MOLECULAR PROBES FOR STEROID RECEPTORS

类固醇受体分子探针

• 批准号: 2518239
• 负责人: JOHN A. KATZENELLENBOGEN
• 金额: $ 23.16万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2518239
• 关键词: X ray crystallography; affinity labeling; chemical binding; chemical stability; chimeric proteins; circular dichroism; computer simulation; electrospray ionization mass spectrometry; estrogen receptors; estrogens; high performance liquid chromatography; ligands; model design /development; nuclear magnetic resonance spectroscopy; physical model; protein engineering; protein folding; protein purification; protein sequence; protein structure function; proteolysis; site directed mutagenesis; structural biology; thermodynamics

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.

雌激素受体是一种多域，配体激活 转录因子；而DNA结合的结构 NMR和X射线研究的详细知名度是 激素结合结构域的结构迄今未知。这 该项目的总体目标是确定3-D结构 该域使用在协同作用中运行的三种方法 时尚：（a）激素结合域的表达 化学纯净的雌激素受体（ER-HBD） 构象的均匀形式，（b）使用特定的 化学探针和定义片段的表达 结构和功能，以及（c）计算的应用 建模方法。作为特定目的我，我们将使用ptrxfus 大肠杆菌中的向量表达ER N304-S554序列（该序列 代表ER-HBD的核心），我们将使用电喷雾 电离质谱（ESI-MS）以证明身份和 表达材料的化学纯度，事实证明是 在许多情况下是一个问题。在特定目标2中，我们将使用此 通过 亲和力标签以识别HBD中的配体接触位点，并 通过残留特定的修改以识别暴露的位点，并 我们将通过表达HBD来探测ER-HBD的核心结构 片段和亚域的结构元素，以下结构 通过圆形二色性（CD）形成和配体功能 结合。作为特定目标3，我们将使用高级计算 同源性建模方法预测和完善3-D ER-HBD的结构模型，使用基于能量和模式 识别方法和测试与我们的结果一致性 自己的研究；我们将使用此模型评估配体结合 基于结构设计的特异性并计划特异性 通过子域交换进行重新设计实验。作为特定目标 4，我们将准备同位素标记的配体和ER-HBD核心 NMR研究的结构片段，可以合作完成 与A. J. Wand，生物化学系。 X射线分析为 与A. Wang一起完成。细胞和结构生物学，以及 化学，我们将使用化学上纯净的制剂 构象同质，并在 蛋白质或复合配体。我们预计这些研究， 结合了合成化学，生化，分子 生物学，（通过协作）高级计算 建模，NMR和X射线结构分析将导致 在我们对结构的理解方面的重大进展 雌激素受体及其与雌激素的相互作用。