In vitro folding and assembly of the Escherichia coli ATP-binding cassette transporter, BtuCD

TY - JOUR

T1 - In vitro folding and assembly of the Escherichia coli ATP-binding cassette transporter, BtuCD

AU - Di Bartolo, Natalie D

AU - Hvorup, Rikki N

AU - Locher, Kaspar P

AU - Booth, Paula J

PY - 2011/5/27

Y1 - 2011/5/27

N2 - Studies on membrane protein folding have focused on monomeric α-helical proteins and a major challenge is to extend this work to larger oligomeric membrane proteins. Here, we study the Escherichia coli (E. coli) ATP-binding cassette (ABC) transporter that imports vitamin B(12) (the BtuCD protein) and use it as a model system for investigating the folding and assembly of a tetrameric membrane protein complex. Our work takes advantage of the modular organization of BtuCD, which consists of two transmembrane protein subunits, BtuC, and two cytoplasmically located nucleotide-binding protein subunits, BtuD. We show that the BtuCD transporter can be re-assembled from both prefolded and partly unfolded, urea denatured BtuC and BtuD subunits. The in vitro re-assembly leads to a BtuCD complex with the correct, native, BtuC and BtuD subunit stoichiometry. The highest rates of ATP hydrolysis were achieved for BtuCD re-assembled from partly unfolded subunits. This supports the idea of cooperative folding and assembly of the constituent protein subunits of the BtuCD transporter. BtuCD folding also provides an opportunity to investigate how a protein that contains both membrane-bound and aqueous subunits coordinates the folding requirements of the hydrophobic and hydrophilic subunits.

AB - Studies on membrane protein folding have focused on monomeric α-helical proteins and a major challenge is to extend this work to larger oligomeric membrane proteins. Here, we study the Escherichia coli (E. coli) ATP-binding cassette (ABC) transporter that imports vitamin B(12) (the BtuCD protein) and use it as a model system for investigating the folding and assembly of a tetrameric membrane protein complex. Our work takes advantage of the modular organization of BtuCD, which consists of two transmembrane protein subunits, BtuC, and two cytoplasmically located nucleotide-binding protein subunits, BtuD. We show that the BtuCD transporter can be re-assembled from both prefolded and partly unfolded, urea denatured BtuC and BtuD subunits. The in vitro re-assembly leads to a BtuCD complex with the correct, native, BtuC and BtuD subunit stoichiometry. The highest rates of ATP hydrolysis were achieved for BtuCD re-assembled from partly unfolded subunits. This supports the idea of cooperative folding and assembly of the constituent protein subunits of the BtuCD transporter. BtuCD folding also provides an opportunity to investigate how a protein that contains both membrane-bound and aqueous subunits coordinates the folding requirements of the hydrophobic and hydrophilic subunits.

KW - ATP-Binding Cassette Transporters

KW - Catalytic Domain

KW - Escherichia coli

KW - Escherichia coli Proteins

KW - Hydrophobic and Hydrophilic Interactions

KW - Protein Denaturation

KW - Protein Folding

KW - Urea

U2 - 10.1074/jbc.M110.176891

DO - 10.1074/jbc.M110.176891

M3 - Article

C2 - 21345797

SN - 0021-9258

VL - 286

SP - 18807

EP - 18815

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 21

ER -