STRUCTURE OF PLASMA SEX STEROID-BINDING PROTEIN

血浆性类固醇结合蛋白的结构

• 批准号: 2197051
• 负责人: PHILIP H PETRA
• 金额: $ 13.34万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-04-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2197051
• 关键词: X ray crystallography; affinity labeling; chemical synthesis; chimeric proteins; conformation; crystallization; dihydrotestosterone; dimer; enzyme linked immunosorbent assay; estradiol; globulins; glycosylation; hormone binding protein; molecular cloning; mutant; protein engineering; protein purification; protein sequence; protein structure function; sex hormones; site directed mutagenesis; solutions; species difference; structural biology; testosterone

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.

这项研究的目的是了解 类固醇-蛋白质相互作用，在类固醇中起作用生化过程 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）血浆的SBP作为蛋白质 模式来实现这一目标。 SBP是唯一的非催化性 大多数物种，包括人类， 其特异性结合睾酮（T）、双氢睾酮（DHT）和 17 β-雌二醇（E2）具有高亲和力。由于兔SBP仅结合 雄激素，这两种蛋白质将被分离和比较，以揭示 负责区分雄激素和 雌激素类。化学方法、蛋白质化学、分子生物学 生物学和X射线衍射将用于完成生物化学 两种蛋白质的表征，包括其类固醇结合位点 和三维结构的解决方案。该项目由 三个具体目标：（1）具体目标1是完成 物理化学表征和进行解决的三个- 空间结构(2)具体目标2是表征类固醇- 通过亲和标记和定点诱变结合位点， 鉴定在类固醇结合中起作用的氨基酸侧链 过程(3)具体目标3是在细菌和昆虫中表达SBP 细胞产生大量的去糖基化重组SBP， 结晶试验和结构分析。最近发现， 去糖基化的SBP是完全活性的，并且更容易结晶。 这项研究的长期目标是详细描述 可能的，非催化类固醇结合蛋白的结构， 以了解类固醇-蛋白质相互作用的分子基础。 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基， 类固醇核。所取得的成果应能促进对 合成类固醇或其他药物的设计，可以与自然竞争， 激素，因此可以提供机会， 类固醇依赖性疾病的治疗方法。此外，委员会认为， 这些结果将对最近发现的 收缩压受体，这已牵连在特定的吸收性 类固醇激素进入靶细胞。