NMR STUDIES OF HFH PROTEINS

转录因子的功能依赖于正确的 启动子和增强子区域中的靶DNA位点的识别。 虽然人们已经做了大量的研究来研究它们的相互作用， 许多问题仍然存在。 其中之一是保守的DNA结合基序 介导不同DNA位点的识别。 肝细胞核 因子3（HNF-3）和叉头干蜱（fkh）相关蛋白（HFH） 组成了一个大的蛋白质家族， 被称为“有翼螺旋”基序来识别它们的靶DNA位点。 虽然HFH蛋白具有几乎不变的识别 序列，它们表现出不同的DNA结合特异性。 这 不能用特定的相互作用模型来解释， 保守氨基酸和DNA碱基。 根据生化数据， 提出了HFH-3/fkh的DNA结合特异性 同源物是由DNA的不同呈现介导的 识别螺旋 为了详细检验这一假设，我们建议 运用分子生物学和现代核磁共振技术， 研究HFH-1的结构和DNA结合特异性， HFH-2：两个不同的HFH家族成员。 我们将决定， 比较HFH-1和HFH-2的DNA结合结构域的结构。 我们试图了解20个氨基酸序列对 识别序列的结构表示。 我们将比较 HFH-1和HFH-2的DNA复合物的结构和研究 HFH蛋白中的相同氨基酸残基是否与 DNA. 我们还将研究HFH蛋白的动力学性质 和它们的DNA复合体 我们特别感兴趣的是 接触残基和C-末端的动态翼序列。 我们 将研究基因突变的影响， 识别螺旋对DNA结合特异性的影响。 初始目标 将是与识别序列相邻的20个氨基酸的区域， 其影响HFH蛋白的DNA结合特异性。 的 蛋白质-DNA相互作用的研究不仅对 理解转录和基因调控，但也是至关重要的 用于分子建模和蛋白质设计。

The functions of transcription factors depend upon correct recognition of target DNA sites in promotor and enhancer regions. Although intensive research has been done to study their interactions, many questions remain. One of them is how conserved DNA binding motif mediates recognition of divergent DNA sites. The hepatocyte nuclear factor 3 (HNF-3) and Drysophila forkhead (fkh) related proteins (HFH) constitute a large family of proteins and use a modified helix turn known as the "winged helix" motif to recognize their target DNA sites. Although the HFH proteins have almost invariable recognition sequences, they exhibit divergent DNA binding specificity. This cannot be explained by the model of specific interactions between conserved amino acids and DNA bases. Based on biochemical data, a hypothesis is proposed that DNA binding specificity of the HFH-3/fkh homologues is mediated by different presentation of the DNA recognition helix. To test this hypothesis in detail, we propose to apply both molecular biology and modern heteronuclear NMR methods to study the structures and the DNA binding specificity of HFH-1 and HFH-2: two divergent HFH family members. We will determine and compare the structures of the DNA binding domains of HFH-1 and HFH-2. We seek to understand the effect of the 20 amino acid sequence on structural presentation of the recognition sequence. We will compare the structures of the DNA complexes of HFH-1 and HFH-2 and study whether the same amino acid residues in the HFH proteins interact with DNA. We will also study the dynamics properties of the HFH proteins and their DNA complexes. We are particularly interested in the DNA contact residues and the dynamic wing sequence in the C-terminus. We will study the effect of mutations designed to alter presentation of the recognition helix on DNA binding specificity. The initial target will be a 20 amino acid region adjacent to the recognition sequence, which influences the DNA binding specificity of the HFH proteins. The study of protein-DNA interactions is not only important for understanding transcription and gene regulation, but is also critical for molecular modeling and protein design.