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Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization.

Galián C, Manon F, Dezi M, Torres C, Ebel C, Lévy D, Jault JM - PLoS ONE (2011)

Bottom Line: Functional reconstitution using different lipid compositions induced an 8-fold increase of the ATPase activity (k(cat)∼5 s(-1)).We further validated that the quality of the purified BmrC/BmrD heterodimer is suitable for structural analyses, as its reconstitution at high protein densities led to the formation of 2-D crystals.Electron microscopy of negatively stained crystals allowed the calculation of a projection map at 20 Å resolution revealing that BmrC/BmrD might assemble into oligomers in a lipidic environment.

View Article: PubMed Central - PubMed

Affiliation: Université Joseph Fourier, Institut de Biologie Structurale, Grenoble, France.

ABSTRACT
Optimized protocols for achieving high-yield expression, purification and reconstitution of membrane proteins are required to study their structure and function. We previously reported high-level expression in Escherichia coli of active BmrC and BmrD proteins from Bacillus subtilis, previously named YheI and YheH. These proteins are half-transporters which belong to the ABC (ATP-Binding Cassette) superfamily and associate in vivo to form a functional transporter able to efflux drugs. In this report, high-yield purification and functional reconstitution were achieved for the heterodimer BmrC/BmrD. In contrast to other detergents more efficient for solubilizing the transporter, dodecyl-ß-D-maltoside (DDM) maintained it in a drug-sensitive and vanadate-sensitive ATPase-competent state after purification by affinity chromatography. High amounts of pure proteins were obtained which were shown either by analytical ultracentrifugation or gel filtration to form a monodisperse heterodimer in solution, which was notably stable for more than one month at 4°C. Functional reconstitution using different lipid compositions induced an 8-fold increase of the ATPase activity (k(cat)∼5 s(-1)). We further validated that the quality of the purified BmrC/BmrD heterodimer is suitable for structural analyses, as its reconstitution at high protein densities led to the formation of 2-D crystals. Electron microscopy of negatively stained crystals allowed the calculation of a projection map at 20 Å resolution revealing that BmrC/BmrD might assemble into oligomers in a lipidic environment.

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Purification of BmrC/BmrD using different detergents.(a) BmrC/BmrD solubilization by non-ionic and zwitterionic detergents was performed at 2 mg/ml protein and 10 mg/ml detergent and assessed by Coomassie Blue stained SDS-PAGE of the soluble (S) and insoluble (P) material obtained from 15 µg of total protein sample (T). Note that the strong densities observed in some of the pellet bands probably arise from difficulties encountered while resuspending the pellet that led to inhomogeneous samples. Black arrowhead, BmrD; grey arrowhead, BmrC. (b) The intensities of the bands corresponding to the S and T samples were quantitated using Image Gauge software (Fuji Film Science Lab) and the solubilization yields were calculated as the ratio of each protein in the supernatant (S) relative to its amount in the membrane fraction (T). (c, d) Protein purified by nickel affinity chromatography in the presence of the indicated detergents was analyzed by (c) Coomassie Blue stained SDS-PAGE (5 µg per lane, except for DDM : 4 µg) and (d) size-exclusion chromatography performed on a Superdex 200 10/300 GL column using 0.05% (w/v) DDM in the elution buffer. Elution volumes of proteins used for column calibration are indicated above: ferritin (F, 440 kDa), aldolase (AD, 158 kDa), albumin (AB, 67 kDa), Vo: void volume. The intensities of the BmrC and BmrD bands in the polyacrylamide gel in c were quantitated and the ratio BmrC∶BmrD (ratio C∶D) was indicated under each lane. Black arrowhead, BmrD; grey arrowhead, BmrC.
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pone-0019677-g001: Purification of BmrC/BmrD using different detergents.(a) BmrC/BmrD solubilization by non-ionic and zwitterionic detergents was performed at 2 mg/ml protein and 10 mg/ml detergent and assessed by Coomassie Blue stained SDS-PAGE of the soluble (S) and insoluble (P) material obtained from 15 µg of total protein sample (T). Note that the strong densities observed in some of the pellet bands probably arise from difficulties encountered while resuspending the pellet that led to inhomogeneous samples. Black arrowhead, BmrD; grey arrowhead, BmrC. (b) The intensities of the bands corresponding to the S and T samples were quantitated using Image Gauge software (Fuji Film Science Lab) and the solubilization yields were calculated as the ratio of each protein in the supernatant (S) relative to its amount in the membrane fraction (T). (c, d) Protein purified by nickel affinity chromatography in the presence of the indicated detergents was analyzed by (c) Coomassie Blue stained SDS-PAGE (5 µg per lane, except for DDM : 4 µg) and (d) size-exclusion chromatography performed on a Superdex 200 10/300 GL column using 0.05% (w/v) DDM in the elution buffer. Elution volumes of proteins used for column calibration are indicated above: ferritin (F, 440 kDa), aldolase (AD, 158 kDa), albumin (AB, 67 kDa), Vo: void volume. The intensities of the BmrC and BmrD bands in the polyacrylamide gel in c were quantitated and the ratio BmrC∶BmrD (ratio C∶D) was indicated under each lane. Black arrowhead, BmrD; grey arrowhead, BmrC.

