LINK PROTEIN DOMAIN STRUCTURE BY NMR

软骨蛋白聚糖聚集体主要由以下复合物组成： 透明质酸和蛋白聚糖，其通过 所谓的链接蛋白（LP）。LP包含几个不同的功能 特异性结合蛋白质和透明质酸的结构域。有两 相邻的串联重复的透明质酸结合结构域（通常 称为蛋白聚糖串联重复序列或PTR结构域）。虽然 祖先PTR结构域（TSG-6）溶液结构已被 报道，其与HA和金属离子的相互作用的细节是 基本上没有特征。尚未确定LP PTR结构域的结构。 报道，它们与HA和金属离子的相互作用并不完全 表征了因此，该计划的长期目标是 确定牛（和人）连接的两个PTR结构域的结构 蛋白预计除了三维的 溶液结构，金属构象效应信息 离子和透明质酸结合将获得， 将确定所涉及的残留物。 这个可行性项目的目标是表达，作为重组 E.大肠杆菌和昆虫细胞表达系统， 用于配体结合和NMR结构分析的100个残基PTR结构域。 具体而言，我们将：1）表达重组牛PTR-1、PTR-2和重组牛PTR-1基因。 连接的PTR 1-PTR 2结构域并优化它们的（再）折叠; 2）表征 并定量它们的金属和HA结合特性;以及3）获得 最佳NMR溶液条件，并开始详细的NMR结构 通过高场多维质子NMR光谱分析。初始 试图定位这些蛋白质的HA和金属离子结合位点 也将作出。 这些研究将最终导致对分子/ 透明质酸-PTR结构域相互作用的原子细节， 这对于合理设计治疗方法是有用的， 增强软骨内环境稳定。这项工作的结果也将是 直接适用于研究聚集蛋白聚糖中类似的PTR结构域， 和与连接蛋白形成三元复合物的多功能蛋白聚糖分子 哈哈。

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.