CONTROL MECHANISM OF HEMOPROTEIN REACTIVITY

这项拟议的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， 可逆结合O2的蛋白质，作为功能原型。在 拟议的研究，位于氧气中的特定氨基酸的突变 结合血红素腔，如His64、Val68、Leu29、Phe43、Phe46、Leu89和 Ser92，将通过定点突变获得，突变的肌红蛋白 将在大肠杆菌中表达的蛋白质用于结构和功能 学习。血红素腔中的氨基酸替代调节(At) 最不重要的三个因素：空间拥挤、氢键和 地方性极性。我们将用这些血红素口袋准备肌红蛋白突变体 氨基酸被大小相近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 我们准备将被检查的电子顺磁共振，核子 磁共振，X射线结晶学，拉曼散射，电子- 核双共振、X-射线光谱和配体的动力学 有约束力的。为了做到这一点，我们正在结合研究的资源和技能 凯斯西部储备大学、赖斯大学、康奈尔大学、康奈尔大学的小组。的 加州大学戴维斯分校罗马，西北大学，普林斯顿大学。和 斯坦福同步辐射实验室。为了有效地利用 蛋白质工程与先进光谱学的融合力量 技巧。血红素口袋的结构将按顺序勾勒出来 确定血红素口袋氨基酸替代对(I)的影响 结合配体的几何构型，(Ii)配体结合的动力学， 以及(Iii)与血红素铁的配基可及性。拟议的研究将 进一步推进首席研究员目前突变的肌红蛋白 研究并将提供对功能的结构性解释 由突变引起的改变。因此，有关职能角色的信息 血红素口袋氨基酸在控制血红素铁反应性中的作用 就会得到肌红蛋白。在本申请中提出的研究 将有助于确定结构与功能的关系 一般的血色素蛋白，并将提供必要的重要信息 生物医用人工血球蛋白的最终制备 重要的是，例如以血红蛋白为基础的血液替代品。

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.