权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

通过组合集成和单分子光谱研究自插入膜蛋白的双层插入机制

基本信息

批准号：
276454827
负责人：
Professor Dr. Sandro Keller
金额：
--
依托单位：
Institut für Molekulare Biowissenschaften
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2015
资助国家：
德国
起止时间：
2014-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/276454827?language=en
关键词：
Bilayer Insertion Mechanisms Self Inserting

项目摘要

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.

这一联合提案解决了一个基本问题，即特定的膜蛋白如何在不需要转座子或外部能量来源的情况下自我插入脂质双分子层。为此，我们打算将一套综合的光学光谱方法与单分子Förster共振能量转移（smFRET）实验相结合，研究折叠和未折叠状态的结构动力学以及两种自插入蛋白的膜插入机制。第一种蛋白质是Mistic，一种螺旋束蛋白，对细菌生物膜的形成至关重要。尽管Mistic正在被用于帮助其他膜蛋白的生产和膜插入，但其自插入能力仍然难以捉摸。我们将探索疏水厚度和偶极电势等膜特性对Mistic折叠和稳定性的影响，揭示其折叠态的环境依赖动力学及其膜插入机制。因此，我们期望更好地理解它作为触发生物膜形成的膜传感器的假定生理功能，以及它作为膜插入伴侣的生物技术用途。第二种感兴趣的蛋白质是外膜磷脂酶A (OmpLA)，这是一种细菌β -桶，已被证明在完整膜蛋白的折叠研究中非常有用。鉴于该蛋白作为体外研究模型系统的重要性，我们的目标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结合是由方法上的协同和互补驱动的。这个联合项目在很大程度上取决于两个申请人小组之间的密切互动，因为它包含了广泛的不同但互补的方法和途径，包括蛋白质生产，纯化，smFRET的位点特异性双标记，自动圆二色性和荧光猝灭光谱，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。