PICOSECOND TIME-RESOLVED WAXS OF PROTEINS IN SOLUTION

溶液中的皮秒时间分辨蛋白质蜡

• 批准号: 8172009
• 负责人: Philip Anfinrud
• 金额: $ 4.38万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8172009
• 关键词: Affinity; Bacterial Proteins; Biological Models; Brain; Comparative Study; Computer Retrieval of Information on Scientific Projects Database; Crystallography; Fingerprint; Funding; Grant; Hemoglobin; Institution; Kinetics; Lasers; Ligation; Light; Literature; Lung; Molecular Conformation; Myoglobin; Oxygen; Pattern; Physiologic pulse; Protein Conformation; Protein Dynamics; Proteins; Reporting; Research; Research Personnel; Resources; Roentgen Rays; Sampling; Scapharca inaequivalvis dimeric hemoglobin; Solutions; Source; Structure; Techniques; Time; Tissues; United States National Institutes of Health; base; millisecond; neuroglobin; oxygen transport; photolysis; response; sensor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We aim to employ the technique of picosecond time-resolved wide-angle scattering (WAXS) to investigate the dynamics of proteins in solution. This technique can be employed on proteins that are naturally light-sensitive or can be made light sensitive and undergo a reversible photocycle when excited by light. We use a laser pulse to excite a sample of protein solution, probe it with a time-delayed X-ray pulse and record its diffraction pattern.We plan to investigate the kinetics of the T/R transition of hemoglobin (Hb). When transporting oxygen from the lungs to the tissues Hb switches between a high affinity and (R) and low affinity (T) conformation. The structure of both state are well known by static crystallography, but the rate with which the transition occurs in still much in debate. Dynamic studies based on spectroscopic techniques, reported in the literature, are based only local spectroscopic markers and give only give indirect information about the overall protein conformation. We aim to use the WAXS pattern of Hb as an 'fingerprint' that can be matched to known X-ray structures, to track which fraction of Hb is in T and R state as function of time. We use HbCO as substitute for HbO2 because it can be photolyzed with a laser flash with high efficiency. The CO rebinds to the Hb within a few milliseconds. Since Hb shows both an ultrafast tertiary response to the photolysis and a time delayed quaternary one, we plan to perform a comparative study with myoglobin (Mb). We would expect in the case of Mb the same ultrafast tertiary response as Hb, but it lacks a quaternary transition. As a simpler model system for Hb, we plan to study the dimeric hemoglobin of Scapharca inaequivalvis (HbI).HbI has only three different ligation states, compared to twelve in the case of Hb.Further proteins we plan to investigate are neuroglobin, myoglobin-like protein found in the brain acting as an oxygen sensor, and the Photoactive Yellow Protein (PYP),bacterial photosensor.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们的目标是采用皮秒时间分辨广角散射（WAXS）技术来研究溶液中蛋白质的动力学。该技术可用于天然光敏感的蛋白质，或可使其对光敏感并在被光激发时经历可逆光循环的蛋白质。我们使用激光脉冲激发蛋白质溶液样本，用延时 X 射线脉冲对其进行探测并记录其衍射图。我们计划研究血红蛋白 (Hb) 的 T/R 转变动力学。当将氧气从肺部输送到组织时，血红蛋白在高亲和力 (R) 和低亲和力 (T) 构象之间切换。这两种状态的结构都是静态晶体学所熟知的，但转变发生的速率仍然存在很大争议。文献中报道的基于光谱技术的动态研究仅基于局部光谱标记，并且仅给出有关整体蛋白质构象的间接信息。我们的目标是使用 Hb 的 WAXS 图案作为可与已知 X 射线结构相匹配的“指纹”，以跟踪 Hb 的哪一部分作为时间的函数处于 T 和 R 状态。我们使用 HbCO 作为 HbO2 的替代品，因为它可以通过激光闪光进行高效光解。 CO 在几毫秒内与 Hb 重新结合。由于 Hb 对光解表现出超快的三级响应和延迟的四级响应，因此我们计划与肌红蛋白 (Mb) 进行一项比较研究。我们期望 Mb 具有与 Hb 相同的超快三级响应，但它缺乏四级转变。 作为一个更简单的 Hb 模型系统，我们计划研究 Scapharca inaequivalvis (HbI) 的二聚体血红蛋白。HbI 只有三种不同的连接状态，而 Hb 则有十二种。我们计划研究的其他蛋白质包括神经红蛋白、肌红蛋白-类似于大脑中发现的充当氧传感器的蛋白质，以及光活性黄色蛋白（PYP），细菌光传感器。