STRUCTURE OF PLASMA SEX STEROID-BINDING PROTEIN

这项研究的目的是了解人类免疫缺陷的分子基础 类固醇-蛋白质相互作用--类固醇的生化过程 激素作用和类固醇代谢。性类固醇结合蛋白， 选择人(HSBP)和兔(RSBP)血浆中的SBP作为蛋白质 实现这一目标的模型。SBP是唯一的非催化性 包括人类在内的大多数物种血浆中的类固醇结合蛋白， 它特异性地结合睾酮(T)、双氢睾酮(DHT)，以及 17β-雌二醇(E_2)，具有高亲和力。由于兔SBP只结合了 雄激素，这两种蛋白质将被分离并进行比较，以揭示 负责区分雄激素和雄激素的分子决定因素 雌激素。化学方法、蛋白质化学方法、分子方法 生物学，并用X射线衍射法完成生化 这两种蛋白质的特性，包括它们的类固醇结合部位 和三维结构的求解。这个项目是由 三个具体目标：(1)具体目标1是完成 物理化学表征和承接解决三个问题- 空间结构。(2)具体目标2是确定类固醇的特征- 亲和标记结合位点和定点突变 确定在类固醇结合中起作用的氨基酸侧链 进程。(3)特异目的3：在细菌和昆虫中表达SBP 用于生产大量脱糖重组SBP的细胞 结晶试验和结构分析。最近发现， 脱糖SBP是完全活性的，更容易结晶。 这项研究的长期目标是尽可能详细地描述 可能，非催化类固醇结合蛋白的结构在 以了解类固醇-蛋白质相互作用的分子基础。 在这些研究中获得的结构信息应适用于 其他类固醇结合蛋白，因为它们都识别相同的碱性 类固醇核。所获得的结果应该会刺激对 设计合成类固醇或其他可与天然药物竞争的药物 荷尔蒙，从而提供了开发治疗药物的机会 激素依赖型疾病的治疗方法。此外， 这一结果将对最近发现的 SBP受体与性的特异性摄取有关 类固醇激素进入靶细胞。

The purpose of this research is to understand the molecular basis of steroid-protein interaction, a biochemical process functioning in steroid hormone action and steroid metabolism. The Sex Steroid-Binding Protein, SBP, of human (hSBP) and rabbit (rSBP) plasma have been chosen as protein models to accomplish this objective. SBP is the sole noncatalytic sex steroid-binding protein in the plasma of most species, including humans, which specifically binds testosterone (T), dihydrotestosterone (DHT), and 17beta-estradiol (E2) with high affinity. Since rabbit SBP only binds the androgens, both proteins will be isolated and compared to reveal the molecular determinants responsible for distinguishing androgens from estrogens. The methods of chemistry, protein chemistry, molecular biology, and X-ray diffraction will be used to complete the biochemical characterization of both proteins, including their steroid-binding sites and solution of the three-dimensional structure. The project is composed of three specific aims: (1) Specific Aim 1 is to complete the physicochemical characterization and undertake solution of the three- dimensional structure. (2) Specific Aim 2 is to characterize the steroid- binding site by affinity labeling and site-directed mutagenesis to identify amino acid side-chains that function in the steroid-binding process. (3) Specific Aim 3 is to express SBP in bacteria and insect cells to produce large amounts of deglycosylated recombinant SBP for crystallization trials and structure analysis. It was recently found that deglycosylated SBP is fully-active and is easier to crystallize. The long-term goal of this research is to describe, in as much detail as possible, the structure of a noncatalytic steroid-binding protein in order to understand the molecular basis of steroid-protein interaction. The structural information obtained in these studies should apply to other steroid-binding proteins since they all recognize the same basic steroid nucleus. The results obtained should stimulate research on the design of synthetic steroids or other drugs that can compete with natural hormones and thus could provide opportunities for developing therapeutic approaches in the treatment of steroid-dependent diseases. Furthermore, the results will have an important impact on the recent discovery of the SBP receptor which has been implicated in the specific uptake of sex steroid hormones into target cells.