Genetic Dissection of the Specificity of Leucine Zipper Dimerization

亮氨酸拉链是当一对蛋白质分子中的一对短的α -螺旋以一种称为盘绕线圈的结构结合时形成的。亮氨酸拉链和其他盘绕线圈在自然界中是丰富的；在典型基因组中编码的所有蛋白质中，约有10%存在它们，包括许多重要的转录因子。该项目解决了关于亮氨酸拉链的两个互补问题。首先，在酵母转录因子GCN4的特定亮氨酸拉链的背景下，亚基如何相互作用，控制拉链组装的特异性和稳定性。不同的相互作用将被设计到GCN4亮氨酸拉链中，并通过生物物理分析进行表征，包括平衡和停止流动的圆二色性、沉积平衡和x射线晶体学。此外，新的亮氨酸拉链组件的生物物理分析将基于分析超离心和荧光相关光谱实施。其次，该项目将探索整个基因组中盘绕线圈的多样性。利用强大的遗传方法，该项目将检查来自酵母基因组的数百种自然发生的、预测的线圈之间相互作用的特异性，这些线圈必须共存于同一个细胞中。由于它们的小尺寸和简单，重复的结构，亮氨酸拉链为研究蛋白质如何折叠的一般问题提供了有用的模型。通过将特定相互作用的研究与许多拉链特性的全基因组研究相结合，该项目将测试我们对蛋白质折叠蛋白的理解是否足以阐明大量广泛分布的一类重要蛋白质的生物学。

Leucine zippers are formed when a pair of short alpha -helices in a pair of protein molecules bind in aconfiguration called a coiled coil. Leucine zippers and other coiled coils are abundant in nature;they are found in about 10% of all proteins encoded in typical genomes, including many importanttranscription factors. The project addresses two complementary questions about leucine zippers.First, how do the subunit interactions in the context of a specific leucine zipper from the yeasttranscription factor GCN4, control the specificity and stability of zipper assembly. Differentinteractions will be engineered into the GCN4 leucine zipper and characterized by biophysicalassays including equilibrium and stopped-flow circular dichroism, sedimentation equilibrium, andX-ray crystallography. In addition, new biophysical assays for leucine zipper assembly will beimplemented based on analytical ultracentrifugation and fluorescence correlation spectroscopy.Second, the project will explore the diversity of coiled coils in a whole genome. Using powerfulgenetic methods, the project will examine the specificity of interactions among hundreds ofnaturally occurring, predicted coiled-coils from the yeast genome that must coexist in the same cell.Because of their small size and simple, repetitive structures, leucine zippers have provided usefulmodels for studying general issues in how proteins fold. By combining research on specificinteractions with genome-wide studies of the properties of many zippers, this project will testwhether our understanding of protein foldin is sufficient to illuminate the biology of a large andwidely distributed class of important proteins.