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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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DDM-purified BmrC/BmrD is monodisperse.(a) Size-exclusion chromatography of DDM-purified protein (∼0.3 mg) was performed as indicated in fig. 1d. Inset: Silver-stained SDS-PAGE of protein eluted at 10 ml. Black arrowhead, BmrD; grey arrowhead, BmrC. Vo: void volume. (b–d) Samples of nickel-affinity purified BmrC/BmrD (0.85 mg/ml) kept frozen at −80°C after flash freezing in liquid N2, were thawed and analyzed by sedimentation velocity measurements at 20°C and 42000 revolutions per minutes. (b) Superposition of selected sedimentation profiles obtained at 278 nm and the corresponding fitted curves using continuous size distribution analysis. (c) Superposition of the residuals. (d) c(s) curves for the stock protein (continuous line) and the protein diluted 4 times in dialysis buffer (dashed line).
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pone-0019677-g002: DDM-purified BmrC/BmrD is monodisperse.(a) Size-exclusion chromatography of DDM-purified protein (∼0.3 mg) was performed as indicated in fig. 1d. Inset: Silver-stained SDS-PAGE of protein eluted at 10 ml. Black arrowhead, BmrD; grey arrowhead, BmrC. Vo: void volume. (b–d) Samples of nickel-affinity purified BmrC/BmrD (0.85 mg/ml) kept frozen at −80°C after flash freezing in liquid N2, were thawed and analyzed by sedimentation velocity measurements at 20°C and 42000 revolutions per minutes. (b) Superposition of selected sedimentation profiles obtained at 278 nm and the corresponding fitted curves using continuous size distribution analysis. (c) Superposition of the residuals. (d) c(s) curves for the stock protein (continuous line) and the protein diluted 4 times in dialysis buffer (dashed line).

Mentions: Analysis by size-exclusion chromatography (SEC) of the whole complex purified after a 6 hours induction shows an elution profile with a more symmetrical peak (cf. Fig. 2a). This sharper, Gaussian-like peak, reveals that the reduction of the induction time not only increased the purification yield, but also improved the homogeneity of the purified proteins [36]. Silver staining of the material eluted in this peak shows that the BmrC/BmrD complex was relatively pure as low molecular weight contaminants were removed and only a very faint contaminant remained with an apparent molecular mass of ∼100 kDa. Comparison of the elution volume of the major peak with that of protein standards of known size allows an estimation of the molecular mass of the BmrC/BmrD detergent complex of ∼310 kDa (more precisely the Stokes' radius RS is ∼6.1 nm). This mass is about twice the protein part of a BmrC/BmrD/detergent complex, given that the theoretical molecular masses of 6×His-tagged BmrC and BmrD proteins are 67 and 77 kDa, respectively. A comparable molecular mass (300±30 kDa) was obtained by this approach for the homodimeric BmrA transporter (66.2 kDa for the monomer), which was indeed shown to be a homodimer in a DDM solution using analytical ultracentrifugation [21].


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)

DDM-purified BmrC/BmrD is monodisperse.(a) Size-exclusion chromatography of DDM-purified protein (∼0.3 mg) was performed as indicated in fig. 1d. Inset: Silver-stained SDS-PAGE of protein eluted at 10 ml. Black arrowhead, BmrD; grey arrowhead, BmrC. Vo: void volume. (b–d) Samples of nickel-affinity purified BmrC/BmrD (0.85 mg/ml) kept frozen at −80°C after flash freezing in liquid N2, were thawed and analyzed by sedimentation velocity measurements at 20°C and 42000 revolutions per minutes. (b) Superposition of selected sedimentation profiles obtained at 278 nm and the corresponding fitted curves using continuous size distribution analysis. (c) Superposition of the residuals. (d) c(s) curves for the stock protein (continuous line) and the protein diluted 4 times in dialysis buffer (dashed line).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3094339&req=5

pone-0019677-g002: DDM-purified BmrC/BmrD is monodisperse.(a) Size-exclusion chromatography of DDM-purified protein (∼0.3 mg) was performed as indicated in fig. 1d. Inset: Silver-stained SDS-PAGE of protein eluted at 10 ml. Black arrowhead, BmrD; grey arrowhead, BmrC. Vo: void volume. (b–d) Samples of nickel-affinity purified BmrC/BmrD (0.85 mg/ml) kept frozen at −80°C after flash freezing in liquid N2, were thawed and analyzed by sedimentation velocity measurements at 20°C and 42000 revolutions per minutes. (b) Superposition of selected sedimentation profiles obtained at 278 nm and the corresponding fitted curves using continuous size distribution analysis. (c) Superposition of the residuals. (d) c(s) curves for the stock protein (continuous line) and the protein diluted 4 times in dialysis buffer (dashed line).
Mentions: Analysis by size-exclusion chromatography (SEC) of the whole complex purified after a 6 hours induction shows an elution profile with a more symmetrical peak (cf. Fig. 2a). This sharper, Gaussian-like peak, reveals that the reduction of the induction time not only increased the purification yield, but also improved the homogeneity of the purified proteins [36]. Silver staining of the material eluted in this peak shows that the BmrC/BmrD complex was relatively pure as low molecular weight contaminants were removed and only a very faint contaminant remained with an apparent molecular mass of ∼100 kDa. Comparison of the elution volume of the major peak with that of protein standards of known size allows an estimation of the molecular mass of the BmrC/BmrD detergent complex of ∼310 kDa (more precisely the Stokes' radius RS is ∼6.1 nm). This mass is about twice the protein part of a BmrC/BmrD/detergent complex, given that the theoretical molecular masses of 6×His-tagged BmrC and BmrD proteins are 67 and 77 kDa, respectively. A comparable molecular mass (300±30 kDa) was obtained by this approach for the homodimeric BmrA transporter (66.2 kDa for the monomer), which was indeed shown to be a homodimer in a DDM solution using analytical ultracentrifugation [21].

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