Surface-tethered planar membranes containing the ß-barrel assembly machinery: a platform for investigating bacterial outer membrane protein folding.
Surface-tethered planar membranes containing the ß-barrel assembly machinery: a platform for investigating bacterial outer membrane protein folding.
复制标题
包含“桶”组装机械的表面束缚平面膜：用于研究细菌外膜蛋白质折叠的平台。
DOI：
10.1016/j.bpj.2021.10.033
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发表时间：
2021
影响因子：
3.4
通讯作者：
Hall SCL
中科院分区：
文献类型：
--
作者：
Hall SCL
革兰氏阴性菌的外膜提供了一个强大的物理化学屏障，保护细胞免受自然环境的影响，并作为抗微生物材料的第一道防线。位于外膜内的蛋白质负责一系列生物功能，包括控制流入和流出。这些外膜蛋白（OMP）最终通过β-桶组装机（BAM）复合物插入并折叠在膜内。Bam复合物折叠和插入OMP的确切机制仍不清楚。在这里，我们已经开发了一个平台，调查BAM介导的OMP插入。通过用铜螯合自组装单层衍生化金表面，我们能够组装包含在磷脂双层内重构的完整BAM复合物的平面系统。这种界面蛋白质束缚的双层极化中子反射仪的结构表征显示不同的区域与已知的高分辨率模型的BAM复杂。此外，通过监测与OMP插入石英晶体微天平与耗散监测相关的质量变化，我们能够证明该系统的功能，通过插入两个不同的OMP内的膜，perpectin，和OmpT。该平台在研究BAM介导的OMP插入机制以及OMP在磷脂双层环境中的功能和活性方面具有很好的应用前景。
The outer membrane of Gram-negative bacteria presents a robust physicochemical barrier protecting the cell from both the natural environment and acting as the first line of defense against antimicrobial materials. The proteins situated within the outer membrane are responsible for a range of biological functions including controlling influx and efflux. These outer membrane proteins (OMPs) are ultimately inserted and folded within the membrane by theβ-barrel assembly machine (Bam) complex. The precise mechanism by which the Bam complex folds and inserts OMPs remains unclear. Here, we have developed a platform for investigating Bam-mediated OMP insertion. By derivatizing a gold surface with a copper-chelating self-assembled monolayer, we were able to assemble a planar system containing the complete Bam complex reconstituted within a phospholipid bilayer. Structural characterization of this interfacial protein-tethered bilayer by polarized neutron reflectometry revealed distinct regions consistent with known high-resolution models of the Bam complex. Additionally, by monitoring changes of mass associated with OMP insertion by quartz crystal microbalance with dissipation monitoring, we were able to demonstrate the functionality of this system by inserting two diverse OMPs within the membrane, pertactin, and OmpT. This platform has promising application in investigating the mechanism of Bam-mediated OMP insertion, in addition to OMP function and activity within a phospholipid bilayer environment.