CONTROL MECHANISM OF HEMOPROTEIN REACTIVITY

血蛋白反应性的控制机制

• 批准号: 2190221
• 负责人: MASAO IKEDA-SAITO
• 金额: $ 19.38万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2190221
• 关键词: Raman spectrometry; X ray crystallography; X ray spectrometry; aminoacid; chemical binding; chemical reaction; chemical substitution; electron spin resonance spectroscopy; heme; hydrogen bond; ligands; myoglobin; nuclear magnetic resonance spectroscopy; nucleic acid sequence; protein structure function; site directed mutagenesis

The objective of the proposed research is to elucidate the molecular mechanism which controls reactivity of hemoproteins. We use myoglobin, the protein which reversibly binds O2, as a functional prototype. In the proposed research, mutations of specific amino acids located in the oxygen binding heme cavity, such as His64, Val68, Leu29, Phe43, Phe46, Leu89 and Ser92, will be attained by site-directed mutagenesis, and mutant myoglobin proteins will be expressed in E. coli for the structural and functional studies. The amino acid replacements in the heme cavity modulate (at least) three important factors: steric crowding, hydrogen bonding, and local polarity. We will prepare myoglobin mutants with these heme pocket amino acids replaced by other residues with similar size but different polarity, or those with different size but similar polarity. Each mutant we prepare will be examined by electron paramagnetic resonance, nuclear magnetic resonance, X-ray crystallography, Raman scattering, electron- nuclear double resonance, X-ray spectroscopy, and kinetics of ligand binding. To do this we are combining resources and skills of the research groups at Case Western Reserve Univ., Rice Univ., Cornell Univ., Univ. of California, Davis, Univ. of Rome, Northwestern Univ., Princeton Univ. and Stanford Synchrotron Radiation Lab. for an efficient utilization of the power of merging protein engineering with advanced spectroscopic techniques. The structure of the heme pocket will be delineated in order to determine the effects of heme pocket amino acid substitutions upon (i) the geometry of the bound ligands, (ii) the dynamics of ligand binding, and (iii) ligand accessibility to the heme iron. The proposed study will further advance the principal investigator's current mutant myoglobin research and will provide structural interpretation of functional alterations induced by mutations. Thus, information about functional roles of heme pocket amino acids in controlling the reactivity of the heme iron in myoglobin will be obtained. The research proposed in this application will help to ascertain the structure-function relationships of hemoproteins in general, and will provide vital information necessary for the eventual preparation of artificial hemoproteins of biomedical importance, such as hemoglobin-based blood substitutes.

拟议研究的目的是阐明分子 控制血红素蛋白反应性的机制。我们使用肌红蛋白， 可逆地结合 O2 的蛋白质，作为功能原型。在 拟议的研究，位于氧中的特定氨基酸的突变 结合血红素腔，例如 His64、Val68、Leu29、Phe43、Phe46、Leu89 和 Ser92，将通过定点诱变获得，突变肌红蛋白 蛋白质将在大肠杆菌中表达，用于结构和功能 研究。血红素腔中的氨基酸替代物调节（在 至少）三个重要因素：空间拥挤、氢键和 局部极性。我们将用这些血红素口袋制备肌红蛋白突变体 氨基酸被大小相似但不同的其他残基取代 极性，或大小不同但极性相似的那些。每个突变体 我们准备将通过电子顺磁共振、核磁共振检查 磁共振、X射线晶体学、拉曼散射、电子 核双共振、X射线光谱和配体动力学 绑定。为此，我们正在整合研究资源和技能 凯斯西储大学、莱斯大学、康奈尔大学、大学的小组的 加利福尼亚州戴维斯大学罗马大学、西北大学、普林斯顿大学和 斯坦福同步辐射实验室。为了有效利用 蛋白质工程与先进光谱技术相结合的力量 技术。血红素袋的结构将按顺序描绘 确定血红素口袋氨基酸取代对 (i) 的影响 结合配体的几何形状，(ii) 配体结合的动力学， (iii) 配体对血红素铁的可及性。拟议的研究将 进一步推进首席研究员目前的突变肌红蛋白 研究并将提供功能的结构解释 突变引起的改变。因此，有关功能角色的信息 血红素口袋氨基酸在控制血红素铁反应性中的作用 将获得肌红蛋白。本申请中提出的研究 有助于确定结构与功能的关系 一般血红蛋白，并将提供必要的重要信息 生物医学人工血红蛋白的最终制备 重要性，例如基于血红蛋白的血液替代品。