Conformational dynamics of OMPs, periplasmic chaperones and BamA

外膜蛋白作为受体、转运体、离子通道、脂肪酶和孔蛋白维持革兰氏阴性细菌的生存能力。外膜蛋白的折叠和组装涉及周质伴侣和多蛋白机制的同步作用，这些机制将高度疏水的多肽以折叠能力的状态护送到目标外膜。在过去的几十年里，人们已经确定了生物发生过程中的蛋白质及其生化功能。然而，所有这些分子机器的作用机制和耐人寻味的能力，在没有典型的细胞能量来源三磷酸腺苷的情况下，但仅仅基于热力学原理，还没有被很好地理解。在这一应用中，我们旨在了解周质伴侣Skp和SurA与大的外膜蛋白的特定相互作用，以及这两种伴侣的特定识别位点和协同作用。我们将使用时间分辨的单分子FRET来监测这些动力学和结构，这使我们能够揭示从纳秒到分钟时间尺度的动力学和从2到8纳米的空间信息。我们进一步的目标是揭示BAMA的侧门动力学，以及这些动力学是如何通过人工脂双层的重组和保留接近天然的脂成分而受到脂类环境的影响。我们将进一步研究侧门是如何受到其他BAM蛋白、抗生素和OMP蛋白插入的影响的。在环境的影响下，与其他BAM蛋白或抗生素直接相互作用，以及在插入OMP蛋白期间。

Outer membrane proteins maintain the viability of Gram-negative bacteria as receptors, transporters, ion channels, lipases, and porins. Folding and assembly of outer membrane proteins involves synchronized action of periplasmic chaperones and multi-protein machineries, which escort the highly hydrophobic polypeptides to their target outer membrane in a folding competent state. Over the past decades proteins and their biochemical function along the biogenesis pathway have been identified. Yet, the mechanisms of action and the intriguing ability of all these molecular machines to work without the typical cellular energy source of ATP, but solely based on thermodynamic principles are not well understood. In this application we aim to understand specific interactions of the periplasmic chaperones Skp and SurA with large outer membrane proteins, specific recognition sites and synergies of both chaperones. We will monitor these dynamics and structures using time-resolved single-molecule FRET, which allows us to reveal kinetics from the nanosecond to the minute timescale and spatial information from 2 to 8 nanometer. We further aim to reveal insights on the lateral gate dynamics of BamA and how those dynamics are influenced by the lipidic environment through reconstitution in artificial lipid bilayers and by preserving near native lipid compositions. We will further study how the lateral gate is influenced by other BAM proteins, by antibiotics and during insertion of OMP proteins. of the environment, direct interactions with other BAM proteins or antibiotics, and during insertion of OMP proteins.