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: 9808474
• 负责人: James Hu
• 金额: $ 30万
• 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: 1998
• 资助国家: 美国
• 起止时间: 1998-08-15 至 2002-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9808474&HistoricalAwards=false
• 关键词: Genetic; Dissection; Specificity; Leucine; Zipper

HU MCB 9808474 1. Technical How proteins recognize other proteins is of fundamental importance in virtually every biological process. Leucine zippers, which form a variety of small alpha-helical oligomers, provide an ideal system to study the forces determining the stability and specificity of protein-protein interactions. This study builds on earlier genetic studies to examine how amino acid sequences determine the dimerization specificities of leucine zippers. Previously, mutant zippers with altered dimerization specificities were isolated with changes at the a, e, and g positions of the characteristic heptad repeat (abcdefg)n. Mutants with substitutions at the a positions fell into mutually exclusive classes and clear rules predicted their specificities. In contrast, the dimerization specificities of mutants altered at the e and g positions were complex and inconsistent with simple predictive models involving intersubunit ionic interactions. Two classes of questions will be addressed. First, how do the thermodynamic stabilities of homodimers and heterodimers determine the specificities of these mutant and wild-type zippers? Thermodynamic parameters for homodimeric and heterodimeric leucine zipper peptides will be measured by circular dichroism and fluorescence methods. Second, how do sequence patterns at the a, e, and g positions affect dimerization specificity? New mutants will be isolated using selections and screens for particular patterns of homodimer and heterodimer formation that were not found in earlier studies. The specificities of mutants will be are compared to naturally occurring leucine zippers, evaluated in a common assay. These data should provide a critical test of models and algorithms designed to predict dimerization specificity. 2. Non-technical A variety of small alpha-helical peptides is enriched in the amino acid leucine, and such a family of structures is hence named leucine zippers. These sequences pr ovide an ideal system to understand the forces determining the stability and specificity of protein-protein interactions, as many proteins dimerize by interacting through their leucine zippers. In this study, mutated sequences with changed dimerization specificities or with substitutions are compared, using circular dichroism and fluorescence methods. The questions to be addressed are: (1) how do the thermodynamic stabilities of homodimers and heterodimers determine the specificities of these mutant and wild-type zippers? and (2) how do sequence patterns at specific positions affect dimerization specificity? These results will provide a critical test of models and algorithms designed to predict dimerization specificity of proteins.

Hu MCB 9808474 1。技术蛋白如何识别其他蛋白质在几乎每个生物过程中都至关重要。 亮氨酸拉链形成各种小α-螺旋寡聚物，它提供了一个理想的系统，可以研究确定蛋白质 - 蛋白质相互作用的稳定性和特异性的力。这项研究以早期的遗传研究为基础，以检查氨基酸序列如何确定亮氨酸拉链的二聚化特异性。 以前，在特征性Heptad重复（ABCDEFG）n的A，E和G位置变化时，将具有变化二聚化特异性的突变型拉链分离出来。 A位置取代的突变体属于相互排斥的类别，明确的规则预测了其特殊性。 相比之下，在E和G位置改变的突变体的二聚化特异性与涉及亚基离子相互作用的简单预测模型不一致。将解决两个类别的问题。 首先，同二聚体和异二聚体的热力学稳定性如何确定这些突变体和野生型拉链的特异性？ 同二聚体和异二聚体亮氨酸拉链肽的热力学参数将通过圆形二色性和荧光方法测量。 其次，A，E和G位置的序列模式如何影响二聚化特异性？ 在早期研究中未发现的特定模式和异二聚体形成的特定模式的选择和筛选将分离出新的突变体。 将突变体的特异性与常见测定中评估的自然存在的亮氨酸拉链进行比较。 这些数据应对旨在预测二聚化特异性的模型和算法进行关键测试。 2。非技术的各种小α-螺旋肽富集在氨基酸亮氨酸中，因此，这种结构系列被称为亮氨酸拉链。这些序列是一个理想的系统，可以理解确定蛋白质 - 蛋白质相互作用的稳定性和特异性的力，因为许多蛋白质通过通过其亮氨酸拉链相互作用来降低。 在这项研究中，使用圆形二分法和荧光方法比较具有变化二聚化特异性或取代的突变序列。要解决的问题是：（1）同二聚体和异二聚体的热力学稳定性如何确定这些突变体和野生型拉链的特异性？ （2）特定位置处的序列模式如何影响二聚化特异性？ 这些结果将对旨在预测蛋白质二聚化特异性的模型和算法进行关键测试。