Genetic Dissection of the Specificity of Leucine Zipper Dimerization

当一对蛋白质分子中的一对短α-螺旋结合成卷曲螺旋时，亮氨酸拉链就形成了。亮氨酸拉链和其他卷曲螺旋在自然界中是丰富的，它们存在于典型基因组中编码的所有蛋白质的约10%中，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：第一，在酵母转录因子GCN 4的特定亮氨酸拉链的背景下，亚基相互作用如何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成GCN 4亮氨酸拉链，并通过包括平衡和停流圆二色性、沉降平衡和X射线晶体学在内的生物药理学测定来表征。此外，还将基于分析超速离心和荧光相关光谱技术实施新的亮氨酸拉链组装生物物理测定。其次，该项目将探索整个基因组中卷曲螺旋的多样性。利用强大的遗传学方法，该项目将研究数百个自然发生的、预测的、必须在同一细胞中共存的酵母基因组卷曲螺旋之间相互作用的特异性。由于亮氨酸拉链的小尺寸和简单重复的结构，它们为研究蛋白质如何折叠的一般问题提供了有用的模型。通过结合对特异性相互作用的研究和对许多拉链性质的全基因组研究，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一个大而广泛分布的重要蛋白质类的生物学。

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.