inward-facing IF) et ouvert vers l'extérieur (outward-facing OF) du transporteur, nous avons développé un protocole reproductible et quantitatif induisant l'état OF. Nous avons enregistré des spectres bidimensionnels RMN à l'état solide avec différents temps de mélange (20 et 200 ms) afin de suivre les changements des déplacements chimiques et d'identifier les résidus par des corrélations séquentielles. L'apparition très apparente de nouveaux signaux concomitants à la grande amplitude des perturbations de déplacement chimique (CSP) met en évidence l'importante flexibilité et les changements conformationnels de la protéine en présence d'ATP: Mg2+. Afin d'identifier les résidus apparaissant dans les spectres, nous avons utilisé le remplacement paramagnétique du co-facteur Mg2+ par du Mn2+. Cette méthode a révélé que les acides aminés apparaissant dans les spectres sont situés à proximité du site de liaison de l'ATP. En outre, les mesures EPR ont confirmé l'état fermé de la protéine en identifiant la distance correspondant à 1,8 nm entre deux atomes de Mn2+. Nous avons étudié les différences conformationnelles entre l'état IF et OF de BmrA. L'observation de nombreux CSP, ainsi que l'apparition de nouveaux signaux sont observés pour un mutant ne pouvant pas hydrolyser l'ATP, indiquant que l'hydrolyse n'est pas nécessaire pour la transition IF à OF dans BmrA. Nous avons également analysé le mécanisme lié au motif X-loop décrit comme étant impliqué dans la communication entre deux domaines de la protéine. Nous avons observé pour une protéine mutante dans laquelle le transport est aboli mais qui reste ATPase active, une transition incomplète puisque seul un sous-ensemble de CSPs est observé, ainsi qu'un manque de rigidification. Ces mesures suggèrent que la flexibilité semble être le point central dans la transmission des changements conformationnels nécessaires de la partie motrice à la partie d'exportation de molécules. Ces observations montrent que ce système serait semblable à un moteur tournant à plein régime qui ne serait pas connecté de manière rigide à un arbre de transmission le reliant au système de transport / ATP binding cassette (ABC) transporters can translocate a variety of molecules by coupling drug/lipid efflux with an ATP-Mg2+ fuelled engine. They are found in all forms of life and they are involved in a number of drug resistances including anti-cancer drugs and antibiotics. My studies focus on the drug exporter BmrA (130 kDa) from Bacillus subtilis as a model system and homologue of the human P-glycoprotein that is involved in multidrug resistance in cancer. We show that the reconstitution of this protein in lipids from Bacillus subtilis at a lipid-protein ratio of 0.5 m/m allows an optimal protein insertion into lipid bilayer as well as it complies with the two central NMR requirements: high signal-to-noise in the spectra and sample stability over a time period of years. The obtained spectra point to a well-folded protein and a highly homogenous preparation, as witnessed by the narrow resonance lines and the signal dispersion typical of the expected secondary structure distribution of the membrane protein. In the same time, we adapted the GRecon method used in electron microscopy studies for membrane protein reconstitution to the needs of solid-state NMR sample preparation. We followed in detail the reconstitution of the ABC transporter BmrA by dialysis as a reference, and established optimal reconstitution conditions using the combined sucrose/cyclodextrin/lipid gradient characterizing GRecon. NMR spectra recorded on a sample produced by GRecon showed a highly similar fingerprint as those recorded previously on samples reconstituted by dialysis. GRecon sample preparation presents a gain in time of nearly an order of magnitude for reconstitution. In order to study the inward-facing (IF) and the outward-facing (OF) state of the transporter, we developed a reproducible and quantitative protocol of ATP:Mg2+:VO43- addition inducing the OF state. We used selectively labelled samples obtained by the addition of natural abundance residues in the bacterial medium in order to reduce the number of signals in the spectra of this large protein. We recorded solid-state NMR two-dimensional spectra with different mixing times (20 and 200 ms) in order to follow chemical shift changes and identify residues by sequential correlations. The very noticeable apparition of new signals concomitant with the large amplitude of chemical shift perturbations (CSPs) highlight the important flexibility and conformational changes of the protein in presence of ATP:Mg2+:VO43- substrate. In order to identify the residues appearing in the spectra, we use paramagnetic replacement by Mn2+ of the Mg2+ acting as a co-factor in the active site. The paramagnetic relaxation enhancements caused the Mn2+ revealed that the amino acids appearing in the spectra are located in proximity to the ATP binding pocket. Besides, EPR measurements confirmed the closed state of the protein by identifying the corresponding 1.8 nm distances between two Mn2+. We investigate on the conformational differences identified between the IF and OF state in the ABC transporter BmrA reconstituted in its natural lipids. The observation of numerous CSPs, as well as the apparition new signals are observed for a hydrolysis-incompetent mutant on addition of ATP, indicating that hydrolysis is not required for the IF to OF transition in BmrA. We also analyze the mechanistic of the X-loop motif described to be involved in the communication between two domains of the protein. We observe for a mutant protein in which transport is abolished, but which remains ATPase active, an incomplete transition since only a subset of CSPs is observed, as well as lack of rigidification. This suggests that the change in dynamics might be central for transmitting the relevant conformational changes to the part of the protein driving transport, concomitant of an engine which is turning an input shaft, but which fails to connect in a rigid manner, trough adequate gears, with the output shaft driving the pump