Mentions: Membrane preparations highly enriched in BmrC/BmrD were incubated with a sampling of commonly used non-ionic and zwitterionic detergents. The non-ionic detergents tested were n-dodecyl-ß-D-maltoside (DDM) and its thio-derivative n-dodecyl-ß-D-thiomaltoside (DOTM), n-octyl-ß-D-glucoside (OG) and 6-O-(n-heptylcarbamoyl)-methyl-α-D-glucopyranoside (HECAMEG). As shown in Figure 1a–b, DDM performed best among the tested non-ionic detergents, although the yield was rather low with less than 30% of the protein being solubilized. Similar extraction yields were obtained with DOTM (not shown). In contrast, OG was unable to solubilize BmrC/BmrD. The zwitterionic detergents tested were lauryldimethylamine oxide (LDAO), n-dodeclyphosphocholine (FC-12), n-Hexadecylphosphocholine (FC-16), n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Z3-12), and and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). With the exception of CHAPS, all solubilized BmrC/BmrD more efficiently than the non-ionic detergents. The two fos-cholines had by far the best extraction efficiencies, close to 100%. Additionally, a mixture of 0.1% DDM and 1% polyoxyethylene-8-dodecylether (C12E8) that was successfully used to solubilize Sav1866 [17], was also tried but did not give any improvement as compared to DDM alone (not shown). All mild detergents (i.e. with an extraction efficiency <40%) solubilized BmrD slightly better than BmrC (Fig. 1b). Given the excess of BmrC in the membrane fraction, this observation suggests that excess subunits that are not assembled into heterodimers are misfolded in the membrane and therefore less readily solubilized (see also the Discussion section). In contrast, LDAO and Z3.12 appeared to solubilize the BmrC subunit more efficiently than BmrD. This effect was even more pronounced using the stronger detergents (i.e. FC12 and FC16) that solubilized almost entirely both proteins. This second category of detergents thus maintained an excess of BmrC subunit in the solubilized fraction.


Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization.

Galián C, Manon F, Dezi M, Torres C, Ebel C, Lévy D, Jault JM - PLoS ONE (2011)

Purification of BmrC/BmrD using different detergents.(a) BmrC/BmrD solubilization by non-ionic and zwitterionic detergents was performed at 2 mg/ml protein and 10 mg/ml detergent and assessed by Coomassie Blue stained SDS-PAGE of the soluble (S) and insoluble (P) material obtained from 15 µg of total protein sample (T). Note that the strong densities observed in some of the pellet bands probably arise from difficulties encountered while resuspending the pellet that led to inhomogeneous samples. Black arrowhead, BmrD; grey arrowhead, BmrC. (b) The intensities of the bands corresponding to the S and T samples were quantitated using Image Gauge software (Fuji Film Science Lab) and the solubilization yields were calculated as the ratio of each protein in the supernatant (S) relative to its amount in the membrane fraction (T). (c, d) Protein purified by nickel affinity chromatography in the presence of the indicated detergents was analyzed by (c) Coomassie Blue stained SDS-PAGE (5 µg per lane, except for DDM : 4 µg) and (d) size-exclusion chromatography performed on a Superdex 200 10/300 GL column using 0.05% (w/v) DDM in the elution buffer. Elution volumes of proteins used for column calibration are indicated above: ferritin (F, 440 kDa), aldolase (AD, 158 kDa), albumin (AB, 67 kDa), Vo: void volume. The intensities of the BmrC and BmrD bands in the polyacrylamide gel in c were quantitated and the ratio BmrC∶BmrD (ratio C∶D) was indicated under each lane. Black arrowhead, BmrD; grey arrowhead, BmrC.
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Related In: Results  -  Collection

