Structural Studies of Scaffords involved in Endocytosis and Signal Transduction

该项目的长期目标是从机制上了解细胞膜重塑和细胞骨架重排是如何在高度调控的内吞过程中达到顶峰的。为了实现这一点，我将解决Bin/双体素/RVS蛋白(例如内亲素、两体素或突触素)的结构，该蛋白在内吞作用中具有良好的已知功能。表达和纯化这些蛋白质的方案已经建立，这让我有时间专注于该项目的结构方面。我将记录电子冷冻显微镜图像，并使用尖端图像处理策略来重建膜结合蛋白质的结构。虽然概念上很简单，但手头的任务并不是微不足道的，而且代表了技术上可能的界限。一旦第一个样品的结构确定进行，我将继续努力纯化上述蛋白质的相互作用伙伴，如N-WASP或Dynamin(这些蛋白质的表达系统已在当地建立)，并进行这些高度相关的复合体的结构测定，最好是在膜结合状态下。预期的结果将为膜重塑的机制提供关键的见解，从而有助于当代结构生物学和生物物理学中最不具代表性的领域之一。

The longterm goal of this project is to gain mechanistic understanding of how membrane remodeling and cytoskeletal rearrangements are orchestrated to culminate in the highly regulated process of endocytosis. To achieve this, I will solve the structure of a Bin/Amphiphysin/rvs protein (e.g. endophilin,amphiphysin or syndapin) protein with a well documented function in endocytosis. The protocols for expression and purification of these proteins are already established, leaving me time to focus on the structural aspects of the project. I will record electron cryomicroscopic images and use cutting-edge image processing strategies to reconstruct the structures of the membrane-bound proteins. While conceptually simple, the tasks at hand are not trivial and represent a boundary of what is technologically possible. Once structure determination of the first sample is underway, I will extend my efforts to purify interaction partners of the above-mentioned protein, like N-WASP, or dynamin (expression systems for these proteins have already been established locally), and pursue structure determination of these highly relevant complexes, preferably in a membrane-bound sate. The anticipated results will provide critical insights into the mechanisms of membrane remodeling, thus contributing to one of the most underrepresented fields in contemporary structural biology and biophysics.