Genetic Dissection of the Specificity of Leucine Zipper Dimerization

亮氨酸拉链二聚化特异性的遗传解析

• 批准号: 0111000
• 负责人: James Hu
• 金额: $ 36.6万
• 依托单位: Texas A&M Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至 2005-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0111000&HistoricalAwards=false
• 关键词: Genetic; Dissection; Specificity; Leucine; Zipper

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.

当一对蛋白质分子中的一对短α螺旋以称为卷曲线圈的结构结合时，就形成了亮氨酸拉链。亮氨酸拉链和其他卷曲螺旋在自然界中含量丰富；它们存在于典型基因组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。首先，在酵母转录因子 GCN4 的特定亮氨酸拉链背景下，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到 GCN4 亮氨酸拉链中，并通过生物物理测定进行表征，包括平衡和停流圆二色性、沉降平衡和 X 射线晶体学。此外，还将基于分析超速离心和荧光相关光谱法对亮氨酸拉链组装进行新的生物物理测定。其次，该项目将探索整个基因组中卷曲线圈的多样性。该项目将使用强大的遗传方法，检查数百个来自酵母基因组的自然发生的、预测的卷曲螺旋之间相互作用的特异性，这些卷曲螺旋必须共存于同一细胞中。由于亮氨酸拉链尺寸小且结构简单、重复，因此为以下方面提供了有用的模型：研究蛋白质如何折叠的一般问题。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的理解是否足以阐明大量且广泛分布的一类重要蛋白质的生物学特性。