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pone-0019677-g001: Purification of BmrC/BmrD using different detergents.(a) BmrC/BmrD solubilization by non-ionic and zwitterionic detergents was performed at 2 mg/ml protein and 10 mg/ml detergent and assessed by Coomassie Blue stained SDS-PAGE of the soluble (S) and insoluble (P) material obtained from 15 µg of total protein sample (T). Note that the strong densities observed in some of the pellet bands probably arise from difficulties encountered while resuspending the pellet that led to inhomogeneous samples. Black arrowhead, BmrD; grey arrowhead, BmrC. (b) The intensities of the bands corresponding to the S and T samples were quantitated using Image Gauge software (Fuji Film Science Lab) and the solubilization yields were calculated as the ratio of each protein in the supernatant (S) relative to its amount in the membrane fraction (T). (c, d) Protein purified by nickel affinity chromatography in the presence of the indicated detergents was analyzed by (c) Coomassie Blue stained SDS-PAGE (5 µg per lane, except for DDM : 4 µg) and (d) size-exclusion chromatography performed on a Superdex 200 10/300 GL column using 0.05% (w/v) DDM in the elution buffer. Elution volumes of proteins used for column calibration are indicated above: ferritin (F, 440 kDa), aldolase (AD, 158 kDa), albumin (AB, 67 kDa), Vo: void volume. The intensities of the BmrC and BmrD bands in the polyacrylamide gel in c were quantitated and the ratio BmrC∶BmrD (ratio C∶D) was indicated under each lane. Black arrowhead, BmrD; grey arrowhead, BmrC.
Mentions: Membrane preparations highly enriched in BmrC/BmrD were incubated with a sampling of commonly used non-ionic and zwitterionic detergents. The non-ionic detergents tested were n-dodecyl-ß-D-maltoside (DDM) and its thio-derivative n-dodecyl-ß-D-thiomaltoside (DOTM), n-octyl-ß-D-glucoside (OG) and 6-O-(n-heptylcarbamoyl)-methyl-α-D-glucopyranoside (HECAMEG). As shown in Figure 1a–b, DDM performed best among the tested non-ionic detergents, although the yield was rather low with less than 30% of the protein being solubilized. Similar extraction yields were obtained with DOTM (not shown). In contrast, OG was unable to solubilize BmrC/BmrD. The zwitterionic detergents tested were lauryldimethylamine oxide (LDAO), n-dodeclyphosphocholine (FC-12), n-Hexadecylphosphocholine (FC-16), n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Z3-12), and and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). With the exception of CHAPS, all solubilized BmrC/BmrD more efficiently than the non-ionic detergents. The two fos-cholines had by far the best extraction efficiencies, close to 100%. Additionally, a mixture of 0.1% DDM and 1% polyoxyethylene-8-dodecylether (C12E8) that was successfully used to solubilize Sav1866 [17], was also tried but did not give any improvement as compared to DDM alone (not shown). All mild detergents (i.e. with an extraction efficiency <40%) solubilized BmrD slightly better than BmrC (Fig. 1b). Given the excess of BmrC in the membrane fraction, this observation suggests that excess subunits that are not assembled into heterodimers are misfolded in the membrane and therefore less readily solubilized (see also the Discussion section). In contrast, LDAO and Z3.12 appeared to solubilize the BmrC subunit more efficiently than BmrD. This effect was even more pronounced using the stronger detergents (i.e. FC12 and FC16) that solubilized almost entirely both proteins. This second category of detergents thus maintained an excess of BmrC subunit in the solubilized fraction.

Bottom Line: Functional reconstitution using different lipid compositions induced an 8-fold increase of the ATPase activity (k(cat)∼5 s(-1)).We further validated that the quality of the purified BmrC/BmrD heterodimer is suitable for structural analyses, as its reconstitution at high protein densities led to the formation of 2-D crystals.Electron microscopy of negatively stained crystals allowed the calculation of a projection map at 20 Å resolution revealing that BmrC/BmrD might assemble into oligomers in a lipidic environment.

View Article: PubMed Central - PubMed

Affiliation: Université Joseph Fourier, Institut de Biologie Structurale, Grenoble, France.

ABSTRACT
Optimized protocols for achieving high-yield expression, purification and reconstitution of membrane proteins are required to study their structure and function. We previously reported high-level expression in Escherichia coli of active BmrC and BmrD proteins from Bacillus subtilis, previously named YheI and YheH. These proteins are half-transporters which belong to the ABC (ATP-Binding Cassette) superfamily and associate in vivo to form a functional transporter able to efflux drugs. In this report, high-yield purification and functional reconstitution were achieved for the heterodimer BmrC/BmrD. In contrast to other detergents more efficient for solubilizing the transporter, dodecyl-ß-D-maltoside (DDM) maintained it in a drug-sensitive and vanadate-sensitive ATPase-competent state after purification by affinity chromatography. High amounts of pure proteins were obtained which were shown either by analytical ultracentrifugation or gel filtration to form a monodisperse heterodimer in solution, which was notably stable for more than one month at 4°C. Functional reconstitution using different lipid compositions induced an 8-fold increase of the ATPase activity (k(cat)∼5 s(-1)). We further validated that the quality of the purified BmrC/BmrD heterodimer is suitable for structural analyses, as its reconstitution at high protein densities led to the formation of 2-D crystals. Electron microscopy of negatively stained crystals allowed the calculation of a projection map at 20 Å resolution revealing that BmrC/BmrD might assemble into oligomers in a lipidic environment.

Show MeSH
Related in: MedlinePlus