Dahlman-Wright K, Baumann H, McEwan I J, Almlöf T, Wright A P, Gustafsson J A, Härd T
Centre for Structural Biochemistry, Karolinska Institutet, Novum Huddinge, Sweden.
Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1699-703. doi: 10.1073/pnas.92.5.1699.
A 58-amino acid polypeptide containing the functional core region, the tau 1 core, of the major transactivation domain of the human glucocorticoid receptor has been expressed in Escherichia coli and purified to homogeneity. The polypeptide retains 60-70% of the activity of the intact domain when assayed in vivo or in vitro. This report describes a structural characterization of the tau 1 core peptide fragment. Circular dichroism spectroscopy shows that the tau 1 core and a larger fragment encompassing the intact tau 1 domain are largely unstructured in water solution under a variety of pH conditions. The tau 1 core, however, acquires a significant alpha-helical structure when analyzed in the presence of trifluoroethanol, an agent that favors secondary structure formation in regions that have propensity for alpha-helical conformation. Two- and three-dimensional NMR spectroscopy of 15N-labeled tau 1 core, in the presence of trifluoroethanol, has allowed sequential assignment of 1H and 15N resonances and identification of three protein segments with alpha-helical character. Potentially helix-breaking proline substitutions, in proposed alpha-helical regions, lead to reduced activity, suggesting that alpha-helices are important for transactivation in vivo.
一种包含人糖皮质激素受体主要反式激活结构域功能核心区域（tau 1核心）的58个氨基酸的多肽已在大肠杆菌中表达并纯化至同质。在体内或体外测定时，该多肽保留了完整结构域60 - 70%的活性。本报告描述了tau 1核心肽片段的结构特征。圆二色光谱表明，在多种pH条件下，tau 1核心以及包含完整tau 1结构域的更大片段在水溶液中基本无结构。然而，当在三氟乙醇存在下进行分析时，tau 1核心获得了显著的α - 螺旋结构，三氟乙醇是一种有利于在具有α - 螺旋构象倾向的区域形成二级结构的试剂。在三氟乙醇存在下，对15N标记的tau 1核心进行二维和三维核磁共振光谱分析，已实现1H和15N共振的顺序归属，并鉴定出三个具有α - 螺旋特征的蛋白质片段。在提议的α - 螺旋区域中潜在的破坏螺旋的脯氨酸取代导致活性降低，这表明α - 螺旋对体内反式激活很重要。
