STUDIES OF HEMOGLOBIN S USING A RECOMBINANT DNA STRATEGY

本研究的目的是进一步探讨 使用生物化学、生物物理学和生物化学方法， 重组DNA技术。我们还将尝试定义人类是否 生物化学、生物物理和重组DNA技术。我们还将 试图确定人胚胎Hb（Gower-Hb 2，α 2 ε 2） 能抑制Hb S聚合。这项研究的长期目标是 了解HbS的聚合机制，并产生 有助于开发抗镰状化药物的基本知识 在未来在上一个资助期内，我们的实验室取得了 在进一步了解其机制方面取得了相当大的进展 聚合在分子水平上使用生物化学，生物物理和 重组DNA技术。我们已经成功地描述了各种 在β 6、β 7、β 85、β 87和 Hb S中的β 88位置以及Hb F和δ 6中的γ 6和γ 87位置 和HbA/2的δ 87。我们还 成功地在细菌中表达单个可溶性人β链。 这些系统与基于PCR的诱变相结合将促进 产生足够量的血红蛋白用于 这个grant。本申请的具体目的如下：（1） 进一步了解血红蛋白中β 6瓦尔的强疏水相互作用 S聚合物，我们将进一步阐明Phe beta85和Leu的关键作用 受体口袋里的beta88我们将定义EF的结构 各种重组Hb S变体的受体口袋， 通过X射线分析在酵母中β 85和β 88位置的取代， 晶体我们将研究β 85位氨基酸置换的效果， 和β 88位置上的聚合物结构。我们 将进一步研究各种氨基酸替代的效果， 疏水相互作用的受体和供体位点 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 β 85和Leu β 88的关键作用， 在受体口袋里。(2)我们将建立方法来证明 在聚合之前使用各种物理方法 方法.我们还将表征在HbS之前的核形成 聚合法蛋白质-蛋白质相互作用与 还将研究成核。(3)我们还将评估抗- 胚胎Hb的ε-珠蛋白的镰状化特性与 Hb F的γ-珠蛋白链，基于高极性赖氨酸的存在， ε 87而不是β 87 Thr或γ 87 Gln。我们将表达人类 胚胎血红蛋白（α 2 ε 2）。 我们测试ε-球蛋白抗镰状化特性的理由是 该基因的转录控制在转基因小鼠中是自主的;并且 这种基因在成年期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 镰状细胞病分子病理学的基本知识， 为制定战略奠定基础的可能性， 抑制脱氧Hb S聚合。

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.