NMR STUDY OF HFH PROTEINS

我们的长期目标是了解蛋白质-DNA识别的过程 转录调控。转录因子在许多生物学过程中起着关键作用。 通过调节细胞特异性基因表达的重要过程。的 转录因子的功能依赖于对靶的正确识别 启动子和增强子区域中的DNA位点。虽然密集的研究 虽然已经研究了它们之间的相互作用，但仍然存在许多问题。之一 它们是保守的DNA结合基序如何介导不同的 DNA位点。肝细胞核因子3（HNF-3）与果蝇叉头 (fkh)相关蛋白（HFH）构成蛋白质的大家族， 被称为“有翼螺旋”基序的修饰的螺旋转角螺旋， 它们的靶DNA位点/虽然，HFH蛋白几乎不变 在识别序列中，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HNF-3/fkh的DNA结合特异性 同源物是由不同的DNA识别介导的 螺旋。为了详细检验这一假设，我们建议同时应用 分子生物学和现代核磁共振方法来研究 结构和HFH蛋白的DNA结合特异性。我们将 确定并比较HFH-1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-1的DNA复合物的结构，并研究是否 HFH蛋白中的相同氨基酸残基与DNA相互作用。我们将 研究了HFH蛋白及其DNA的动力学性质 配合物我们对DNA接触残基特别感兴趣， C端的动态翼序列。我们将研究 设计用于改变DNA上识别螺旋呈递的突变 结合特异性最初的目标将是一个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， 对分子建模和蛋白质设计也至关重要。

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.