title: In vitro folding and assembly of the Escherichia coli ATP-binding cassette transporter, BtuCD.
abstract: 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.
authors.affiliation: Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
authors.forename: Natalie D
authors.initials: ND
authors.lastname: Di Bartolo
authors.forename: Rikki N
authors.initials: RN
authors.lastname: Hvorup
authors.forename: Kaspar P
authors.initials: KP
authors.lastname: Locher
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Booth
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D011489:Protein Denaturation; D017510:Protein Folding; D014508:Urea / Q000737:chemistry
chemical_list: D018528:ATP-Binding Cassette Transporters; C050021:BtuC peptide, E coli; C050022:BtuD peptide, E coli; D029968:Escherichia coli Proteins; D014508:Urea