MOLECULAR PROBES FOR STEROID RECEPTORS

雌激素受体是一个多结构域，配体激活的 转录因子;而DNA结合的结构 从NMR和X射线研究中详细了解了结构域， 激素结合结构域的结构至今未知。的 该项目的总体目标是确定三维结构， 这一领域，使用三种方法，在协同运作， 方式：（a）表达的激素结合结构域的 雌激素受体（ER-HBD）在化学纯和 构象均匀的形式，（B）使用特定的 化学探针和确定片段的表达以研究 结构和功能，以及（c）计算的应用 建模方法作为特定目标I，我们将使用pTrxFus E.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 离子化质谱法（ESI-MS），以证明鉴别， 表达材料的化学纯度，已被证明是 在很多情况下是个问题。在具体目标2中，我们将使用 材料，以获得ER-HBD的拓扑信息，通过 亲和标记以鉴定HBD中的配体接触位点， 通过残基特异性修饰来识别暴露位点，和 我们将通过表达HBD来探测ER-HBD的核心结构， 片段和亚结构域结构元件，以下结构 通过圆二色性（CD）形成和通过配体功能 约束力作为具体目标3，我们将使用先进的计算 同源建模方法来预测和改进三维 ER-HBD的结构模型，使用基于能量和模式 识别方法和测试结果的一致性， 我们将使用该模型来评估配体结合 基于结构的设计和计划的特异性 通过子域交换重新设计实验。作为具体目标 4、制备同位素标记的配体和ER-HBD核心 用于核磁共振研究的结构片段， 名为. J. Wand，部门生物化学。对于X射线分析， 完成A。Wang，Depts.细胞和结构生物学，以及 化学，我们将使用化学纯的制剂， 构象均匀，并在分子中含有重原子。 蛋白质或复合配体。我们预计这些研究， 它结合了联合收割机合成化学、生物化学、分子 生物学和（通过合作）先进的计算 建模，核磁共振和X射线结构分析，将导致 在我们理解的结构的重大进展， 雌激素受体及其与雌激素的相互作用。

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.