Structural dynamics of membrane-bound BAX oligomers: intramolecular flexibility vs population heterogeneities

膜结合 BAX 低聚物的结构动力学：分子内灵活性与群体异质性

• 批准号: 319282808
• 负责人: Professorin Dr. Enrica Bordignon
• 金额: --
• 依托单位: Arbeitsgruppe EPR-Spektroskopie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/319282808?language=en
• 关键词: Structural; dynamics; membrane; bound; BAX

Bax is a executioner member of the Bcl-2 family that plays a key role in the regulation of apoptosis by mediating the permeabilization of the mitochondrial outer membrane (MOM). However, the molecular mechanism of MOM permeabilization remains one of the key questions in the field. For a proper understanding, knowledge about the structure of Bax involved in the process is required. Despite recent advances in the structure of the functional state of Bax for MOM permeabilization, important questions remain open. Our previous data indicate that the C-terminal piercing domain of Bax is highly dynamic and involved in protein/protein interactions within the oligomers. However, precisely this conformational flexibility has so far precluded a proper understanding of the structural organization of full length, active Bax oligomers in the membrane environment. In addition, the clamp model that we proposed requires further validation in terms of protein topology with respect to the membrane. Here we intend to go beyond of the state of the art by combining single molecule imaging and electron paramagnetic resonance in order to tackle these questions. The main strength of this proposal is the use of both optical and magnetic resonance methodologies to the same end.The main aim of this project is to understand the structural organization of active, membrane-embeded Bax oligomers with a focus on the intramolecular flexibility of the piercing domain and the conformational heterogeneities at the population level in the context of oligomeric assemblies. For this purpose, we will use a biophysical approach that combines state-of-the-art electron paramagnetic resonance techniques with single molecule FRET microscopy. We will address the following questions:A) Orientation of Bax dimers with respect to the pore and the membrane planeB) Structural organization of the piercing domain of active Bax: intramolecular dynamics vs population heterogeneities and orientation of helices within the dimerC) Photocaged cysteines as tools for specific orthogonal labeling in one protomer: towards a structural model of Bax oligomers in the membraneThe experiments planned in this proposal include approaches at the forefront of structural research of membrane proteins and will open new possibilities for their characterization in the native environment of the lipid bilayer. The structural organization of Bax during MOM permeabilization and its link to function will be key to achieve a comprehensive understanding of how the Bcl-2 proteins regulate apoptosis, which is a fundamental question in biology.

Bax是Bcl2家族中的一个执行者成员，通过调节线粒体外膜的通透性，在细胞凋亡的调节中发挥关键作用。然而，MOM通透性的分子机制仍然是该领域的关键问题之一。为了正确理解，需要了解过程中涉及的BAX结构的知识。尽管BAX对MOM通透性的功能状态的结构最近取得了进展，但重要的问题仍然悬而未决。我们以前的数据表明，Bax的C末端穿透结构域是高度动态的，参与了寡聚体中的蛋白质/蛋白质相互作用。然而，准确地说，到目前为止，这种构象灵活性阻碍了对全长、活性Bax低聚物在膜环境中的结构组织的正确理解。此外，我们提出的钳制模型需要进一步验证蛋白质相对于膜的拓扑结构。在这里，我们打算通过结合单分子成像和电子顺磁共振来解决这些问题，从而超越最先进的状态。这一建议的主要优点是同时使用光学和磁共振方法。该项目的主要目的是了解活性的、膜包埋的Bax低聚物的结构组织，重点是在低聚物组装的背景下，穿刺域的分子内灵活性和群体水平的构象异质性。为此，我们将使用一种生物物理方法，将最先进的电子顺磁共振技术与单分子FRET显微镜相结合。我们将解决以下问题：a)Bax二聚体相对于孔和膜平面的取向B)活性Bax穿透域的结构组织：分子内动力学与群体异质性和二聚体内螺旋的取向C)光笼化半胱氨酸作为特定的正交标记物的工具在膜中：建立膜中Bax低聚体的结构模型本建议中计划的实验包括膜蛋白结构研究的前沿方法，并将为其在脂质双层的自然环境中的表征开辟新的可能性。Bax在MOM通透性过程中的结构组织及其与功能的联系将是全面理解Bcl2蛋白如何调控细胞凋亡的关键，这是生物学中的一个基本问题。