NMR STUDY OF HFH PROTEINS

肝细胞核因子3（hepatocyte nuclear factor 3，HNF-3）和果蝇叉头蛋白（Drosophila forkhead，fkh）同源物组成了一个蛋白质大家族，它们利用翅状螺旋基序识别同源DNA位点。该家族成员存在于广泛的组织中，并在细胞类型特异性基因表达中发挥重要作用。有趣的是，尽管HNF-3/fkh同源物在其主要DNA识别螺旋（H3）中具有几乎不变的氨基酸序列，但它们表现出不同的DNA结合特异性。先前的数据已经证明，紧接在H3之前的氨基酸序列可以在不同的HNF-3/fkh家族成员中采用替代构象，并且这些替代构象调节H3的呈递以及DNA结合特异性。然而，这些替代构象如何改变不同HNF-3/fkh家族成员之间的DNA接触模式尚未完全了解。因此，结合分子生物学和现代核磁共振技术将被应用于研究这个问题。HNF-3/fkh同源物是高度动态的DNA结合蛋白。然而，它们的动力学性质如何与它们的DNA结合过程相关尚不清楚。因此，不同的HNF-3/fkh蛋白和它们的DNA复合物之间的内部运动的系统调查是必要的。本研究将使用弛豫NMR技术。先前的结果表明，HNF-3/fkh家族成员产生的大多数DNA接触位于双链AT富集序列的一条链上。因此，一个问题是，这种独特的DNA接触模式是否是自然界保持高度保守的主要DNA接触螺旋的另一个原因？结合分子生物学和核磁共振技术将被用来研究DNA接触残基和HNF-3/fkh成员的结合特异性之间的关系。本研究所获得的信息将有助于我们了解HNF-3/fkh蛋白在组织特异性基因调控和发育调控中的作用。

The hepatocyte nuclear factor 3 (HNF-3) and Drosophila forkhead (fkh) homologues constitute a large family of proteins and utilize a winged helix" motif to recognize their cognate DNA sites. The family members are present in a wide range of tissues and ply important roles in cell- type-specific gene expression. Interestingly, although the HNF-3/fkh homologues have almost invariable amino acid sequences in their principal DNA recognition helix (H3), they exhibit divergent DNA binding specificity. Previous data have demonstrated that the amino acid sequence immediately prior to H3 can adopt alternative conformations among different HNF-3/fkh family members, and that these alternative conformations regulate the presentation of H3 as well as DNA binding specificity. However, how these alternative conformations can change DNA contact patterns among different HNF-3/fkh family members are not fully understood. Thus, a combination of molecular biology and modern NMR techniques will be applied to investigate this question. HNF-3/fkh homologues are highly dynamic DNA binding proteins. However how their dynamic properties are related to their DNA binding process is not clear. Thus, a systematic investigation of internal motions among different HNF-3/fkh proteins and their DNA complexes are necessary. Relaxation NMR techniques will be used for this study. Previous results indicated that most DNA contacts made by an HNF-3/fkh family member are on one strand of a double stranded AT rich sequence. Thus, a question is whether this unique DNA contact mode is another reason for nature to keep the highly conserved principal DNA contact helix? A combination of molecular biology and NMR techniques will be used to study the relationship between the DNA contact residues and binding specificity in HNF-3/fkh members. The information obtained from this research will help us to understand the roles of the HNF-3/fkh proteins in tissue specific gene regulation and developmental regulation.