Cristallisation du transporteur ABC BmrA de Bacillus subtilis : développement d’une nouvelle méthode de dosage des détergents par Matrix-Assisted Laser Desorption Ionization (MALDI)

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-detergentNotre 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-détergen

Crystallization of BmrA, bacterial ABC transporter : development of a new detergents dosage assay by Matrix-Assited Laser Desorption Ionization (MALDI)

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-détergentOur 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-detergen

Two different centered monoclinic crystals of the E. coli outer-membrane protein OmpF originate from the same building block

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Diffraction anisotropy falloff in the direction of the detergent belt for two centered monoclinic crystals of OmpF

This data article describes the anisotropy of diffraction observed for the centered monoclinic crystals of OmpF reported in “Two different centered monoclinic crystals of the E. coli outer-membrane protein OmpF originate from the same building block (Chaptal et al., 2016 [1])”. The datasets intensity falloff as a function of resolution are provided along with reflections along the (h,l) and (k,l) planes. A comparison with the crystal packing in the real cell is also provided, with the correspondence to the reciprocal vectors. These data can be retrieved from the Protein Data Bank under accession codes PDB: 4jfb and PDB: 4d5u. Keywords: Diffraction anisotropy, Membrane protei

Stubborn Contaminants: Influence of Detergents on the Purity of the Multidrug ABC Transporter BmrA

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The cleavage of the His₆-tag at the <i>N</i>-terminus of BmrA allows the elimination of AcrB by a second Ni²⁺–NTA chromatographic step.

<p><i>A</i>, The E504Q BmrA mutant was incubated with 2 mM ATP and MgCl₂ during 30 min 23°C, and trypsin (10 µg/mL) was added. At the time indicated, an aliquot was withdrawn, mixed with the SDS-PAGE loading buffer and kept on ice before being submitted to electrophoresis. After 6 h of incubation with trypsin, the ‘lower’ band migrating just below the full-length BmrA was identified by Edman sequencing and shown to correspond to BmrA lacking its <i>N</i>-terminal extremity and starting at Leu(12)-Lys(13)-Pro(14)-Phe(15)-Phe(16)… Therefore the main cut with trypsin occurred between Lys(11) and Leu(12) and is indicated by a red dashed arrow. <i>B</i>, After solubilization with DDM of membrane containing overexpressed E504Q BmrA mutant, extracted and non-extracted materials were separated by an ultracentrifugation and the supernatant was loaded onto a Ni²⁺ high trap chelating column followed by a PD-10 desalting column. The recovered E504Q BmrA mutant was incubated with 2 mM ATP and MgCl₂ during 30 min and was submitted to trypsin digestion as in <i>A</i>. The incubation time was chosen to clearly see both the full-length, uncut protein, and BmrA with its <i>N</i>-terminal being cleaved (inset). The mixture was then submitted to a second Ni²⁺-High Trap chelating column as before and the chromatogram of the protein eluted from the column and monitored at 280 nm is shown. The first peak, fractions 26–34, corresponded to the unbound proteins and the second peak, fractions 67–74, to the proteins eluted with 250 mM imidazole. <i>C</i>, the different fractions obtained in <i>B</i> were analyzed by 10% SDS-PAGE. The two last lanes correspond to fractions 26–34 and 83–90 which were pooled separately and concentrated on a centricon (MWCO 50 kDa) before being submitted to electrophoresis. The Red arrow indicates the position of AcrB.</p

SDS-PAGE of the purified BmrA.

<p><b><i>A</i></b>, 10% SDS-PAGE of 15 µg of purified BmrA in the detergents as indicated in the figure. The numbered bands were cut out and their trypsin digested products were analyzed by LC-MS/MS (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114864#pone-0114864-t001" target="_blank">table 1</a>). <b><i>B</i></b>, 10% SDS-PAGE of 5 µg of purified BmrA in the detergents as indicated in the figure. *BL21(DE3)Δ<i>acrAB</i>, Δ<i>acrEF</i> was used for the overexpression of BmrA. Please note that samples for FC12 in panels <b><i>A</i></b> and <b><i>B</i></b> were obtained from two different purifications protocols (see the text).</p

Impurity identification of purified BmrA in the various detergents.

a<p>band numbering correspond to the bands cut out from the 10% SDS-PAGE in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114864#pone-0114864-g005" target="_blank">Fig. 5</a>. The apparent molecular weights as estimated by migration on the gel are indicated in parenthesis. n.d., no protein detected. The score is a protein quality identification index, considering the number of peptide sequences and MS/MS spectra that have been identified for each protein.</p><p>Impurity identification of purified BmrA in the various detergents.</p

Extraction of BmrA in <i>E. coli</i> membrane with 1% (w/v) detergents.

<p>After solubilization with the indicated detergent, extracted and non-extracted materials were separated by ultracentrifugation, and the supernatant (S) and pellet (P) were resolved on a 10% SDS-PAGE. Fifteen μl of soluble and insoluble fractions were loaded (∼75 µg of protein). Positive control experiment was carried out with sodium dodecyl sulfate (SDS) and negative control was carried out without detergent (buffer alone). Mb: the membrane fraction. Red arrow indicates the position of BmrA.</p

Purification and crystallization of BmrA protein in FC12.

<p><b><i>A</i></b>, elution of FC12 solubilized BmrA protein from a Q sepharose fast flow column. The absorbance of the protein was monitored at 280 nm and BmrA was eluted from the column with 600 mM NaCl. BmrA fractions were resolved on 10% SDS-PAGE. Lane 1, loading step. Lane 2, washing step. Lanes 3 and 4, protein eluted during the NaCl gradient. Lane 5, pool. Red arrows indicate the positions of the BmrA dimer and monomer. <b><i>B</i></b>, elution of BmrA from a Ni²⁺ high trap chelating column. SDS-PAGE, Lane 6, peak fractions. Lane 7, pool. Lane 8, concentrated pool. <b><i>C</i></b>, crystals obtained at 20°C by the vapor-diffusion hanging drop method with 2 µl of 10 mg/ml purified BmrA and 2 µl of the reservoir solution (18% polyethylene glycol 1500, 10% 2-methyl-2,4-pentanediol and 100 mM Tris-HCl pH 8.0).</p