NMR STUDY OF HFH PROTEINS

肝细胞核因子3(HNF-3)和果蝇叉头蛋白(FKH)同源物组成了一个蛋白质大家族，并利用一个有翼的螺旋基序来识别它们的同源DNA位点。该家族成员广泛存在于多种组织中，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)上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。以往的数据表明，H3之前的氨基酸序列可以在不同的HNF-3/FKH家族成员之间采用替代构象，这些替代构象调节H3的呈现以及DNA结合的特异性。然而，这些不同的构象如何改变不同的HNF-3/FKH家族成员之间的DNA接触模式还不完全清楚。因此，将结合分子生物学和现代核磁共振技术来研究这一问题。HNF-3/FKH同源物是高度动态的DNA结合蛋白。然而，它们的动态性质如何与它们的DNA结合过程相关尚不清楚。因此，有必要系统地研究不同HNF-3/FKH蛋白及其DNA复合体之间的内部运动。松弛核磁共振技术将用于这项研究。以往的研究结果表明，HNF-3/FKH家族成员的大多数DNA接触是在双链AT富含序列的一条链上。因此，一个问题是，这种独特的DNA接触模式是否是大自然保持高度保守的主DNA接触螺旋的另一个原因？我们将结合分子生物学和核磁共振技术来研究HNF3/FKH分子中DNA接触残基与结合专一性之间的关系。本研究获得的信息将有助于我们理解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.