Bilayer-Insertion Mechanisms of Self-Inserting Membrane Proteins by Combined Ensemble and Single-Molecule Spectroscopy

这一联合提案解决了一个基本问题，即特定的膜蛋白如何自插入脂质双层，而不需要translocons或外部能源。为此，我们打算结合联合收割机一套全面的合奏光学光谱方法与单分子Förster共振能量转移（smFRET）的实验，研究折叠和未折叠状态的结构动力学，以及两个自我插入蛋白质的膜插入机制。第一种蛋白质是Mistic，一种细菌生物膜形成所必需的螺旋束蛋白。虽然Mistic被用来帮助其他膜蛋白的生产和膜插入，但它的自我插入能力仍然难以捉摸。我们将探讨膜的性质，如疏水性的厚度和偶极电位，这已被假设为调制Mistic的折叠和稳定性的影响，并揭示其折叠状态的环境依赖性动力学和膜插入的机制。因此，我们期望达到一个更好的理解其假定的生理功能作为膜传感器触发生物膜的形成，以及其作为膜插入分子伴侣的生物技术用途。第二个感兴趣的蛋白质是外膜磷脂酶A（OmpLA），一种细菌β-桶，已被证明在完整膜蛋白的折叠研究中非常有用。鉴于这种蛋白质作为体外研究模型系统的重要性，我们的目的是更好地了解其在变性条件下的未折叠状态及其（耦合？）折叠和膜插入，特别强调预结构或构象选择的作用。这两种结构和功能不同的膜蛋白的组合是由方法的协同作用和互补性的动机。该联合项目关键取决于两个申请人小组之间的密切互动，因为它包括广泛多样但互补的方法和途径，包括蛋白质生产，纯化和smFRET的位点特异性双标记，系综水平的自动圆二色谱和荧光猝灭光谱，使用定制仪器的单分子实验，以及计算分析方法的开发和验证。

This joint proposal addresses the fundamental question of how particular membrane proteins self-insert into lipid bilayers without requiring translocons or external energy sources. To this end, we intend to combine a comprehensive set of ensemble optical-spectroscopic methods with single-molecule Förster resonance energy transfer (smFRET) experiments to study the structural dynamics of both folded and unfolded states as well as the membrane-insertion mechanisms of two self-inserting proteins. The first protein is Mistic, a helical-bundle protein essential for bacterial biofilm formation. Although Mistic is being exploited to aid in the production and membrane insertion of other membrane proteins, its self-insertion capability remains elusive. We will explore the effects of membrane properties such as hydrophobic thickness and dipole potential, which have been hypothesized to modulate the fold and stability of Mistic, and unveil the environment-dependent dynamics of its folded state and its mechanism of membrane insertion. We thus expect to arrive at a better understanding of both its presumed physiological function as a membrane sensor for triggering biofilm formation as well as its biotechnological use as a membrane-insertion chaperone. The second protein of interest is outer membrane phospholipase A (OmpLA), a bacterial beta-barrel that has proven highly useful in folding studies on integral membrane proteins. In light of the importance of this protein as a model system for in vitro investigations, we aim at a better understanding of its unfolded state under denaturing conditions and its (coupled?) folding and membrane insertion, with particular emphasis on the role of prestructuring or conformational selection. The combination of these two structurally and functionally distinct membrane proteins is motivated by methodological synergism and complementarity. This joint project crucially depends on close interactions between the groups of the two applicants, since it comprises a wide range of diverse but complementary methods and approaches, including protein production, purification, and site-specific double labeling for smFRET, automated circular dichroism and fluorescence quenching spectroscopy at the ensemble level, single-molecule experiments using a custom-built instrument, and the development and validation of computational analysis methods.