NOVEL NMR STUDIES OF HEME PROTEINS INVOLVED IN ANEMIA

几种类型的贫血疾病，包括一些相对 常见、严重的溶血性疾病是积聚的结果。 高铁血红蛋白在红细胞中的异常高水平。这些 病理情况是两种疾病之间不平衡的结果 相互竞争的过程，内源性的血红蛋白自氧化速率 并被细胞色素b5重新还原。这项建议是针对 获得与这些病理直接相关的基本化学知识 通过使用聚焦阵列的发散刀刃来改善条件 新的顺磁性多维异核核磁共振 血红素蛋白，高效表达的重组方法 系统和新的电化学方法。新型异核材料 所提出的多维核磁共振方法特别适合于研究 血红素蛋白，更重要的是对血红素蛋白的研究 复合体。这些方法最近已被应用于研究 细胞色素b5-细胞色素c复合体，一个重要的远程模型 生物电子转移。我们的目的是扩大这些研究 以细胞色素b5-肌红蛋白复合体为模型的研究 生理相关细胞色素b5-的溶液结构 血红蛋白复合体。尽管组成蛋白质的结构 (细胞色素b5，血红蛋白和肌红蛋白)是已知的，结构 络合物抵制了结晶学的表征。现代核磁共振 本文提出的方法和对这些方法的新扩展提供了 获得对这些对象的方向的约束的独特能力 与生理相关的复合体和溶液中的蛋白质 条件。我们还计划研究血红素蛋白的作用机制 控制中点潜力和重组能量。这些研究 将使我们能够更定量地解释 自然界中加速自氧化或改变再还原速率 血红蛋白的突变体。中点电位的初步测量 已经用薄层细胞制造了细胞色素b5。小说 使用表面修饰电极的电化学方法应 允许直接测量组件和组件的电子转移速率 因此，可以更深入地了解影响重组的因素 分子间电子转移的能量和速率 生理复合体。最后，一个远距离的目标与 上述具体建议涉及现场定向药物设计 旨在缓解贫血的影响。一旦我们获得了足够的 关于控制中点电位的蛋白质因素的信息， 自氧化和再还原的速率我们希望设计出新的 可以稳定突变的血红蛋白或改变 细胞色素b5的中点电位促进再还原 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.