Pernstich Christian, Senior Laura, MacInnes Katherine A, Forsaith Marc, Curnow Paul
School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK; Bristol Centre for Functional Nanomaterials, University of Bristol, Bristol BS8 1TD, UK.
Protein Expr Purif. 2014 Sep;101:68-75. doi: 10.1016/j.pep.2014.05.011. Epub 2014 Jun 5.
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.
氢离子同向转运体家族的整合膜蛋白在芳香族化合物的微生物代谢中发挥着重要作用。在此，我们表明，来自不动杆菌属ADP1菌株的4-羟基苯甲酸转运蛋白PcaK能够在大肠杆菌中成功过表达，并通过亲和层析进行纯化。在添加了胆固醇半琥珀酸酯的n-十二烷基-β-D-麦芽糖苷去污剂中，亲和纯化的PcaK是一种稳定的、单分散的同三聚体。纯化后的蛋白具有α-螺旋二级结构，并且可以在合成蛋白脂质体中重构为功能状态。当通过施加电化学质子梯度（Δμ‾H(+)）或膜电位（ΔΨ）而不是单独通过ΔpH为蛋白脂质体供能时，观察到了不对称底物转运。PcaK在转运4-羟基苯甲酸和3,4-二羟基苯甲酸时相对于密切相关的化合物具有选择性，这证实了先前关于底物特异性的报道。然而，PcaK在转运2-羟基苯甲酸时也表现出意外的偏好。这些结果为进一步详细研究该转运蛋白家族的结构和功能提供了基础。
