Establishing a common function for ferlin proteins in membrane fusion using novel genetic code expansion and single molecule techniques.

膜运输被认为是真核细胞的一个重要和独特的特征，细胞内运输和分泌事件在稳态和信号转导中起着重要作用。虽然许多关于膜运输的知识来自酵母的研究，但在多细胞生物中，有一些重要的蛋白质参与了酵母中没有发现的膜运输。 这些蛋白质中的一个家族是ferlin，这是一个进化上古老的运输蛋白家族，与越来越多样化的生理活动有关，包括生育，声音编码，肌肉发育和受损细胞膜的修复。这种生理作用的多样性使得为该家族建立共同的潜在分子功能具有挑战性。该项目将解决我们对Ferlin家族蛋白质知识的这一空白，并在此过程中增加我们对生育，听力和肌肉发育的理解。该项目还将开发本科生实验室研究培训机会和实验室课程，重点是膜生物学，并为研究生提供跨学科研究的最先进培训。该项目测试了ferlin作为钙敏感支架的共同功能，用于调节胞吐和胞吞作用。由于蛋白质的大尺寸、其跨膜结构域以及铁蛋白典型的大量内源性半胱氨酸而带来的挑战，排除了许多通常用于测试这种假设功能的传统重组蛋白测定法。为了克服这些挑战，已经开发了一种新的单分子蛋白质-蛋白质相互作用测定，其允许探测大的多价膜蛋白的接触。 此外，将利用遗传密码扩展技术将环境敏感的荧光非天然氨基酸掺入到ferlin中。这些技术将被用来确定ferlin活性的基础功能。拟议中的研究将定义一组潜在的分子水平功能，这些功能将费林蛋白结合起来，尽管它们具有不同的生理作用。这些信息将允许将ferlin整合到膜运输和细胞信号传导的更大画面中。一种新的单分子荧光技术的发展将提供一种方法，为大型多域膜蛋白的研究，从而有应用超越ferlins.This奖项的研究反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

Membrane trafficking is regarded as a crucial and distinctive characteristic of eukaryotic cells, with both intracellular transport and secretion events playing an essential role in homeostasis and signaling. While much of what is known about membrane trafficking comes from studies in yeast, there are important proteins involved in membrane trafficking in multicellular organisms not seen in yeast. One family of these proteins are the ferlins, an evolutionarily ancient family of trafficking proteins linked to an increasingly diverse list of physiological activities, including fertility, the encoding of sound, muscle development, and repair of damaged cell membranes. This diversity of physiological roles has made establishing a common underlying molecular function for this family challenging. This project will address this gap in our knowledge of the Ferlin family proteins, and in so doing add to our understanding of fertility, hearing, and muscle development. The proposed work will also develop undergraduate laboratory research training opportunities and lab classes focused on membrane biology and provide graduate students with state-of-the-art training in interdisciplinary research.The project tests the idea that ferlins share a common function as calcium sensitive scaffolds for regulated exocytosis and endocytosis. Challenges due to the large size of the proteins, their transmembrane domains, and the high number of endogenous cysteines typical of ferlins have precluded many of the traditional recombinant protein assays typically used to test this hypothetical function. To overcome these challenges a novel single molecule protein-protein interaction assay that allows for probing the contacts of large multivalent membrane proteins has been developed. In addition, genetic code expansion techniques will be exploited to incorporate environmentally sensitive fluorescent unnatural amino acids into the ferlins. These technologies will be exploited to determine the functions that underlie ferlin activity. The proposed studies will define a set of underlying molecular-level functions that unite the ferlins, despite their disparate physiological roles. This information will allow integrating ferlins into the larger picture of membrane trafficking and cell signaling. The development of a novel single molecule fluorescence technique will provide a method for the study of large multi-domain membrane proteins and thus have applications beyond the study of ferlins.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.