STRUCTURE/FUNCTION STUDIES OF GLUCOCORTICOID RECEPTOR

糖皮质激素受体的结构/功能研究

• 批准号: 6233601
• 负责人: E B THOMPSON
• 金额: $ 24.59万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-15 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6233601
• 关键词: Baculoviridae; X ray crystallography; binding sites; circular dichroism; corticosteroid receptors; fluorescence spectrometry; intermolecular interaction; nuclear magnetic resonance spectroscopy; protein folding; protein structure function; recombinant proteins; site directed mutagenesis; surface plasmon resonance; tertiary amine

Lack of knowledge of the structure of its major transactivation domain (AF1) has hampered full understanding of the mechanism by which the glucocorticoid receptor functions. It is presumed that AF1 interacts directly, or through intermediary proteins, with the "transcription machinery" complex of proteins. This requires surface-to-surface interactions between proteins and the structure to produce them. When expressed separately as a recombinant protein, however, AR displays little or no structure and binds weakly at best with a few such proteins. We have made two observations that offer exciting promise for understanding how AF1 obtains the structure necessary for its presumed function and for discovering its natural binding partners. First, we found that in the presence of an osmolyte, trimethylamine oxide (TMAO), the AF1 of the human GR acquires native-like structure and shows greatly enhanced binding to certain other proteins. We will use TMAO and other osmolytes to study the structure of AF1 and to find its binding partners. Second, we have shown that when a two-domain fragment of the GR containing AF1 and the DNA-binding domain is bound to its cognate DNA binding site, AR seems to acquire structure and be able to bind certain other proteins. In this project, we will determine the protein and DNA parameters that control this event. We will compare the AF1 structure and the protein binding partners uncovered by the two techniques. One of the nuclear proteins shown to bind AF1 under the conditions we employ appears to be a member of the nuclear receptor co- activator family of proteins. Methods used will include circular dichroism, intrinsic fluorescence emission spectroscopy, peptide mapping, thermodynamically rigorous studies of protein:DNA binding NMR and if crystals are obtained, X-ray diffraction.

缺乏对其主要反式激活结构域（AF1）的结构的了解，这阻碍了人们对糖皮质激素受体功能的机制的完全理解。假定AF1直接或通过中间蛋白与蛋白质的“转录机械”复合物相互作用。这需要蛋白质与结构之间的地面相互作用才能产生它们。但是，当分别表示为重组蛋白时，AR几乎没有或没有结构，并且最多与几种此类蛋白质结合。我们做出了两次观察，为了解AF1如何获得其假定功能所需的结构并发现其自然绑定伙伴所必需的结构，这给了人们两个令人兴奋的希望。首先，我们发现，在存在渗透液，三甲胺氧化物（TMAO）的情况下，人类GR的AF1获得了类似天然的结构，并显示出与某些其他蛋白质的结合大大增强。我们将使用tmao和其他渗透压研究AF1的结构并找到其结合伙伴。其次，我们已经表明，当包含AF1的GR和DNA结合结构域的两个域片段与其同源DNA结合位点结合时，AR似乎可以获取结构并能够结合某些其他蛋白质。在此项目中，我们将确定控制此事件的蛋白质和DNA参数。我们将比较两种技术发现的AF1结构和蛋白质结合伙伴。在我们采用的条件下，显示结合AF1的一种核蛋白似乎是蛋白质的核受体共激活剂家族的成员。所使用的方法将包括圆形二色性，固有的荧光发射光谱，肽映射，热力学上严格的蛋白质研究：DNA结合NMR以及如果获得晶体，X射线衍射。