The biological membrane surrounding the living cell provides a sealed barrier that tightly regulates the interactions with the outside environment. A large number of integral membrane proteins mediate these interactions and are involved in a wide variety of biological processes. An increasing number of studies have led to the conclusion that lipids provide more than a hydrophobic solvent for membrane proteins, and that interactions between lipids and proteins are required to allow protein function. ABC transporters are one of the most important family of membrane proteins. However, the importance of their lipidic environment is largely unknown. Only a few studies showed that their activity was dependent on the lipidic composition of the surrounding bilayer. The bacterial ABC transporter HorA was used as a model to probe the influence of the lipidic environment on that class of membrane proteins.<p><p> HorA is a multidrug transporter expressed in Lactobacillus brevis, a Gram-positive beer spoilage bacterium. It turned out that phosphatidylethanolamine (PE) was indispensable to maintain both the activity and the structural integrity of HorA.<p> Surprisingly, replacement of PE by the chemically related PC (phosphatidylcholine) did not led to the suppression of HorA activity, but to an unexpected phenotype. Whereas the cytoplasmic domains of HorA were still able to hydrolyze ATP, the membrane parts of the transporter were unable to use that energy to mediate substrate transport. Using several biophysical methods particularly adapted to the study of reconstituted systems, we showed that the structure of HorA is strongly altered by this lipid replacement. In particular, the structural organization of the transmembrane domains of the protein is strongly affected.<p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Notre projet vise à déterminer la structure 3D du transporteur BmrA de Bacillus subtilis. La protéine a été purifiée dans six détergents différents. L'utilisation de foscholine 12, a conduit à cristalliser OmpF, une porine de la membrane externe d'E. coli. Nous montrons que les conditions de cristallisation influencent directement l'empilement cristallin d'OmpF. Le protocole de purification de BmrA, optimisé en utilisant du triton X100 à l'extraction puis un mélange β-D-dodecyl maltoside-cholate pour les étapes chromatographiques nous a permis d'obtenir à 4°C des cristaux, pour lesquels nous avons vérifié qu'ils sont constitués de BmrA. Ces cristaux ont permis d'obtenir un jeu complet jusqu'à 7 Å. Ces données de diffraction constituent une avancée significative pour résoudre à court terme la structure 3D de BmrA. Nous avons développé une nouvelle méthode de dosage des détergents qui est basée sur la détermination par spectrométrie de masse de type MALDI du ratio d'isotopes deutérés/ protonés. La méthode a été validée avec la FC12, le DDM, le β-OG, le LMNG, le CHAPS, le cholate et des détergents calix[4]aréniques, en mesurant la concentration de ces détergents dans différentes conditions d'extraction/purification, de concentration, dialyse et gel filtration, de différentes protéines membranaires. Cette méthode nous a permis (i) d'estimer la taille de la ceinture de détergent associée à BmrA et d'autres protéines membranaires (ii) de moduler cette taille en fonction de mélange de détergents et (iii) d'apporter des informations sur le comportement des complexes protéine-détergent / Our project aims to determine the 3D structure of BmrA from Bacillus subtilis. The protein was purified in six different detergents. Using foscholine 12, led to crystallize OmpF, an outer membrane porin of E. coli. We show that the crystallization conditions directly influence the crystal packing of OmpF. The BmrA purification protocol optimized by using Triton X100 at the extraction and a mixture β-D-dodecyl-maltoside cholate for chromatographic steps allowed us to get to 4°C crystals, for which we verified they consist of BmrA. These crystals have yielded full data to 7 Å. These diffraction data are a significant advance in the short term to resolve the 3D structure of BmrA. We have developed a new detergents dosage assay which is based on the determination by MALDI-type mass ratio of deuterated isotopes / protonated. The method was validated with the FC12, the DDM, the β-OG, the LMNG, CHAPS, cholate detergents and calix [4] aréniques by measuring the concentration of these detergents in different conditions of extraction/ purification, concentration, dialysis and gel filtration, of different membrane proteins. This method allowed us (i) to estimate the size of the detergent belt associated to BmrA and other membrane proteins (ii) to modulate this size in terms of the detergent mixture and (iii) to provide information on the behavior of complex protein-detergent