Structure/Function Studies of Lipid Binding Proteins

这项建议的核心重点是建立一个 生物识别的结构基础。 大分子 两种蛋白质之间的识别是一个组合过程 通过各种各样的集体互动， 蛋白质表面的氨基酸。 我们的研究旨在 解决蛋白质结构的问题， 蛋白质相互作用 两个核心问题将是 在这项研究中解决：1）小分子 配体对蛋白质结构的影响，2）配体上的结构域是什么？ 蛋白质表面对于蛋白质-蛋白质 互动 我们的实验系统围绕着 脂肪细胞酸结合蛋白，其与 脂肪酸及其被胰岛素受体磷酸化。 与载体蛋白结合的脂肪酸允许其 胰岛素受体的酪氨酰磷酸化。 全脂肪酸结合蛋白块的磷酸化 脂肪酸交换和脂质从 结合域 蛋白质的去磷酸化， 酪氨酸磷酸酶允许配体释放，并使 脂肪酸结合蛋白的基础状态。 协会 脂质结合蛋白与胰岛素受体激酶 被认为是通过与酸性和酸性的相互作用而发生的 与酪氨酰磷酸化位点相邻的结构域和 第二个结构域，称为SH 2结构域，假定发挥作用。 在激酶识别中的主要作用。 我们假设 结合分子和结构生物学，我们将定义 这些结构域在蛋白质-蛋白质识别中。 具体 在批准期间，我们建议检查分子 这两种蛋白质的识别： 1. 确定全脂肪细胞脂肪的结构 酸结合蛋白的X射线晶体学。 检查脂肪酸对酪氨酸-19的影响 和SH 2结构域上。 2. 突变ALBP的SH 2结构域。创造一个主导的 S101 E、T103 L和V118 E。突变。 研究SH 2结构域在激酶中的作用 识别. 创建一个Y19 F，显性SH 2突变， 测试AS和激酶抑制剂。 3. 使Y19 F、占优势的SH 2 ALBP与 受体的可溶性激酶结构域和ATP， 形成三元复合物。 绘制分子图谱 两种蛋白质之间的相互作用。 这项研究最重要的方面将是 在分子水平上描述小分子如何 影响蛋白质结构以及如何将其转化为 蛋白质识别的差异。 于某一特定 我们将确定结合脂肪酸如何影响 结合蛋白V以及它如何转化成一种 与胰岛素受体激酶有利的相互作用。 也 我们将能够在分子水平上评估 酸性和SH 2结构域对底物蛋白的贡献 在酪氨酸激酶相互作用中。 从长远来看， 这里获得的信息将使我们能够现场设计特定的 ALBP在脂质代谢中的作用及其 调节是磷酸化。

The central focus of this proposal is the establishment of a structural basis for biological recognition. Macromolecular recognition between two proteins is a combinatorial process bought about by the collective interactions of a variety of amino acids on a protein surface. Our studies are designed to address the issues of protein structure and the basis for protein-protein interaction. Two central issues will be addressed within this study: 1) What effects do small molecule ligands have upon protein structure and, 2) What domains on a protein surface are most critical for protein-protein interaction. Our experimental system revolves around the fatty acid binding protein of adipose cells, its interaction with fatty acid, and its phosphorylation by the insulin receptor. Fatty acid binding to the carrier protein allows for its tyrosyl phosphorylation by the insulin receptor. Phosphorylation of the holo fatty acid binding protein blocks fatty acid exchange and the release of the lipid from the binding domain. Dephosphorylation of the protein by a specific tyrosine phosphatase allows for ligand release and returns the fatty acid binding protein to the basal state. The association of the lipid binding protein with the insulin receptor kinase is thought to occur via interactions with both and acidic domain adjacent to the site of tyrosyl phosphorylation and a second domain, termed the SH2 domain, postulated to play a primary role in kinase recognition. We hypothesize that by a combinant of molecular and structural biology we will define these domains in protein-protein recognition. Specifically during the granting period we propose to examine molecular recognition by these two proteins by: 1. Determine the structure of the holo adipocyte fatty acid binding protein by X-ray crystallography. Examine the effect of the fatty acid on tyrosine-19 and on the SH2 domain. 2. Mutagenize the SH2 domain of ALBP. Create a dominant by generation of S101E, T103L and V118E. mutations. Examine the role of the SH2 domain in kinase recognition. Create a Y19F, dominant SH2 mutation, test as and inhibitor of the kinase. 3. Co-crystalize the Y19F, dominant SH2 ALBP with the soluble kinase domain of the receptor and ATP to form a ternary complex. Map the molecular interaction sites between the two proteins. The most significant aspects of this study will be the description at the molecular level how small molecules will affect protein structure and how that can be translated into differences in protein-protein recognition. For our particular system we will determine how the bound fatty acid influences the binding protein V and how that translates into a more favorable interaction with the insulin receptor kinase. Also we will be able, at the molecular level, to assess the relative contributions of acidic and SH2 domains on substrate proteins in tyrosine kinase interactiolns. In the long-term, the information gained here wil allow us to design specific in-situ tests of the role of the ALBP in lipid metabolism and its regulation be phosphorylation.