STRUCTURE/FUNCTION STUDIES OF RETINOL BINDING PROTEINS

维生素A是各种生物过程所必需的。这些研究 在拨款建议的正文中描述的是针对 了解细胞视黄醇结合蛋白在视黄醇中的作用 吸收、新陈代谢和运输。我们的假设是这些 蛋白质不仅仅是被动的储存库，它可以防止非特异性 疏水性维生素进入细胞膜的分配： 蛋白质本身参与蛋白质与蛋白质之间的相互作用 代谢维甲酸的酶，因此调节视黄醇 新陈代谢和功能。通过定点突变和各种 我们将研究细胞视黄醇的以下几个方面 结合蛋白(CRBP)与细胞视黄醇结合蛋白类型 II(CRBP(II))：(1)细胞内维生素A的运输蛋白 将视黄醇输送到代谢酶中。这是一个 需要蛋白质-蛋白质识别。我们将确定 载体蛋白参与了这种识别。(2)配体如何进入 CRBP和CRBP(II)的结合腔。X射线结构是 对这两种蛋白质都有效，显然构象 改变必须伴随着配体的进入和退出。我们描述了旨在 来确定这种构象变化的性质。(3)CRBP 区分视黄醇和视网膜，而CRBP(II)不区分。我们 将决定这种功能差异的结构基础。(4) 水在配基结合中的作用。CRBP和CRBP的结合腔 CRBP(II)除了含有特定结合的水分子外，还含有特定结合的水分子 配位体视黄醇。我们假设有一个功能目的，在 除了明显的结构用途外，还包括水 结合部位的分子。

Vitamin A is required for a variety of biological processes. The studies described within the body of the grant proposal are directed at understanding how cellular retinol binding proteins function in retinol uptake, metabolism, and transport. It is our hypothesis that these proteins are more than just passive reservoirs which prevent non-specific partitioning of the hydrophobic vitamin into cellular membranes: The proteins themselves are involved in protein-protein interactions with the enzymes which metabolize retinoids, and as a consequence regulate retinol metabolism and function. With site-directed mutagenesis and a variety of techniques we will examine the following aspects of cellular retinol binding protein (CRBP) and cellular retinol binding protein type II(CRBP(II)): (1) How the intracellular transport proteins for vitamin A deliver retinol to the metabolizing enzymes. This is a process which requires protein-protein recognition. We will determine the sites on the carrier proteins involved in this recognition. (2) How the ligand enters the binding cavities of CRBP and CRBP(II). The X-ray structures are available for both proteins and it is apparent that a conformational change must accompany ligand entry and exit. We describe experiments aimed at determining the nature of this conformational change. (3) CRBP discriminates between retinol and retinal, whereas CRBP(II) does not. We will determine the structural basis for this functional difference. (4) The role of water in ligand binding. The binding cavities of CRBP and CRBP(II) contain specifically bound water molecules in addition to the ligand retinol. We postulate that there is a functional purpose, in addition to the obvious structural purpose, for the inclusion of water molecules in the binding site.