Analysis and Design of Coiled Coil Partnering

项目摘要 这项拟议工作的广泛的长期目标是了解蛋白质如何建立相互作用 在各个领域的级别上的专用性，并在可用于计算的模型中捕捉这一点 预测和设计。这个问题是人类健康问题的核心。相互之间的不正当互动 突变或错误调节的蛋白质可能导致疾病，并加深对这一现象的理解 发生的情况很重要。此外，设计新的蛋白质或多肽来特异性地抑制天然蛋白质 复合体将为未来的治疗提供一条途径。相互作用专用性问题可以简化为 聚焦于一个普遍存在但结构简单的蛋白质-蛋白质相互作用主题：阿尔法螺旋螺旋线圈。 盘状螺旋存在于所有物种的蛋白质组中，并与各种不同的 功能。它们的序列和结构特性使它们易于进行计算分析，并且 盘绕线圈也便于用生物物理方法进行实验研究。拟议的研究 包括收集系统的实验交互数据，并使用它来开发和测试各种 预测相互作用的计算技术。计算方法也将被开发用于 设计了盘绕状多肽，并对设计进行了实验表征。具体的 这项建议的目的是：(1)测试和改进预测盘管结构、能量和 相互作用，(2)扩展现有的螺旋线圈设计能力，以处理更广泛的目标，(3)到 在动物物种中广泛测量bZIP盘绕线圈的相互作用，并开发一个模型来研究蛋白质如何- 蛋白质相互作用的特异性可以进化，以及(4)确定2- 组分受体组氨酸激动酶，并操纵这些以产生异种特性的激动酶。这个 目标1和目标2的预测和设计方法将主要使用基于结构的技术。身体上 激励能量函数将用于评估，还将与更多经验相结合进行测试 接近了。目标3涉及开发一种新的蛋白质-蛋白质相互作用分析方法，并将其用于 衡量&gt；10,000个可能的联想。这些数据将被用来分析bZIP的演变 转录因子的相互作用，也将是有价值的测试目标1的计算方法。 目的4检验原核生物组氨酸激酶主要是同源相关的假说，并提出 用于确定其相互作用特异性的决定因素的计算和实验技术。 这四个目标共同构成了结构建模、生物物理和 生物信息学分析和实验测试旨在了解盘绕线圈相互作用的特异性。

Project Summary The broad, long-term objectives of the proposed work are to understand how proteins establish interaction specificity at the level of individual domains and to capture this in models that can be used for computational prediction and design. The problem is central to issues of human health. Improper interactions among mutated or mis-regulated proteins can lead to disease, and achieving a deeper understanding of how this occurs is important. Further, the design of novel proteins or peptides to specifically inhibit native protein complexes would provide a route to future therapies. The interaction specificity problem can be simplified by focusing on a ubiquitous yet structurally simple protein-protein interaction motif: the alpha-helical coiled coil. Coiled coils occur throughout the proteomes of all species and are associated with a wide variety of functions. Their sequence and structural properties make them tractable for computational analysis, and coiled coils are also convenient for experimental study using biophysical methods. The proposed research involves collecting systematic experimental interaction data and using it to develop and test diverse computational techniques for predicting interactions. Computational methods will also be developed for designing coiled-coil-like peptides, and experiments are proposed to characterize the designs. The specific aims of this proposal are: (1) To test and improve methods for predicting coiled-coil structures, energies and interactions, (2) To extend existing coiled-coil design capabilities to treat a broader range of targets, (3) To measure bZIP coiled-coil interactions broadly across animal species and to develop a model of how protein- protein interaction specificity can evolve, and (4) To identify interaction specificity determinants of 2- component receptor histidine kinases and manipulate these to generate heterospecific kinases. The prediction and design methods of Aims 1 and 2 will use primarily structure-based techniques. Physically motivated energy functions will be used for evaluation, and also tested in conjunction with more empirical approaches. Aim 3 involves the development of a new protein-protein interaction assay and its use to measure >10,000 possible associations. These data will be applied to analyze the evolution of bZIP transcription factor interactions and will also be valuable for testing the computational methods of Aim 1. Aim 4 tests the hypothesis that prokaryotic histidine kinases primarily homoassociate, and proposes computational and experimental techniques for identifying determinants of their interaction specificity. Together, the four aims comprise an integrated program of structural modeling, biophysical and bioinformatic analysis and experimental testing aimed at understanding coiled-coil interaction specificity.