Structural studies of PAS domain signaling mechanisms

描述（由申请人提供）：PAS （Per-ARNT-Sim）蛋白相互作用域广泛分布于生物学中，它们存在于9000多种蛋白质中，包括酶、转录因子和离子通道。在这些背景下，PAS结构域参与其功能所需的分子内和分子间相互作用的组合。有趣的是，许多这些相互作用可以通过结合辅因子或人工配体改变周围蛋白质结构的环境变化来调节。PAS功能的结构和生化机制及其调控是本研究的重点，将继续我们在这一领域的前期研究。考虑到利用这些结构域的设置的多样性，我们将通过比较三种不同类型的含PAS蛋白的代表性成员的生物物理和生化研究来确定PAS调节的一般特征。这些包括：1)。利用基于fmn的PAS结构域控制DNA结合活性以响应蓝光的光感受器。光谱学和结构研究将用于检查感官PAS结构域的构象变化如何调节活动。2）. 细菌组氨酸（“传感器”）激酶，其中PAS结构域感知各种环境条件。PAS结构域和全长蛋白的生物物理和生化研究将用于解决激活和灭活突变如何调节激酶活性。这些变化的功能效应将通过体外酶和体内信号分析来探讨。3)。真核转录因子，包括人bHLH/PAS蛋白。将我们的工作扩展到这些蛋白质中的几个，我们将使用光谱工具来研究可以通过表面突变人为触发转换构象的结构域。我们还将研究这些结构域如何利用转录辅激活子募集的生化和结构研究同时结合多个蛋白质靶标。这项研究将为PAS结构域使用的信号机制提供基本的见解，特别是它们如何与酶和非酶效应物一起工作。这将提高我们对pas蛋白的理解，并提出这些蛋白的工程原理。

DESCRIPTION (provided by applicant): PAS (Per-ARNT-Sim) protein interaction domains are widely distributed through biology as documented by their presence in a diverse group of over 9000 proteins, including enzymes, transcription factors and ion channels. Within these contexts, PAS domains participate in a combination of intra- and intermolecular interactions necessary for their function. Intriguingly, many of these interactions can be regulated by environmental changes in bound cofactors or artificial ligands that alter the surrounding protein structure. The structural and biochemical mechanisms of PAS function and this regulation are the focus of the proposed research, continuing our prior studies in this area. Given the diversity of settings utilizing these domains, we will determine general features of PAS regulation by comparative biophysical and biochemical studies of representative members of three different classes of PAS-containing proteins. These include: 1). Photoreceptors that use FMN-based PAS domains to control DNA binding activity in response to blue light. Spectroscopic and structural studies will be used to examine how conformational changes in a sensory PAS domain regulate activity. 2). Bacterial histidine ("sensor") kinases, where PAS domains sense a variety of environmental conditions. Biophysical and biochemical studies of PAS domains and full length proteins will be used to address how activating and inactivating mutations regulate kinase activity. The functional effects of these changes will be probed with in vitro enzymatic and in vivo signaling assays. 3). Eukaryotic transcription factors, including human bHLH/PAS proteins. Extending our work on several of these proteins, we will use spectroscopic tools to investigate a domain that can be artificially triggered to switch conformation by surface mutations. We will also examine how these domains can simultaneously bind multiple protein targets using biochemical and structural studies of transcriptional coactivator recruitment. This research will provide fundamental insights into the signaling mechanisms used by PAS domains, particularly in how they work in conjunction with enzymatic and non-enzymatic effectors. This will improve our understanding of PAS-containing proteins and suggest engineering principles for these proteins. Relevance to public health: PAS domains play a central role in human disease, as point mutations in these elements have been correlated with several cancers and cardiac irregularities. These domains are also essential to several bacterial pathways, providing links to microbial pathogenesis. Improved understanding of PAS structure/function links may provide novel routes for diagnosis and intervention in disease processes.