Characterization of BamA reconstituted into a solid-supported lipid bilayer as a platform for measuring dynamics during substrate protein assembly into the membrane
Characterization of BamA reconstituted into a solid-supported lipid bilayer as a platform for measuring dynamics during substrate protein assembly into the membrane
复制标题
BamA 的表征，重组为固体支持的脂质双层，作为底物蛋白组装到膜期间测量动力学的平台
DOI：
10.1016/j.bbamem.2020.183317
复制
发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Shen Hsin-Hui
中科院分区：
文献类型：
--
作者：
Ding Yue;Shiota Takuya;Le Brun Anton P.;Dunstan Rhys A.;Wang Bo;Hsu Hsien-Yi;Lithgow Trevor;Shen Hsin-Hui
在革兰氏阴性菌中，多蛋白β-Barrel组装机复合体是一种纳米机器，在将β-Barrel蛋白组装到外膜(OM)的过程中发挥着至关重要的作用。这种多蛋白复合体的核心成分BAMA是一种进化上保守的蛋白质，它携带五个从外膜突出的多肽运输相关(POTRA)结构域。BAMA对于伴随蛋白质插入细菌细胞的OM表面是必不可少的。在这项工作中，我们在金衬底上重建了一层含有BAMA的薄膜，并利用耗散石英微天平和中子反射仪在纳米水平上表征了各组分的结构和在不同情况下的运动。在N-十二烷基β-D-麦芽糖苷(DDM)中纯化的BAMA首先被工程到N-N-α，N-α-双(羧甲基)-L-赖氨酸修饰的金表面上，然后去除DDM并进行双层组装。然后，该系统被用于监测底物膜蛋白的结合和插入。数据表明，BAMA的总伸长为120°，膜的嵌入对BAMA的形态没有影响。然而，底物的加入使BAMA的周质Potra结构域进一步远离膜表面。Bama Potra结构域的这种动态行为与细菌细胞内膜和外膜之间的周质空间运输底物聚集的模型一致。这项研究为今后的各种研究，特别是在膜蛋白生物发生领域的应用提供了可靠的工具。
In Gram–negative bacteria, the multi-protein β-barrel assembly machine (BAM) complex is a nanomachine playing a vital role in the process of assembling β-barrel proteins into the outer membrane (OM). The core component of this multiprotein complex, BamA, is an evolutionarily conserved protein that carries five polypeptide-transport-associated (POTRA) domains that project from the outer membrane. BamA is essential for chaperoning the insertion of proteins into the OM surface of bacterial cells. In this work, we have reconstituted a membrane containing BamA on a gold substrate and characterized structure of each component and movement in different situation at the nanoscale level using quartz-crystal microbalance with dissipation and neutron reflectometry (NR). The purified BamA in n-dodecyl β-D-maltoside (DDM) was first engineered onto a nickel-NTA (Nα, Nα-bis-(carboxymethyl)-l-lysine) modified gold surface followed by DDM removal and bilayer assembly. The system was then used to monitor the binding and insertion of a substrate membrane protein. The data shows the total reach of BamA was 120 Å and the embedding of membrane had no effect on the BamA morphology. However, the addition of the substrate enabled the periplasmic POTRA domain of BamA to extend further away from the membrane surface. This dynamic behaviour of BamA POTRA domains is consistent with models invoking the gathering of transported substrates from the periplasmic space between the inner and outer membranes in bacterial cells. This study provides evidence that NR is a reliable tool for diverse investigations in the future, especially for applications in the field of membrane protein biogenesis.