title: Expression, purification and reconstitution of the 4-hydroxybenzoate transporter PcaK from Acinetobacter sp. ADP1.
abstract: The aromatic acid:H(+) symporter family of integral membrane proteins play an important role in the microbial metabolism of aromatic compounds. Here, we show that the 4-hydroxybenzoate transporter from Acinetobacter sp. ADP1, PcaK, can be successfully overexpressed in Escherichia coli and purified by affinity chromatography. Affinity-purified PcaK is a stable, monodisperse homotrimer in the detergent n-dodecyl-β-d-maltopyranoside supplemented with cholesteryl hemisuccinate. The purified protein has α-helical secondary structure and can be reconstituted to a functional state in synthetic proteoliposomes. Asymmetric substrate transport was observed when proteoliposomes were energized by applying an electrochemical proton gradient (Δμ‾H(+)) or a membrane potential (ΔΨ) but not by ΔpH alone. PcaK was selective in transporting 4-hydroxybenzoate and 3,4-dihydroxybenzoate over closely related compounds, confirming previous reports on substrate specificity. However, PcaK also showed an unexpected preference for transporting 2-hydroxybenzoates. These results provide the basis for further detailed studies of the structure and function of this family of transporters.
authors.affiliation: School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Pernstich
authors.affiliation: School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK; Bristol Centre for Functional Nanomaterials, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Laura
authors.initials: L
authors.lastname: Senior
authors.affiliation: School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: MacInnes
authors.affiliation: School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Marc
authors.initials: M
authors.lastname: Forsaith
authors.affiliation: School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK. Electronic address: p.curnow@bristol.ac.uk.
authors.forename: Paul
authors.initials: P
authors.lastname: Curnow
mesh_terms: D000150:Acinetobacter / Q000201:enzymology*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D002788:Cholesterol Esters / Q000737:chemistry; D002846:Chromatography, Affinity; D024881:Drug Resistance, Bacterial / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D062385:Hydroxybenzoates / Q000378:metabolism; D008320:Maltose / Q000031:analogs & derivatives / Q000737:chemistry; D010226:Parabens / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D020156:Salicylic Acid / Q000378:metabolism; D013379:Substrate Specificity; D027981:Symporters / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
chemical_list: C520290:3,4-dihydroxybenzoate; D001426:Bacterial Proteins; D002352:Carrier Proteins; D002788:Cholesterol Esters; D062385:Hydroxybenzoates; D010226:Parabens; D011510:Proteolipids; D027981:Symporters; C411362:dodecyl maltopyranoside; C020485:proteoliposomes; D008320:Maltose; C038193:4-hydroxybenzoic acid; D020156:Salicylic Acid; C013440:cholesteryl succinate
keywords: Enzyme purification; Membrane proteins; Membrane transport; Recombinant protein expression; Reconstitution of membrane transporters