Ultrafast Biophysical Studies Of Proteins

蛋白质的超快生物物理研究

• 批准号: 6983754
• 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/6983754
• 关键词: X ray crystallography; binding sites; bioengineering /biomedical engineering; bioimaging /biomedical imaging; biomedical equipment development; biophysics; computational biology; computer program /software; conformation; electron density; hemoprotein; image processing; intermolecular interaction; ligands; molecular dynamics; myoglobin; photochemistry; protein folding; protein structure function; spectrometry; structural biology; technology /technique development; time resolved data

To gain insights into the function of enzymes, much effort has gone into the determination of their three-dimensional structures. This venture has been highly successful: more than 22,000 proteins structures are now deposited in the Protein Data Bank, of which nearly 4,500 are characterized as enzymes. These static structures provides hints into how proteins function; however, the side chains surrounding the active site 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. We have been working to develop and refine the method of picosecond time-resolved X-ray crystallography, a technique that allows us to literally ?watch? a protein as it functions. This technique is based on the pump-probe method where a 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. 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 ps time resolution and < 2 ? spatial resolution. One of the challenges we have recently dealt with is how to assess structural changes in a visually intuitive fashion. Crystallographers often compare structures by generating difference electron density maps from two different structures. The difference density is portrayed as a contour map with the threshold set at an appropriate density. This approach leaves much to be desired. The electron density differences are not localized on the atoms that move but on their edges, which can make it difficult to assign the difference density ellipsoid to the atom or group of atoms that move. Only differences that exceed some specified threshold are plotted, so this approach is insensitive to small conformational changes. As the magnitude of the structural change diminishes, the difference density dips below the threshold and the feature abruptly disappears, creating a nonlinear response to the change. To surmount these limitations, we developed a novel approach for visualizing structural changes in proteins. Our approach is based on a color-coded rendering of the electron density distribution for the unphotolyzed (magenta) and photolyzed (green) states. Where these two colors overlap, they blend to white. Consequently, the magenta-to-green color gradient depicts the direction of atomic motion, and the brightness of the color gradient correlates with the amplitude of the motion. By mapping the electron density to brightness in a nonlinear fashion, weak features are not obscured by intense features. This nonlinear mapping enhances the dynamic range of structural changes that can be observed in a single image. By stitching a sequence of time-resolved images into a movie, subtle time-dependent changes in the conformation are much more apparent. This approach allows us to intuitively visualize conformational changes at an unprecedented level of detail [Schotte et al., JSB, (2004)]. While we have been engaged in studies of several different protein systems including photoactive yellow protein, here we highlight our efforts to probe ligand migration and correlated structural changes in proteins. Myoglobin, a heme protein found in muscle that reversibly binds small ligands such as O2, CO, and NO has proven to be a useful model system for pursuing these studies. Owing to the photosensitivity of the ligand bond and the reversibility of ligand binding, ligand dissociation can be triggered repeatedly by a laser pulse without damage to thhhe protein. Moreover, mutant forms of this protein can be over expressed in E coli and purified protein can be coaxed to form highly ordered crystals that diffract to atomic resolution. We have studied 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 approximately 1000-fold compared to wild-type MbCO. We have acquired time-resolved structures of this mutant at time delays spanning 100 picoseconds to 3 microseconds [Schotte et al., Science (2004)]. The structural rearrangements triggered by ligand dissociation are striking, and involve correlated motion of the heme and numerous side chains. A comparison of the structural changes occurring in wild-type and L29F MbCO provide a structural explanation for the dramatic differences in the rates at which these two proteins excrete toxic CO [Schotte et al., JSB, (2004)]. Our successes in time-resolved X-ray crystallography couldn?t have been realized were it not for our ability to study the photophysics of protein crystals at the NIH with a state-of-the-art microfocusing femtosecond spectrometer. By analyzing femtosecond time-resolved spectra of photoexcited proteins in crystals, we have been able to develop protocols for attaining more efficient photoexcitation without damaging the chromophore. Currently, our analysis of time-resolved Laue diffraction images employs routines from a hodge-podge of disparate software packages and requires weeks to months of effort to fully process a few days worth of data. To eliminate the data processing inefficiencies inherent in this approach, we have been developing a stand-alone software suite capable of processing time-resolved Laue diffraction images in real time and translating those results into movies of protein motion at atomic resolution. Dr. Eric Henry (LCP) has been assisting us by developing code to automate and/or speed up several critical steps in our data processing algorithm. Preliminary tests suggest that we will be able to analyze diffraction images as fast as they are generated on the beam line. This ?real-time? feedback will help us make much more efficient use of the limited and precious beam time allocated to our research. We have also initiated a collaboration with Dr. Gerhard Hummer (LCP) to perform molecular dynamics simulations of proteins. By comparing experiment and theory at comparable resolution in space and time, we are able to gain a single-molecule perspective into mechanisms of protein function. Our 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.

