MOLECULAR PROBES FOR STEROID RECEPTORS

雌激素受体是一种多结构域、配体激活的受体。 转录因子；而DNA结合的结构 从核磁共振和X射线研究中详细了解了结构域， 到目前为止，激素结合域的结构尚不清楚。这个 本项目的总体目标是确定海床的三维结构 这一领域，使用三种协同工作的方法 时尚：(A)激素结合结构域的表达 雌激素受体(ER-HBD)在化学纯品和 构象均一形式，(B)使用特定的 要研究的化学探针和确定片段的表达 结构和功能，以及(C)计算的应用 建模方法。作为具体目标I，我们将使用pTrxFus 在大肠杆菌中表达ER N304-S554序列(即 代表ER-HBD的核心)，我们将使用电喷雾 电离质谱仪(ESI-MS)用于鉴定和鉴定 表达材料的化学纯度，已被证明是 这在很多情况下都是个问题。在具体目标2中，我们将使用以下内容 材料，以获得ER-HBD的拓扑信息，通过 亲和标记法识别HBD和HBD中的配基接触位点 通过残基特定修饰来识别暴露部位，以及 我们将通过表达HBD来探索ER-HBD的核心结构 片段和亚域结构元素，结构如下 圆二色谱(CD)的形成和配基的作用 有约束力的。作为具体目标3，我们将使用高级计算 预测和精炼3-D的同源建模方法 基于能量和模式的ER-HBD结构模型 识别方法和与我们的结果的一致性测试 自己的研究；我们将使用这个模型来评估配体结合 基于结构的设计的专用性和计划的专用性 通过子域互换来重新设计实验。作为特定的目标 4，我们将制备同位素标记的配体和ER-HBD核心 核磁共振研究的结构片段，将在合作中完成 与A.J.Wand，部门。生物化学。对于要进行的X射线分析 与A·王的关系结束了，部门。细胞和结构生物学，以及 化学，我们将使用化学纯的和 构象均一，并含有重原子 蛋白质或络合配体。我们预计这些研究， 它结合了合成化学，生物化学，分子 生物学，以及(通过协作)高级计算 建模、核磁共振和X射线结构分析，将导致 在我们对结构的理解方面取得了重大进展 雌激素受体及其与雌激素激素的相互作用。

The estrogen receptor is a multidomain, ligand-activated transcription factor; while the structure of the DNA-binding domain is known in detail from NMR and X-ray studies, the structure of the hormone binding domain is so far unknown. The overall goal of this project is to determine the 3-D structure of this domain, using three approaches that operate in a synergistic fashion: (a) the expression of the hormone binding domain of the estrogen receptor (ER-HBD) in chemically pure and conformationally homogeneous form, (b)the use of specific chemical probes and expression of defined fragments to study structure and function, and (c) the application of computational modeling methods. As Specific Aim I, we will use the pTrxFus vector in E. coli to express the ER N304-S554 sequence (that represents the core of the ER-HBD), and we will use electrospray ionization mass spectrometry (ESI-MS) to certify the identity and chemical purity of the expressed material, which has proved to be a problem in many cases. In Specific Aim 2, we will use this material to obtain topological information on the ER-HBD, by affinity labeling to identify ligand contact sites in the HBD and by residue specific modification to identify exposed sites, and we will probe the core structure of the ER-HBD by expressing HBD fragments and subdomain structural elements, following structure formation by circular dichroism (CD) and function by ligand binding. As Specific Aim 3, we will use advanced computational homology modeling approaches to predict and refine a 3-D structural model of the ER-HBD, using energy-based and pattern recognition methods and testing consistency with results from our own studies; we will use this model to evaluate ligand binding specificity for structure-based design and to plan specificity re-engineering experiments by subdomain swapping. As Specific Aim 4, we will prepare isotopically labeled ligands and ER-HBD core structural fragments for NMR studies, to be done in collaboration with A. J. Wand, Dept. Biochemistry. For X-ray analysis to be done with A. Wang, Depts. Cell and Structural Biology, and Chemistry, we will use preparations that are chemically pure and conformationally homogeneous, and contain heavy atoms in the protein or complexed ligand. We anticipate that these studies, which combine synthetic chemical, biochemical, molecular biological, and (through collaborations) advanced computational modeling, NMR and X-ray structural analysis, will lead to significant advances in our understanding of the structure of the estrogen receptor and its interaction with estrogen hormones.