NO DYNAMICS IN HEMEPROTEINS--TRANSIENT INFRARED STUDIES

血红素蛋白没有动力学——瞬态红外研究

• 批准号: 2771014
• 负责人: MARCO A PEREIRA
• 金额: $ 13.4万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2771014
• 关键词: Raman spectrometry; acidity /alkalinity; biomedical equipment development; carbon monoxide; carboxyhemoglobin; charge coupled device camera; chemical binding; conformation; diffusion; heme; hemoglobin; hemoprotein structure; infrared spectrometry; lasers; ligands; molecular dynamics; mutant; myoglobin; nitric oxide; photolysis; stereochemistry; structural biology; temperature; viscosity

The objective of the proposed research is the study of the ligand dynamics of carbonmonoxide(CO) nitric oxide (NO) in hemeproteins and the correlated protein conformational changes using transient mid-infrared absorption, picosecond Raman, and transient visible/near-infrared absorption techniques. The research on the dynamics of the protein cage will probe the amide I infrared optical marker, the iron-histidine Raman line and the 4- and 5- coordination visible/near-infrared absorption spectrum differences in a series of experiments aimed at correlating the barrier fluctuations with the observed ligand dynamics. In the Phe29 mutant myoglobin, the Phe29 residue acts as a very high barrier to ligand diffusion, separating the ligands into two groups: the ones before and after the Phe29 residue. The infrared monitoring of these two species creates a new handle for the study of barrier fluctuations. The optical marker associated with each of these two species will be monitored. This will permit the study of the diffusional process of the outgoing species as a function of pH, temperature and viscosity. Myoglobin under pH3 conditions should present larger barrier fluctuations, while low temperature measurements are likely to freeze those barriers. From these measurements one should be able to infer what are the modulators of NO diffusion. The short lived species will provide information about the near heme environment. During the photodeligation process, both a bleach corresponding to the loss of the bound species and an increase in absorption due to the creation of the new unbound species occur. These changes appear in different spectral regions and they can tell us about the dynamics of recombination, escape rate from the heme pocket, etc. The bleached absorption an isotropy identifies the mean orientation of the bound species (A-states) with respect to the heme, while the unbound species absorption an isotropy identifies orientational details about the B-states. We will create a reliable, user friendly, widely tunable (2--l0 millimicron) highly sensitive, femtosecond transient infrared spectrophotometer. It will incorporate an innovative AC monochromator and a powerful and flexible Dual Ti:Sapphire Regenerative Laser Amplifier. The AC monochromator will allow for full spectrum acquisition at each shot, as well as full spectrum normalization at 4 kHz repetition rate. The proposed experimental schemes are expected to bring a 512-fold improvement upon the existing experimental setups. They will create a femtosecond time- resolution apparatus with FTIR (Fourier Transform Infrared Spectrophotometer) sensitivity.

本研究的目的是研究配体动力学 血红素蛋白中一氧化碳（CO）、一氧化氮（NO）的含量及其相关性 使用瞬时中红外吸收的蛋白质构象变化， 皮秒拉曼和瞬态可见/近红外吸收 技术.对蛋白质笼动力学的研究将有助于探索 酰胺I红外光学标记、铁-组氨酸拉曼线和 4-和5-配位可见/近红外吸收光谱 在一系列旨在将屏障 与观察到的配体动力学的波动。 在Phe 29突变体肌红蛋白中，Phe 29残基作为非常高的 配体扩散的屏障，将配体分成两组： 在Phe 29残基之前和之后的。红外线监测这些 两个物种创造了一个新的处理势垒波动的研究。 与这两个物种中的每一个相关的光学标记将是 监控。这将使研究的扩散过程的 作为pH值、温度和粘度的函数的流出物质。肌红 在pH 3条件下应呈现较大的势垒波动，而低 温度测量可能会冻结这些屏障。从这些 测量应该能够推断出NO的调节剂是什么 扩散短寿命的物种将提供有关近 血红素环境在光刻过程中，漂白剂 对应于结合物种的损失和 由于产生新的未结合物质而发生吸收。这些 变化出现在不同的光谱区域，它们可以告诉我们 重组的动力学，血红素口袋的逃逸率等。 漂白吸收各向同性确定的平均取向 结合物种（A-状态）相对于血红素，而未结合的 物种吸收和各向同性确定有关的取向细节 B州 我们将创建一个可靠的，用户友好的，广泛可调（2-l0 纳米）高灵敏度，飞秒瞬态红外 分光光度计它将采用创新的交流单色器， 一个强大而灵活的双钛宝石再生激光放大器。的 交流单色仪将允许在每次拍摄时进行全光谱采集， 以及在4kHz重复率下的全频谱归一化。拟议 实验计划预计将带来512倍的改善， 现有的实验装置。它们会创造出飞秒级的时间- 傅里叶变换红外光谱仪 分光光度计）灵敏度。