为了深入了解酶的功能，人们在确定其三维结构方面付出了很大的努力。这项事业非常成功：蛋白质数据库中现已存放了超过 22,000 个蛋白质结构，其中近 4,500 个被定性为酶。这些静态结构为蛋白质如何发挥作用提供了线索。然而，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。 我们一直致力于开发和完善皮秒时间分辨 X 射线晶体学方法，这项技术使我们能够真正“观看”。蛋白质的功能。该技术基于泵浦探针方法，其中激光脉冲（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“快照”。蛋白质的结构。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 ps 时间分辨率和 < 2 ? 记录时间分辨大分子结构的光束线。空间分辨率。 我们最近面临的挑战之一是如何以直观的方式评估结构变化。晶体学家经常通过从两种不同结构生成差异电子密度图来比较结构。差异密度被描绘为等值线图，阈值设置为适当的密度。这种方法还有很多不足之处。电子密度差并不集中在移动的原子上，而是集中在其边缘上，这使得很难将差密度椭球分配给移动的原子或原子团。仅绘制超过某个指定阈值的差异，因此该方法对小的构象变化不敏感。随着结构变化幅度的减小，差异密度下降到阈值以下，并且特征突然消失，从而对变化产生非线性响应。为了克服这些限制，我们开发了一种可视化蛋白质结构变化的新方法。我们的方法基于未光解（洋红色）和光解（绿色）状态的电子密度分布的颜色编码渲染。当这两种颜色重叠时，它们会混合成白色。因此，洋红色到绿色的颜色梯度描绘了原子运动的方向，并且颜色梯度的亮度与运动的幅度相关。通过以非线性方式将电子密度映射到亮度，弱特征不会被强特征所掩盖。这种非线性映射增强了可以在单个图像中观察到的结构变化的动态范围。通过将一系列时间分辨图像拼接到电影中，构象中随时间变化的微妙变化变得更加明显。这种方法使我们能够以前所未有的细节直观地可视化构象变化[Schotte et al., JSB, (2004)]。 虽然我们一直致力于包括光敏黄色蛋白在内的几种不同蛋白质系统的研究，但在这里我们重点介绍我们在探测配体迁移和蛋白质中相关结构变化方面所做的努力。肌红蛋白是肌肉中发现的一种血红素蛋白，可可逆地结合 O2、CO 和 NO 等小配体，已被证明是进行这些研究的有用模型系统。由于配体键的光敏性和配体结合的可逆性，配体解离可以通过激光脉冲重复触发，而不损伤蛋白质。此外，这种蛋白质的突变形式可以在大肠杆菌中过度表达，并且可以诱导纯化的蛋白质形成衍射至原子分辨率的高度有序的晶体。我们研究了肌红蛋白（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加快了约 1000 倍。我们已经在 100 皮秒到 3 微秒的时间延迟内获得了该突变体的时间分辨结构 [Schotte et al., Science (2004)]。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。野生型和 L29F MbCO 中发生的结构变化的比较为这两种蛋白质排泄有毒 CO 的速率的显着差异提供了结构解释 [Schotte et al., JSB, (2004)]。 如果不是我们能够在 NIH 使用最先进的微聚焦飞秒光谱仪研究蛋白质晶体的光物理学，我们就不可能在时间分辨 X 射线晶体学方面取得成功。通过分析晶体中光激发蛋白质的飞秒时间分辨光谱，我们已经能够开发出在不损坏发色团的情况下获得更有效的光激发的方案。 目前，我们对时间分辨劳厄衍射图像的分析采用了不同软件包大杂烩的例程，需要几周到几个月的时间才能完全处理几天的数据。为了消除这种方法固有的数据处理效率低下的问题，我们一直在开发一个独立的软件套件，能够实时处理时间分辨劳厄衍射图像，并将这些结果转化为原子分辨率的蛋白质运动电影。 Eric Henry 博士 (LCP) 一直在帮助我们开发代码来自动化和/或加速我们数据处理算法中的几个关键步骤。初步测试表明，我们将能够像光束线上生成衍射图像一样快地分析它们。这？实时？反馈将帮助我们更有效地利用分配给我们研究的有限而宝贵的光束时间。我们还启动了与 Gerhard Hummer 博士 (LCP) 的合作，对蛋白质进行分子动力学模拟。通过在空间和时间上以相当的分辨率比较实验和理论，我们能够获得蛋白质功能机制的单分子视角。我们将光谱、晶体学和计算工具相结合，为在原子水平上探索功能上重要的结构转变铺平了道路，从而实现对蛋白质功能更有意义的机械描述。