Structure and Conformational Changes of Assembled Dynamin

描述（由申请人提供）：Dynamin是一个大的，多结构域的GTPase，对于网格蛋白介导的内吞作用的最后步骤至关重要，是突触囊泡循环的关键调节剂。为了了解它调节这些生物事件的机制，确定动力蛋白如何将核苷酸水解转化为结构变化以促进膜裂变和网格蛋白包被囊泡释放到细胞中是至关重要的。尽管有大量的功能数据，但由于缺乏结构信息，驱动动力学蛋白机械化学活动的构象变化的确切性质仍然不明确。为了更好地了解动力蛋白结构如何影响其功能活性，我们试图利用冷冻电子显微镜、生物化学和图像重建技术的组合来可视化组装动力蛋白的结构和构象变化。这些研究将以足够的分辨率产生动力蛋白的三维重建，使我们能够看到与不同核苷酸状态相关的关键结构变化。电子密集标签的附着将阐明动力蛋白聚合物的整体组织，并将明确定义动力蛋白结构域在这些结构图中的位置。这一知识将大大增加我们对dynamin如何将GTP水解的化学能传递到膜断裂和最终突触前再循环的物理功的理解。身体传递神经冲动的能力高度依赖于一个被称为突触前循环的过程的效率，这一过程维持了大量预先装载神经递质的囊泡，并为神经元对动作电位的快速反应做好准备。GTPase动力蛋白在这一过程中是必不可少的，尽管由于缺乏关于该蛋白的结构信息，其功能的潜在机制仍然未知。我们试图确定动力蛋白结构如何影响其功能，从而确定驱动其生物活性的蛋白质的关键变化。这些发现将最终增强我们对动力蛋白在心理健康中的作用的理解。

DESCRIPTION (provided by applicant): Dynamin is a large, multidomain GTPase essential for the final steps of clathrin mediated endocytosis and is a key regulator of synaptic vesicle recycling. In order to understand the mechanisms by which it modulates these biological events, it is crucial to determine how dynamin translates nucleotide hydrolysis into structural changes to facilitate membrane fission and the liberation of clathrin coated vesicles into the cell. Despite large amounts of functional data, the exact nature of the conformational changes that drive dynamin's mechanochemical activities is still ill-defined due to a lack of structural information. To better understand how dynamin structure influences its functional activities, we seek to visualize the structure and conformational changes of assembled dynamin using a combination of cryo-electron microscopy, biochemistry, and image reconstruction techniques. These studies will yield three-dimensional reconstructions of dynamin at sufficient resolution to allow us to see the key structural changes that are linked to different nucleotide states. Attachment of electron-dense labels will elucidate the overall organization of the dynamin polymer and will unambiguously define the positions of dynamin's domains within these structural maps. This knowledge will greatly increase our understanding of how dynamin transmits the chemical energy of GTP hydrolysis into the physical work of membrane scission and ultimately pre-synaptic recycling. The ability of the body to transmit neurological impulses is highly dependent on the efficiency of a process known as pre-synaptic recycling, which maintains a large pool of vesicles pre-loaded with neurotransmitters and primes the neuron for rapid response to an action potential. The GTPase dynamin is essential for this process, though the underlying mechanisms of its function remain unknown due to a lack of structural information about the protein. We seek to determine how dynamin structure influences its function, thereby establishing the key changes in the protein that drive its biological activities. These findings will ultimately enhance our understanding of dynamin's role in mental health.