STUDIES OF HEMOGLOBIN S USING A RECOMBINANT DNA STRATEGY

本研究的目的是进一步探讨心力衰竭的发病机制。 生化、生物物理和生物化学方法研究脱氧血红蛋白S的聚合 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。我们还将 试图定义人类胚胎Hb(高尔-Hb 2，α2 epsilon 2) 能抑制Hb S的聚合。这项研究的长期目标是 为了了解HBS的聚合机理，并生成 有助于开发抗病毒剂的基本知识 在未来。在之前的资助期间，我们的实验室已经做出了 在进一步了解血管紧张素转换酶的机制方面取得了很大进展 在分子水平上利用生化、生物物理和 重组DNA技术。我们已经成功地描述了各种不同的 带有Beta6、Beta7、Beta85、Beta87和 S在Hb中的β88位，以及Hb F和Delta6中的Gamma6和Gamma87 利用酵母表达系统获得Hb A/2的Delta87。我们还有 成功地在细菌中表达了单一的可溶人β链。 这些系统与基于聚合酶链式反应的突变相结合将有助于 生产足够量的血红蛋白，以满足文献中建议的研究 这笔赠款。本申请的具体目的如下：(1) 进一步了解β6-Val在Hb中的强疏水相互作用 S聚合物我们将进一步阐明Phe、Beta85和Leu的关键作用 接受者口袋里的贝塔88。我们将定义外汇基金的结构 各种重组Hb S变异体的受体口袋 酵母β-85和β-88位基因突变的X射线分析 水晶。我们将研究在Beta85上氨基酸替代的影响 用电子显微镜观察聚合物结构上的β-88位。我们 将进一步研究不同氨基酸替代品的影响 疏水相互作用的受体和供体位置 不同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 在接受者的口袋里。(2)我们将建立方法来论证 使用各种物理方法在聚合前存在核 方法：研究方法。我们还将在HBS之前表征原子核的形成 聚合反应。蛋白质-蛋白质相互作用与蛋白质之间的关系 还将研究原子核的形成。(3)我们还将评估反 胚胎Hb表型珠蛋白镰刀样特性的比较 Hb F的伽马珠蛋白链，基于高极性赖氨酸的存在 而不是β87 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(α2β2)。 我们测试epsilon-珠蛋白抗病特性的基本原理是 在转基因小鼠中，该基因的转录控制是自主的；以及 这种基因在成人生活中的表达可能更容易实现 与胎儿珠蛋白基因相比。这些研究将进一步提供 镰状细胞病的分子病理学基础知识和 有可能为制定战略奠定基础 未来抑制脱氧血红蛋白S聚合的研究。

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.