STUDIES OF HEMOGLOBIN S USING A RECOMBINANT DNA STRATEGY

使用重组 DNA 策略研究血红蛋白 S

• 批准号: 6325933
• 负责人: KAZUHIKO ADACHI
• 金额: $ 17.18万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6325933
• 关键词: Escherichia coli; Saccharomyces cerevisiae; X ray crystallography; circular dichroism; conformation; electron microscopy; genetically modified animals; hemoglobin As; hemoglobin F; hemoglobin Ss; hemoprotein structure; hydropathy; intermolecular interaction; laboratory mouse; nuclear magnetic resonance spectroscopy; oxygen tension; polymerization; protein sequence; protein structure function; recombinant DNA; recombinant proteins; sickle cell anemia; site directed mutagenesis; thermodynamics; video microscopy

The objective of this research is to further investigate the mechanisms of polymerization of deoxy-Hb S using biochemical, biophysical and recombinant DNA technologies. We will also attempt to define whether human biochemical, biophysical and recombinant DNA technologies. We will also attempt to define whether human embryonic Hb (Gower-Hb 2, alpha2 epsilon2) can inhibit Hb S polymerization. The long-range goals of this research are to understand the mechanism of polymerization of HbS and to generate the basic knowledge that will aid in the development of anti-sickling agents in the future. In the previous funding period our laboratory has made considerable progress in further understanding the mechanism of polymerization on the molecular level using biochemical, biophysical and recombinant DNA technologies. We have succeeded in characterizing various recombinant Hbs with substitutions at the beta6, beta7, beta85, beta87 and beta88 positions in Hb S as well as gamma6 and gamma87 in Hb F and delta6 and delta87 of Hb A/2 using a yeast expression system. We have also succeeded in expressing single soluble human beta chains in bacteria. These systems coupled with PCR-based mutagenesis will facilitate production of sufficient amounts of hemoglobin for the studies proposed in this grant. Specific Aims of this application are as follows: (1) To further understand the strong hydrophobic interaction of beta6 Val in Hb S polymers we will further clarify the critical role of Phe beta85 and Leu beta88 in the acceptor pocket. We will define the structure of the EF acceptor pocket of various recombinant Hb S variants containing substitutions at beta85 and beta88 positions in yeast by X-ray analysis of crystals. We will study the effect of amino acid replacement at the beta85 and beta88 positions on polymer structure using electron microscopy. We will further study the effects of the various amino acid replacements at the acceptor and donor sites for hydrophobic interactions on polymerization and polymer structure under various experimental conditions in order to further clarify the critical role of Phe beta85 and Leu beta88 in the acceptor pocket. (2) We will establish methods to demonstrate the presence of nuclei prior to polymerization using a variety of physical methods. We will also characterize formation of nuclei prior to HbS polymerization. The relationship between protein-protein interactions and nuclei formation will also be studied. (3) We will also evaluate anti- sickling properties of epsilon-globin of embryonic Hb compared to the gamma-globin chain of Hb F, based on the presence of a highly polar Lys at epsilon87 instead of beta87 Thr or gamma87 Gln. We will express human embryonic hemoglobin (alpha2 epsilon2) using a yeast expression system. Our rationale for testing epsilon-globin anti-sickling properties is that transcriptional control of this gene is autonomous in transgenic mice; and there expression of this gene in adult life may be much easier to realize compared to the fetal globin genes. These studies will further provide the basic knowledge of the molecular pathology of sickle cell disease and possibility to generate the basis to formulate strategies aimed at inhibition of polymerization of deoxy Hb S in future.

这项研究的目的是进一步研究 使用生化，生物物理和 重组DNA技术。我们还将尝试定义人类是否 生化，生物物理和重组DNA技术。我们也会 尝试定义人类胚胎HB（Gower-HB 2，alpha2 epsilon2）是否存在 可以抑制HB S聚合。这项研究的远程目标是 了解HBS聚合的机制并产生 基本知识将有助于开发反校准代理商 将来。在上一个资金期间，我们的实验室已经做出了 在进一步理解的机制方面取得了长足的进步 使用生化，生物物理和 重组DNA技术。我们已经成功地描述了各种 重组HBS在Beta6，Beta7，Beta85，Beta87和 HB S中的beta88位置以及HB F和Delta6中的γ6和γ87 使用酵母表达系统的HB A/2的Delta87。我们也有 成功地表达了细菌中的单一可溶性人β链。 这些系统加上基于PCR的诱变将有助于 生产足够数量的血红蛋白用于提出的研究 这笔赠款。此应用程序的具体目的如下：（1） 进一步了解HB中β6阀的疏水相互作用 S聚合物我们将进一步阐明PHE Beta85和Leu的关键作用 beta88在受体口袋中。我们将定义EF的结构 包含各种重组HB S变体的受体口袋 通过X射线分析，Beta85和Beta88的替代位置 晶体。我们将研究氨基酸在beta85上的效果 使用电子显微镜在聚合物结构上的β88位置。我们 将进一步研究在 疏水相互作用的受体和供体站点 在各种实验条件下的聚合和聚合物结构 为了进一步阐明PHE BETA85和LEU BETA88的关键作用 在受体口袋里。 （2）我们将建立演示的方法 在聚合之前使用各种物理化的核存在核 方法。我们还将表征HBS之前的核形成 聚合。蛋白质 - 蛋白质相互作用与 还将研究细胞核的形成。 （3）我们还将评估 与 HB F的γ-球蛋白链，基于高度极性LYS的存在 epsilon87而不是beta87 thr或gamma87 gln。我们将表达人类 使用酵母表达系统的胚胎血红蛋白（alpha2 epsilon2）。 我们测试epsilon-Globin抗踢式特性的理由是 该基因的转录控制在转基因小鼠中是自主的。和 在成人生活中，这种基因表达可能会更容易实现 与胎儿球蛋白基因相比。这些研究将进一步提供 镰状细胞疾病的分子病理学的基础知识和 产生基础以制定针对策略的基础的可能性 将来抑制脱氧Hb的聚合。