NMR STUDIES OF RESPIRATORY PROTEINS AND MODEL SYSTEMS

描述：本申请的广泛、长期目标是 用核磁共振波谱研究呼吸道的结构， 蛋白质;特别是O2和CO如何与肌红蛋白和血红蛋白结合 以及模型系统，例如“尖桩栅栏”和“封顶”或 受保护的金属卟啉。 这项工作的健康相关性在于， 充分了解蛋白质如何控制O2与CO结合 对于了解氧气如何在血液中运输很重要， 肌肉，O2亲和力如何在各种呼吸道疾病中改变， 以及从长远来看如何设计血液替代品。 的 具体目标是首先构建一个多探头600 MHz宽带 核磁共振光谱仪，这将允许例行观察， 至少两个不同的样品，同时，在同一个磁体中。 这将增加样品吞吐量，降低整体有效成本 昂贵的核磁共振资源。 第二，CO键合将在 血红素模型系统，使用13 C和17 O化学位移和17 O四重 耦合信息和密度泛函理论（DFT）。 这里的目标 将观察到的光谱参数与结构（配体）相关联 倾斜、弯曲、静电场、血红素褶皱/隆起/鞍形、近端 侧相互作用）。 电子关联和交换效应 将使用DFT形式主义进行处理。 第三个目的是澄清 血红素蛋白中FeCO/远端/近端相互作用的性质 自己 将特别强调获得第一 ~（13）C、~（17）O屏蔽的原理、定量分析及场 肌红蛋白A0、A1和A3底物中的梯度张量， 血红蛋白 第四个目标是研究O2如何与Fe结合， 模型血红素系统，使用为CO开发的方法。最终目标是 研究血红素蛋白本身的Fe-O2相互作用，使用17 O NMR和 DFT。 与CO的早期报道一样，报道的FeOO几何形状变化很大， 从115度到159度。 然而，很可能一个很大的 实际上存在较窄的角度范围，因为能量 与这些扭曲相关的是非常大的。 固体17 O NMR 将使一个假设的测试，配体的几何形状是多 接近130度。 O2稳定的程度将是 再次使用DFT研究（例如，与远端组氨酸的氢键） 方法. 目标是详细了解CO和 O2与血红素蛋白中的Fe结合，以阐明FeCO和FeO 2的主题 几何形状，并提供电场/氢键相互作用的探针。 了解配体如何与金属蛋白结合;例如，O2如何结合 并由血红蛋白稳定，具有相当的长期健康- 相关的影响，因为如果不知道重要的相互作用， 那么改进的疗法将更难设计。

DESCRIPTION: The broad, long term objective of this application is to use NMR spectroscopy to investigate the structures of respiratory proteins; in particular, how O2 and CO bind to myoglobin and hemoglobin as well as model systems, such as "picket fence" and "capped" or protected metalloporphyrins. The health relatedness of this work is that a full understanding of how O2 vs. CO binding is controlled by proteins is important for understanding how O2 is transported in blood and muscle, how O2 affinities are changed in various respiratory diseases, as well as how in the long term, blood substitutes may be designed. The specific aims are first, to construct a multiple probe 600 MHz widebore NMR spectrometer, which will permit routine observations to be made on at least two different samples, simultaneously, in the same magnet. This will increase sample throughput, reducing the overall effective cost of expensive NMR resources. Second, CO bonding will be investigated in heme model systems, using 13C and 17O chemical shift and 17O quadruple coupling information and density functional theory (DFT). The goal here is to relate observed spectroscopic parameters to structure (ligand tilt, bend, electrostatic fields, heme ruffling/doming/saddling, proximal side interactions). The effects of electron correlation and exchange will be handled using the DFT formalism. The third aim is to clarify the nature of FeCO/distal/proximal interactions in heme proteins themselves. Particular emphasis will be placed on obtaining first principles, quantitative analyses of 13C, 17O shielding and field gradient tensors in A0, A1, and A3 substrates of myoglobins and hemoglobins. The fourth goal is to investigate how O2 binds to Fe in model heme systems, using methods developed for CO. The final goal is to study Fe-O2 interaction in heme proteins themselves, using 17O NMR and DFT. As with early reports for CO, reported FeOO geometries vary widely, from ~115 degrees to 159 degrees. However, it is likely that a much narrower range of angles is actually present, since the energies associated with these distortions are very large. Solid-state 17O NMR will enable a test of the hypothesis that the ligand geometries are much closer to 130 degrees. The extent of O2 stabilization will be investigated (e.g. H-bonding to the distal histidine), again using DFT methods. The goal is to obtain a detailed understanding of how CO and O2 bind to Fe in heme proteins, to clarify the topic of FeCO and FeO2 geometries, and to provide probes of E-field/H-bonding interactions. Understanding how ligands bind to metalloproteins; e.g., how O2 binds to and is stabilized by hemoglobin, has considerable long-term health- related implications, since if the important interactions are not known, then improved therapies will be more difficult to engineer.