STRUCTURAL STUDIES OF PAS DOMAIN SIGNALING MECHANISMS

描述(申请人描述)：PAS结构域调节蛋白质/蛋白质 蛋白质之间的相互作用调节着广泛的生物功能， 包括基因表达。几种Pas的蛋白质结合能力 结构域由小的有机配体调节，为这些提供了一种机制 化合物来调节复杂的生物活性。对这些的不当监管 通路是有害的，PAS/毒素的核心作用证明了这一点 络合物在发展中的几种环境致癌作用 毒素。拟议研究将描述如何具体地传递AS域 与配体和其他蛋白质的相互作用以及这些结合事件是如何 整合在一个很小的(100-120个残基)区域内。为了实现这一点，基于核磁共振的 结构研究将在三个一般方面进行：1)。的研究 四种真核蛋白的单一PAS结构域，使用新的方法 快速鉴定这些结构域的可溶片段，以便进行核磁共振研究。结构性 在两个这样的域(12)上使用标准方法获得了信息 和20 kDa)，并表明配体和蛋白质结合位点在 缺少互动伙伴。2)。PAS/配体络合物的研究。 从获得的关于无配体结构域、小分子 文库将通过基于核磁共振的方法进行筛选，以确定潜在的配体。 这些研究的初步数据表明，有几种化合物与 正在调查的PAS域之一，将几个灵活的 以无配体形式观察到的区域。这些构象变化将是 通过考察PAS/配体的结构和动力学进行了更详细的研究 复合体。3)。PAS/蛋白质复合体的研究。其中大部分是由 PAS/PAS异二聚化，便于使用同位素标记来 简化了这些大分子(约30 kDa)配合物的核磁共振波谱。喜忧参半 将使用解析方法，通过对接生成综合体的模型 通过使用多个独立区域的高分辨率结构 在完好的建筑群上获得的低分辨率约束。这些对PAS的看法 不同构象的结构域将为深入了解 它们的调节，并可能打开治疗设计的新方法 可以组装/拆解PAS介导的复合体的试剂。

Description (applicant's description): PAS domains mediate protein/protein interactions among proteins that regulate a wide range of biological functions, including gene expression. The protein-binding capabilities of several PAS domains are modulated by small organic ligands, providing a mechanism for these compounds to regulate complex biological activities. Misregulation of these pathways is deleterious, as demonstrated by the central role of PAS/toxin complexes in developing the carcinogenic effects of several environmental toxins. The proposed research will characterize how PAS domains specifically interact with ligands and other proteins and how these binding events are integrated within a small (100-120 residue) domain. To achieve this, NMR-based structural studies will be conducted in three general areas: 1). Studies of single PAS domains from four eukaryotic proteins, using novel methods to rapidly identify soluble fragments of these domains for NMR studies. Structural information has been obtained using standard methods on two such domains (12 and 20kDa) and suggests that ligand- and protein-binding sites are flexible in the absence of interacting partners. 2). Studies of PAS/ligand complexes. Starting with the information obtained on ligand-free domains, small molecule libraries will be screened by NMR-based methods to identify potential ligands. Preliminary data from these studies demonstrate that several compounds bind to one of the PAS domains under investigation, rigidifying several flexible regions observed in the ligand-free form. These conformational changes will be studied in more detail by examining the structures and dynamics of PAS/ligand complexes. 3). Studies of PAS/protein complexes. Most of these are formed by PAS/PAS heterodimerization, facilitating the use of isotopic labeling to simplify the NMR spectra of these large (about 30kDa) complexes. A mixed resolution approach will be used, generating models of the complex by docking by high resolution structures of the individual domains using multiple low-resolution restraints acquired on the intact complex. These views of PAS domains in different conformations will provide insight into the mechanism of their regulation and possibly open new approaches to the design of therapeutic agents that could assemble/disassemble PAS-mediated complexes.