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

Bax是Bcl-2家族的执行者成员，其通过介导线粒体外膜（mitochondrial outer membrane，ERK）的透化而在细胞凋亡的调节中起关键作用。然而，透化作用的分子机理仍然是该领域的关键问题之一。为了正确理解，需要了解该过程中涉及的Bax结构。尽管最近的进展Bax的功能状态的结构中的药物透化，重要的问题仍然是开放的。我们以前的数据表明，Bax的C-末端刺穿结构域是高度动态的，并参与蛋白质/蛋白质相互作用的寡聚体。然而，正是这种构象的灵活性，到目前为止，排除了适当的理解的全长，活性Bax寡聚体在膜环境中的结构组织。此外，我们提出的钳模型需要进一步验证的蛋白质拓扑结构相对于膜。在这里，我们打算超越最先进的单分子成像和电子顺磁共振相结合，以解决这些问题。该项目的主要优势是使用光学和磁共振方法来达到相同的目的。该项目的主要目的是了解活性的膜嵌入Bax寡聚体的结构组织，重点是在寡聚体组装的背景下，刺穿结构域的分子内灵活性和群体水平上的构象异质性。为此，我们将使用生物物理方法，结合国家的最先进的电子顺磁共振技术与单分子FRET显微镜。我们将回答以下问题：A） Bax二聚体相对于孔和膜平面的取向B） 活性Bax的穿孔结构域的结构组织：分子内动力学与二聚体内螺旋的群体异质性和方向C） Photocaged半胱氨酸作为工具，为特定的正交标记在一个原聚体：对Bax寡聚体的结构模型在membraneThe实验计划在这一建议包括膜蛋白的结构研究的最前沿的方法，并将打开新的可能性，其表征在天然环境中的脂质双层。Bax在细胞透化过程中的结构组织及其与功能的联系将是全面理解Bcl-2蛋白如何调节细胞凋亡的关键，这是生物学中的一个基本问题。

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.