Ultrafast Biophysical Studies Of Proteins

蛋白质的超快生物物理研究

• 批准号: 6810203
• 负责人: Philip A Anfinrud
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6810203
• 关键词: X ray crystallography; bacterial proteins; binding sites; bioengineering /biomedical engineering; biomedical equipment development; biophysics; chemical association; chemical models; electron density; hemoprotein; laser spectrometry; ligands; method development; molecular dynamics; myoglobin; oxygen tension; photolysis; protein folding; protein structure; structural biology; technology /technique development; time resolved data

To better understand how proteins execute their designed function, much effort has gone into the determination of their three-dimensional structures. For example, of the 12,000+ protein structures deposited into the Protein Data Bank, nearly 3,300 are characterized as enzymes. These static structures are enlightening; however, the side chains surrounding the active site of an enzyme are not static spectators but are active participants in the choreographed motions that mediate chemical transformation. To fully understand how an enzyme functions at the molecular level, it is crucial to know the structural changes that ensue as it executes its designed function. With this knowledge, researchers will be better poised to rationally engineer proteins and peptides with therapeutic value. To watch a protein as if functions with atomic spatial resolution, we have developed the technique of picosecond time-resolved X-ray crystallography. This technique is based on the pump-probe method where a picosecond laser pulse (pump) triggers a reaction in a protein crystal and a delayed X-ray pulse (probe) takes a "snapshot" of the protein's structure. These experiments require an intense source of X-ray pulses and synchronized tunable laser pulses. The ID09B time-resolved beamline at the European Synchrotron and Radiation Facility (ESRF) in Grenoble, France is still the only beamline in the world capable of recording time-resolved macromolecular structures with 150 picosecond time resolution. Using this source, we have studied the structural changes that accompany ligand migration in myoglobin (Mb). This protein has proven to be a very useful model system for these investigations: mutant forms can be over expressed in E. coli; it forms highly ordered crystals that diffract to atomic resolution; it reversibly binds small ligands such as O2, CO, and NO; and it can be photolyzed with high efficiency. In particular, we have focused on the L29F mutant of myoglobin (Mb), where the leucine (L) in the 29 position is replaced by phenylalanine (F). According to femtosecond time-resolved IR measurements of photolyzed L29F MbCO, the rate of ligand escape from its primary docking site is accelerated at least 1000-fold compared to wild-type MbCO. We have acquired structures of this mutant at time delays spanning from 100 picoseconds to 3 microseconds. The structural rearrangements triggered by ligand dissociation are striking, and involve correlated motion of the heme and numerous side chains. In particular, to accommodate docked CO, the phenylalanine in position 29 is displaced toward the distal histidine, and this sterically strained intermediate rapidly sweeps CO out of the docking site with a significant population ending up in the Xe4 docking site. On the time scale of some tens of nanoseconds, the CO migrates around the heme to the Xe1 docking site. The correlated structural changes are most clearly unveiled in a movie produced by stitching together the complete series of time-resolved structures. We are currently extending these studies to hemoglobin as well as other mutants of myoglobin. The technique of time-resolved X-ray crystallography is still in its infancy, and much work remains to be done to enhance the quality of data acquired, to develop improved tools to analyze the diffraction data, and to develop improved tools to visualize the 3-D data. Dr. Eric Henry (LCP) is collaborating with us to develop new tools to analyze diffraction images and translate them into 3D electron density maps. Dr. Gerhard Hummer (LCP) is running molecular dynamics simulations to help identify the molecular basis for the structural changes that are observed. Our femtosecond time-resolved spectroscopy lab continues to study the photophysics of various chromophores in protein crystals in order to develop more efficient methods for photoactivation. This combination of spectroscopic, crystallographic, and computational tools are paving the way to explore functionally-important structure transitions at an atomistic level, from which a far more meaningful mechanistic description of protein function will be achieved.

为了更好地了解蛋白质如何执行其设计的功能，人们投入了大量精力来确定其三维结构。例如，在蛋白质数据库中存储的 12,000 多个蛋白质结构中，近 3,300 个被定性为酶。这些静态结构很有启发性。然而，酶活性位点周围的侧链并不是静态的旁观者，而是介导化学转化的精心设计的运动的积极参与者。为了充分了解酶如何在分子水平上发挥作用，了解酶在执行其设计功能时随之发生的结构变化至关重要。有了这些知识，研究人员将能够更好地合理设计具有治疗价值的蛋白质和肽。 为了观察蛋白质在原子空间分辨率下的功能，我们开发了皮秒时间分辨 X 射线晶体学技术。该技术基于泵浦探针方法，其中皮秒激光脉冲（泵浦）触发蛋白质晶体中的反应，而延迟的 X 射线脉冲（探针）拍摄蛋白质结构的“快照”。这些实验需要强大的 X 射线脉冲源和同步可调谐激光脉冲。位于法国格勒诺布尔的欧洲同步加速器和辐射设施 (ESRF) 的 ID09B 时间分辨光束线仍然是世界上唯一能够以 150 皮秒时间分辨率记录时间分辨大分子结构的光束线。利用这个来源，我们研究了肌红蛋白 (Mb) 中伴随配体迁移的结构变化。这种蛋白质已被证明是这些研究中非常有用的模型系统：突变形式可以在大肠杆菌中过度表达；它形成高度有序的晶体，衍射至原子分辨率；它可逆地结合小配体，例如 O2、CO 和 NO；并且可以高效地进行光解。我们特别关注肌红蛋白 (Mb) 的 L29F 突变体，其中 29 位的亮氨酸 (L) 被苯丙氨酸 (F) 取代。根据光解 L29F MbCO 的飞秒时间分辨红外测量，与野生型 MbCO 相比，配体从其主要对接位点逃逸的速率至少加快了 1000 倍。我们在 100 皮秒到 3 微秒的时间延迟内获得了该突变体的结构。由配体解离引发的结构重排是惊人的，并且涉及血红素和众多侧链的相关运动。特别是，为了容纳对接的CO，第29位的苯丙氨酸被移向远端组氨酸，并且这种空间紧张的中间体迅速将CO从对接位点清除，并且大量的CO最终进入Xe4对接位点。在大约几十纳秒的时间尺度上，CO 围绕血红素迁移到 Xe1 对接位点。通过将完整系列的时间分辨结构拼接在一起制作的电影中，相关的结构变化得到了最清晰的揭示。我们目前正在将这些研究扩展到血红蛋白以及肌红蛋白的其他突变体。 时间分辨 X 射线晶体学技术仍处于起步阶段，为了提高所获取数据的质量、开发改进的工具来分析衍射数据以及开发改进的工具来可视化 3 个数据，还有许多工作要做。 -D 数据。 Eric Henry 博士 (LCP) 正在与我们合作开发新工具来分析衍射图像并将其转换为 3D 电子密度图。 Gerhard Hummer 博士 (LCP) 正在进行分子动力学模拟，以帮助确定所观察到的结构变化的分子基础。我们的飞秒时间分辨光谱实验室继续研究蛋白质晶体中各种发色团的光物理学，以开发更有效的光激活方法。这种光谱学、晶体学和计算工具的结合为在原子水平上探索功能重要的结构转变铺平了道路，由此将实现对蛋白质功能更有意义的机械描述。