LINK PROTEIN DOMAIN STRUCTURE BY NMR

软骨蛋白多糖聚集体主要由以下复合体组成 维持和稳定的透明质酸和蛋白多糖 即所谓的链接蛋白(Link Protein，LP)。Lp包含几个不同的泛函 特定结合蛋白质和透明质酸的结构域。有两个 相邻的重复透明质酸结合结构域(通常 在LP中称为蛋白多糖串联重复序列，或PTR结构域)。虽然 祖先PTR结构域(TSG-6)的溶液结构已经被 据报道，其与HA和金属离子相互作用的细节如下 在很大程度上没有特征。Lp PTR结构域的结构尚未确定 但它们与羟基磷灰石和金属离子的相互作用还不完全 特色化的。因此，该计划的长期目标是 从牛(和人)链中确定两个PTR结构域的结构 蛋白。预计除了一个三维的 溶液结构，关于金属构象效应信息 将获得离子和透明质酸的结合，并使蛋白质 将确定涉及的残留物。 这个可行性项目的目标是表达为重组 大肠杆菌和昆虫细胞表达系统中的蛋白质，大约 100个残基PTR结构域用于配体结合和核磁共振结构分析。 具体地说，我们将：1)表达重组牛PTR-1、PTR-2和 加入PTR1-PTR2结构域并优化其(重新)折叠；2)表征 并量化它们的金属和HA结合性能；以及3)获得 优化核磁共振溶液条件，开始详细的核磁共振结构 高场多维质子核磁共振波谱分析。首字母 试图定位这些蛋白质的HA和金属离子结合部位 也将被制作成。 这些研究最终将导致对分子/ 透明质酸-PTR结构域相互作用的原子细节 对合理设计疗法很有用，最终可能 增强软骨的动态平衡。这项工作的结果也将是 直接适用于琼胶凝集素类似PTR结构域的研究 与连接蛋白形成三元复合体的通用型分子 和HA。

The cartilage proteoglycan aggregate is largely composed of complexes of hyaluronic acid and proteoglycans which are maintained and stabilized by the so called Link protein (LP). LP contains several distinct functional domains that specifically bind protein and hyaluronic acid. There are two adjacent tandemly repeated hyaluronic acid binding domains (commonly called the proteoglycan tandem repeats, or PTR, domains) in LP. Although the solution structure of an ancestral PTR domain (TSG-6) has been reported, the details of its interaction with HA and metal ions are largely uncharacterized. No structure of the LP PTR domains have been reported and their interactions with HA and metal ions are not fully characterized. The long range goal of this program is, therefore, to determine the structures of both PTR domains from bovine (and human) link protein. It is anticipated that in addition to a three dimensional solution structure, that information on the conformation effects of metal ion and hyaluronic acid binding will be obtained and that the protein residues involved will be identified. The goals of this feasibility project are to express, as recombinant proteins in E. coli and insect cell expression system, the approximately 100 residue PTR domains for ligand binding and NMR structural analysis. Specifically we will: 1) express recombinant bovine PTR-1, PTR-2 and the joined PTR1-PTR2 domains and optimize their (re)folding; 2) characterize and quantitate their metal and HA binding properties; and 3) obtain optimal NMR solution conditions and begin the detailed NMR structural analysis by high field multidimensional proton NMR spectroscopy. Initial attempts to locate the HA and metal ion binding sites of these proteins will also be made. These studies will eventually lead to the understanding of the molecular/ atomic details of the hyaluronic acid-PTR domain interactions which may be useful for the rational design of therapeutics that could ultimately enhance cartilage homeostasis. The results of this work will also be directly applicable to the study of similar PTR domains in the aggrecan and versican molecules which form a ternary complex with the link protein and HA.