Ultrafast Time-Resolved Crystallography on Scapharca Hb

描述（由申请人提供）：为了具有生物学用途，蛋白质分子依靠结构转变来实现其功能。尽管它们很重要，但通常很难在它们发生时跟踪这种构象重排。时间分辨晶体学的最新进展已经将其能力扩展到纳秒（ns）时间范围内，以实时观察这种结构转变。我们将应用时间分辨晶体学技术对来自Scapharca inaequivalvis的合作二聚血红蛋白进行研究，该蛋白已被证明是研究变构蛋白行为的一个极好的模型系统。这种蛋白质特别适合于这种分析，因为它的性质包括在晶体状态下观察配合配体结合行为的能力和晶体的衍射能力。我们建议使用这种技术来研究配体光解离引起的结构变化。结构变化将由短（10ns）激光脉冲触发，并由150皮秒（ps）至15ns x射线脉冲探测。这种类型的实验可以成功地在第三代同步加速器源进行，如先进光子源（APS - Argonne，美国），欧洲同步辐射设施（ESRF -格勒诺布尔，法国）和SPring8 （Tsukuba，日本），肌红蛋白和光活性黄色蛋白的大量实验已经证明。我们已经在ESRF和APS对这种二聚体血红蛋白的野生型和突变体进行了初步实验，清楚地表明了这种方法的可行性。到目前为止，我们的研究结果表明，结合配体的损失伴随着血红素的形成和蛋白质原子的运动，这表明在配体释放后的1ns到25ns之间的几个时间点上存在重要的瞬态中间体。释放配体的密度表明它遵循活性位点和溶剂之间的替代途径。我们的目标是将时间分辨和超高分辨晶体学与诱变、光谱学和分子动力学模拟相结合，以获得对构象重排介导生物过程变构调节的原理的前所未有的理解。理解这些原理将有助于合理地操纵许多生物过程。

DESCRIPTION (provided by applicant): In order to be biologically useful, protein molecules rely on structural transitions to carry out their function. Despite their importance, it is usually difficult to follow such conformational rearrangements as they occur. Recent advances in time-resolved crystallography have extended its capability into the nanosecond (ns) time ranges required to observe such structural transitions in real time. We will apply time-resolved crystallographic techniques to a cooperative dimeric hemoglobin from Scapharca inaequivalvis that has proven to be an excellent model system for studying allosteric protein behavior. This protein is exceptionally well suited for such an analysis due to properties that include the ability to observe cooperative ligand binding behavior within the crystalline state and the diffracting power of its crystals. We propose to use this technique to investigate structural changes induced by ligand photo-dissociation. Structural changes will be triggered by short (10 ns) laser pulses and probed by 150 picosecond (ps) to 15ns X-ray pulses. This type of experiment can be successfully conducted at third generation synchrotron sources such as the Advanced Photon Source (APS - Argonne, USA), European Synchrotron Radiation Facility (ESRF - Grenoble, France) and SPring8 (Tsukuba, Japan), as extensive experiments on myoglobin and photoactive yellow protein have demonstrated. We have conducted preliminary experiments on wildtype and mutants of this dimeric hemoglobin at ESRF and APS that clearly show the feasibility of this approach. Our results to date show the loss of bound ligand coupled with heme doming and movement of the protein atoms, suggesting important transient intermediates at several time points between l ns and 25ns following ligand release. Density for released ligand suggests it follows an alternate pathway in transit between the active site and solvent. Our goal is to combine time-resolved and ultrahigh resolution crystallography with mutagenesis, spectroscopy and molecular dynamics simulation to gain an unprecedented understanding of principles by which conformational rearrangements mediate allosteric regulation of biological processes. Understanding such principles will aid in the rational manipulation of a number of biological processes.