Genetic Dissection of the Specificity of Leucine Zipper Dimerization

Hu MCB 9808474蛋白质如何识别其他蛋白质在几乎每一个生物过程中都是至关重要的。亮氨酸拉链形成各种小的α -螺旋低聚物，为研究决定蛋白质-蛋白质相互作用的稳定性和特异性的力提供了理想的系统。这项研究建立在早期的遗传研究，以检查氨基酸序列如何决定亮氨酸拉链的二聚化特异性。以前，通过改变特征七重序列（abcdefg）n的a， e和g位置的变化，分离出具有改变二聚化特异性的突变拉链。在a位置上有替换的突变体属于互斥的类别，明确的规则预测了它们的特异性。相比之下，在e和g位置改变的突变体的二聚化特异性是复杂的，与涉及亚基间离子相互作用的简单预测模型不一致。将讨论两类问题。首先，同型二聚体和异型二聚体的热力学稳定性如何决定这些突变型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？新的突变体将通过选择和筛选在早期研究中未发现的同二聚体和异二聚体形成的特定模式来分离。突变体的特异性将与自然发生的亮氨酸拉链进行比较，在常见的分析中进行评估。这些数据应该为预测二聚化特异性的模型和算法提供一个关键的测试。2. 多种小的α -螺旋肽富含氨基酸亮氨酸，这样的结构家族因此被命名为亮氨酸拉链。这些序列提供了一个理想的系统来理解决定蛋白质-蛋白质相互作用的稳定性和特异性的力量，因为许多蛋白质通过它们的亮氨酸拉链相互作用而形成二聚体。在这项研究中，利用圆二色性和荧光方法，比较了二聚化特异性改变或取代的突变序列。需要解决的问题是：(1)同型二聚体和异型二聚体的热力学稳定性如何决定这些突变型和野生型拉链的特异性？(2)特定位置的序列模式如何影响二聚化特异性？这些结果将为预测蛋白质二聚化特异性的模型和算法提供关键测试。

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.