CONTROL MECHANISM OF HEMOPROTEIN REACTIVITY

这项研究的目的是阐明 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 提出的研究，突变的特定氨基酸位于氧 结合血红素腔，如His 64、Val 68、Leu 29、Phe 43、Phe 46、Leu 89和 Ser 92，将通过定点诱变获得，并且突变肌红蛋白 蛋白质将在E.大肠杆菌的结构和功能 问题研究血红素腔中的氨基酸替换调节（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 氨基酸被其他大小相似但不同的残基取代 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，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 约束力为此，我们正在结合研究资源和技能 凯斯西储大学的研究小组，莱斯大学，康奈尔大学，Univ. Of 加州，戴维斯，罗马大学，西北大学，普林斯顿大学和 斯坦福大学同步辐射实验室。为了有效利用 将蛋白质工程与先进的光谱技术相结合的力量 技术.血红素口袋的结构将按顺序描绘 以确定血红素口袋氨基酸取代对（i） 结合配体的几何形状，（ii）配体结合的动力学， 和（iii）配体对血红素铁的可及性。拟定的研究将 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 由突变引起的改变。因此，关于功能角色的信息 血红素口袋氨基酸在控制血红素铁的反应性 将获得肌红蛋白。本申请中提出的研究 将有助于确定结构-功能关系， 血蛋白，并将提供必要的重要信息， 生物医用人工血红素蛋白的最终制备 重要性，如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.