NMR STUDY OF HFH PROTEINS

我们的长期目标是了解在这个过程中蛋白质-DNA的识别 转录调控。转录因子在许多方面发挥着关键作用 通过调节细胞特异性基因表达的重要过程。这个 转录因子的功能依赖于对靶标的正确识别 启动子和增强子区域的DNA位点。尽管密集的研究 虽然已经对它们的相互作用进行了研究，但仍然存在许多问题。其中之一 这就是保守的DNA结合基序如何介导对分歧的识别 DNA站点。肝细胞核因子-3与果蝇叉头 (FKH)相关蛋白(HFH)构成了一个蛋白质大家族并具有广泛的用途 一种被称为“有翼螺旋”基序的修饰的螺旋旋转螺旋来识别 他们的目标DNA位点/虽然，hfh蛋白几乎不变 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 保守的氨基酸和DNA碱基。根据生化数据，一项 假设HNF-3/FKH的DNA结合特异性 DNA识别的不同呈现方式是同源基因的中介 螺旋。为了详细检验这一假设，我们建议应用两种方法 分子生物学和现代异核核磁共振方法研究 HfH蛋白的结构及其与DNA结合的特异性。我们会 确定并比较HfH-1的DNA结合域的结构。 我们试图了解20个氨基酸序列对 识别序列的结构化表示。我们将比较 HfH-1和HfH-1的DNA复合体的结构和研究 HFH蛋白中相同的氨基酸残基与DNA相互作用。我们会 还研究了HFH蛋白及其DNA的动力学性质 复合体。我们对DNA接触残留物和 C-末端的动态翅膀序列。我们将研究这些措施的效果 旨在改变DNA上识别螺旋呈现方式的突变 结合专一性。最初的目标是一个20个氨基酸的区域 邻近识别序列，影响DNA结合 HFH蛋白的特异性。蛋白质-DNA相互作用的研究是 不仅对理解转录和基因调控很重要，而且 对于分子建模和蛋白质设计也是至关重要的。

Our long term goal is to understand protein-DNA recognition in the process of transcription regulation. Transcription factors play key roles in many important processes by regulating cell-specific gene expression. The functions of transcription factors depend on correct recognition of target DNA sites in promoter 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 Drosophila forkhead (fkh) related proteins (HFH) constitute a large family of proteins and use a modified helix turn helix 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 HNF-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 proteins. We will determine and compare the structures of the DNA binding domains of HFH-1. 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-1 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 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 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.