Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, Netherlands Proteomics Centre and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Biochemistry. 2012 Jun 26;51(25):5142-52. doi: 10.1021/bi300451a. Epub 2012 Jun 13.
The ATP-binding-cassette transporter OpuA from Lactococcus lactis is composed of two ATPase subunits (OpuAA) and two subunits (OpuABC) with the transmembrane domain fused to an extracellular substrate-binding protein. Of the almost 1900 homologues of OpuA known to date, a subset has an amino-terminal amphipathic helix (plus extra transmembrane segment) fused to the core of the transmembrane domain of the OpuABC subunit. FRET measurements indicate that the amphipathic α-helix is located close to the membrane surface, where its hydrophobic face interacts with the transport protein rather than the membrane lipids. Next, we determined the functional role of this accessory region by engineering the amphipathic α-helix. We analyzed the consequence of the mutations in intact cells by monitoring growth and transport of glycine betaine under normal and osmotic stress conditions. More detailed studies were performed in hybrid membrane vesicles, proteoliposomes, and bilayer nanodisks. We show that the amphipathic α-helix of OpuA is necessary for high activity of OpuA but is not critical for the biogenesis of the protein or the ionic regulation of transport.
乳球菌（Lactococcus lactis）中的三磷酸腺苷结合盒转运蛋白 OpuA 由两个 ATP 酶亚基（OpuAA）和两个亚基（OpuABC）组成，其跨膜结构域融合到细胞外的底物结合蛋白上。在迄今为止已知的近 1900 个 OpuA 同源物中，有一部分的 N 端具有一个两亲性 α-螺旋（外加额外的跨膜片段），融合到 OpuABC 亚基的跨膜结构域核心上。FRET 测量表明，两亲性 α-螺旋位于靠近膜表面的位置，其疏水面与转运蛋白而不是与膜脂相互作用。接下来，我们通过工程化改造这个两亲性 α-螺旋来确定这个附加区域的功能作用。我们通过监测甘氨酸甜菜碱在正常和渗透胁迫条件下的生长和运输来分析完整细胞中突变的后果。在杂交膜囊泡、蛋白脂质体和双层纳米盘上进行了更详细的研究。我们表明，OpuA 的两亲性 α-螺旋对于 OpuA 的高活性是必需的，但对于蛋白质的生物发生或运输的离子调节不是关键的。
