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: 6241942
• 负责人: SAUL SURREY
• 金额: $ 12.53万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6241942
• 关键词: Escherichia coli; Saccharomyces cerevisiae; acidity /alkalinity; chemical stability; conformation; electron microscopy; genetically modified animals; hemoglobin As; hemoglobin F; hemoglobin Ss; hemoprotein structure; laboratory mouse; mutant; nucleic acid sequence; oxygen tension; polymerization; protein sequence; protein structure function; recombinant DNA; site directed mutagenesis; solubility

We are using a site-directed mutagenesis approach to define structure/function relationships in an effort to determine parameters affecting polymerization, solubility and stability of Hb S in vitro. We have assembled hemoglobins with Ala, Val, Leu and Trp at the beta6 position employing a bacterial expression system and showed that polymer interactions between the the beta6 position of one tetramer and the U-shaped EF helix region on another are greatly influenced by hydrophobicity and stereospecificity of the amino acid side chain at the beta6 position in deoxyhemoglobin. Strong hydrophobic amino acids, in addition to the shape, size and position of the side chair in hemoglobin lead to enhanced polymerization of tetramers in vitro, and also result in decreased solubility. These results suggest that modified hemoglobins can be engineered with altered biochemical properties and that these can be used as tools for structure/function studies for use in defining parameters affecting polymerization, solubility, oxygen binding and stability of hemoglobin molecules. We have also implemented a recently described expression system in which alpha- and beta-globin chains are coexpressed in yeast (S. cerevisiae) and form soluble tetrameric hemoglobin. The alpha- and beta chains assemble into tetramers by incorporating endogenous heme which results in high level production of soluble tetramers. This system will facilitate production of large amounts of assembled tetramers for a variety of studies proposed in this grant. Specific Aims include the following: (1) We will prepare various Hb mutants at beta22, beta23, beta121, alpha6 and alpha23 to define what role these sites play in facilitating polymerization of Hb S. These studies should help clarify the role that various important tetramer contact sites, other than the beta6 position, play in polymerization, solubility and tetramer stability. Identification of amino acids which accelerate polymerization may also help facilitate production of a transgenic animal model for sickle cell disease. (2) We will study the relationship between oxygen affinity and polymerization by engineering Hb mutants at beta23 and beta121, since some naturally-occurring variants at these positions are known to alter 02 affinity. (3) We will also evaluate the effects of pH on polymerization by preparing Hb substitutions at His beta116 and His beta117 which are involved in a contact site on Hb S polymers. (4) Finally, we will continue studies aimed at understanding the molecular basis for inhibition of Hb S polymerization by Hb F. We will use recombinant Hb F and Hb A modified at the gamma87 and beta87 positions, respectively, to study the inhibitory effects on polymerization of Hb S in vitro.

我们正在使用定点诱变方法来定义 结构/功能关系以确定参数 影响 Hb S 体外的聚合、溶解度和稳定性。 我们 已在 beta6 处与 Ala、Val、Leu 和 Trp 组装血红蛋白 采用细菌表达系统的位置并表明聚合物 一个四聚体的 beta6 位置与 另一个U形EF螺旋区域受以下因素影响很大 氨基酸侧链的疏水性和立体特异性 脱氧血红蛋白中 beta6 的位置。 强疏水性氨基酸， 除了血红蛋白中边椅的形状、大小和位置 导致四聚体在体外的聚合增强，并且还导致 溶解度降低。 这些结果表明修饰的血红蛋白 可以通过改变生化特性进行改造，并且这些可以 用作结构/功能研究的工具，用于定义 影响聚合、溶解度、氧结合和的参数 血红蛋白分子的稳定性。 我们还实现了最近描述的表达系统，其中 α 和 β 珠蛋白链在酵母（酿酒酵母）中共表达 并形成可溶性四聚体血红蛋白。 α链和β链 通过掺入内源血红素组装成四聚体，从而产生 高水平生产可溶性四聚体。 该系统将有利于 生产大量组装的四聚体，用于各种 这笔赠款中提出的研究。 具体目标如下： (1) 我们将准备各种Hb beta22、beta23、beta121、alpha6 和 alpha23 的突变体来定义什么 这些位点在促进 Hb S 聚合中发挥的作用。 研究应该有助于阐明各种重要四聚体的作用 除 beta6 位置外的接触位点在聚合中发挥作用， 溶解度和四聚体稳定性。 氨基酸的鉴定 加速聚合也可能有助于促进生产 镰状细胞病转基因动物模型。 （二）我们将研究 通过改造 Hb 来研究氧亲和力与聚合反应的关系 beta23 和 beta121 的突变体，因为一些天然存在的变体 已知这些位置会改变 02 亲和力。 (3) 我们还将评估 通过在 His 处制备 Hb 取代物来研究 pH 对聚合的影响 beta116 和 His beta117 参与 Hb S 的接触位点 聚合物。 （4）最后，我们将继续研究以了解 Hb F 抑制 Hb S 聚合的分子基础。我们 将使用在 gamma87 和 beta87 处修改的重组 Hb F 和 Hb A 分别研究其抑制作用 Hb S 体外聚合。