NOVEL NMR STUDIES OF HEME PROTEINS INVOLVED IN ANEMIA

几类贫血性疾病，包括一些相对 常见的严重溶血性疾病是由于 使高铁血红蛋白在红细胞中的含量过高。 这些 病理状况是两种不平衡的结果， 竞争过程，血红蛋白自氧化的内源性速率 并被细胞色素b5再还原。 这一建议是针对 获得与这些病理学直接相关的基本化学知识 通过使用发散切削刃的聚焦阵列， 顺磁共振新多维异谱NMR 血红素蛋白质，涉及高产率表达的重组方法 系统和新型电化学方法。 小说的异质性 多维核磁共振方法的建议是唯一适合的研究 血红素蛋白质，更重要的是血红素蛋白质的研究 配合物 这些方法最近已被应用于研究 细胞色素b5 -细胞色素c复合物，一个重要的模型， 生物电子转移 我们的目的是扩大这些研究， 细胞色素b5 -肌红蛋白复合物作为模型的研究， 生理学相关的细胞色素b5的溶液结构 血红蛋白复合物 虽然组成蛋白质的结构 （细胞色素b5、血红蛋白和肌红蛋白）是已知的， 复合物抵抗晶体学表征。 现代核磁共振 方法和新的扩展，这些方法提出了这里提供 独特的能力，获得对这些方向的约束， 在生理相关条件下，复合物和溶液中的蛋白质 条件 我们还计划研究血红素蛋白质 控制中点潜力和重组能量。 这些研究 将使更多的定量解释的机制， 加速自氧化或改变自然界中再还原的速率 血红蛋白的突变体 中点电位的初步测量 细胞色素b5的合成已使用薄层细胞制成。 小说 采用表面改性电极的电化学方法应 能够直接测量组分的电子转移速率， 从而可以更深入地了解影响重组因素 能量和电子之间的组件转移率 生理复合体 最后，一个长期目标与 上述具体建议涉及定点药物设计 旨在减轻贫血的影响。 一旦我们获得足够的 关于控制中点电位的蛋白质因子的信息， 自动氧化和再还原的速率，我们希望设计新的 可以稳定突变的血红蛋白或改变血红蛋白的 细胞色素b5的中点电位，以促进 高铁血红蛋白

Several classes of anemic disorders including a number of relatively common, serious hemolytic conditions are the result of the accumulation of methemoglobin to anomalously high levels in red blood cells. These pathological conditions are the result of an imbalance between two competing processes, the endogenous rate of autoxidation of hemoglobin and its re-reduction by cytochrome b5. This proposal is directed at obtaining basic chemical knowledge directly related to these pathological conditions through the use of a focused array of divergent cutting edge technologies; novel multidimensional heteronuclear NMR of paramagnetic heme-proteins, recombinant methods involving high yield expression systems and novel electrochemical methods. The novel heteronuclear multidimensional NMR methods proposed are uniquely suited to the study of heme-proteins and more importantly to the study of heme-protein complexes. These methods have recently been applied to the study of the cytochrome b5 - cytochrome c complex, an important model for long-range biological electron transfer. It is our intent to extend these studies to the study of the cytochrome b5 - myoglobin complex as a model for the solution structure of the physiologically relevant cytochrome b5 - hemoglobin complex. Although the structures of the component proteins (cytochrome b5, hemoglobin and myoglobin) are known, the structure of the complex has resisted crystallographic characterization. Modern NMR methods and novel extensions of those methods proposed here offer the unique capability of obtaining constraints on the orientation of these proteins in complex and in solution under physiologically relevant conditions. We also plan to study the mechanism by which heme-proteins control midpoint potential and reorganizational energies. These studies will then enable more quantitative interpretation of the mechanism of accelerated autoxidation or altered rates of re-reduction in natural mutants of hemoglobin. Preliminary measurements of midpoint potentials of cytochrome b5 have been made using thin layer cells. Novel electrochemical methods employing surface modified electrodes should enable direct measurement of electron transfer rates of components and hence allow greater insights into factors effecting reorganizational energy and rates of electron transfer between components of the physiological complex. Finally, a long-range goal coupled to the specific proposals described above, involves site directed drug design directed at alleviating the anemic effects. Once we have obtained enough information regarding protein factors which control midpoint potential, rates of autoxidation, and re-reduction we hope to design novel pharmaceutical agents which can stabilize mutant hemoglobins or alter the midpoint potential of cytochrome b5 to facilitate re-reduction of methemoglobin.