Structural studies of PAS domain signaling mechanisms

描述（由申请人提供）：PAS（Per-ARNT-Sim）蛋白相互作用结构域在生物学中广泛分布，如其存在于超过9000种蛋白质的不同组中所记录的，包括酶、转录因子和离子通道。在这些情况下，PAS结构域参与其功能所必需的分子内和分子间相互作用的组合。有趣的是，这些相互作用中的许多可以通过改变周围蛋白质结构的结合辅因子或人工配体的环境变化来调节。PAS功能和这种调节的结构和生化机制是拟议的研究的重点，继续我们在这方面的研究。考虑到利用这些域的设置的多样性，我们将通过对三种不同类别的PAS含蛋白的代表性成员进行比较生物物理和生化研究来确定PAS调节的一般特征。这些措施包括：1）。光感受器使用基于FMN的PAS结构域来控制响应蓝光的DNA结合活性。光谱和结构的研究将被用来研究如何在感官PAS域的构象变化调节活动。2）。细菌组氨酸（“传感器”）激酶，其中PAS结构域感测各种环境条件。PAS结构域和全长蛋白的生物物理和生物化学研究将用于解决激活和失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传导试验进行探测。3）。真核转录因子，包括人bHLH/PAS蛋白。扩展我们对这些蛋白质中的几个的工作，我们将使用光谱工具来研究可以通过表面突变人工触发以切换构象的结构域。我们还将研究这些结构域如何同时结合多个蛋白质靶点，使用转录辅激活因子募集的生物化学和结构研究。这项研究将为PAS结构域使用的信号传导机制提供基本见解，特别是它们如何与酶和非酶效应子结合工作。这将提高我们对含PAS蛋白质的理解，并为这些蛋白质的工程原理提出建议。 与公共卫生的相关性：PAS结构域在人类疾病中起着核心作用，因为这些元件中的点突变与几种癌症和心脏不规则性相关。这些结构域对几种细菌途径也是必不可少的，提供了与微生物发病机制的联系。对PAS结构/功能联系的进一步了解可能为疾病过程的诊断和干预提供新的途径。

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.