Ultrafast Biophysical Studies of Biomolecules at the APS

APS 生物分子超快生物物理研究

• 批准号: 10008656
• 负责人: Philip Anfinrud
• 金额: $ 86.93万
• 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/10008656
• 关键词: Acceleration; Active Sites; Area; Biochemical; Biochemical Reaction; Blood Circulation; Blood capillaries; Caliber; Capital; Carboxyhemoglobin; Complement; Crystallization; Crystallography; Data; Data Set; Development; Diffuse; Dimensions; Drops; Enzymes; Equilibrium; Event; Exhibits; Fingerprint; Glass; Hemoglobin; Heterogeneity; High temperature of physical object; Home environment; Human; Image; Infrastructure; Intervention; Kinetics; Knowledge; Lasers; Lead; Life; Lung; Measurement; Methodology; Methods; Microfluidics; Modeling; Molecular Conformation; Motion; Motor; Muramidase; Names; Noise; Optics; Pathway interactions; Pattern; Photons; Physiologic pulse; Physiological; Play; Population; Positioning Attribute; Protein Dynamics; Proteins; Pump; Radiation induced damage; Reaction; Records; Reporting; Resolution; Roentgen Rays; Role; Sampling; Secondary to; Shapes; Signal Transduction; Site; Source; Spectrum Analysis; Speed; Spottings; Structural Protein; Structure; Syringes; System; Temperature; Time; Tissues; Translating; Translations; United States National Institutes of Health; Work; X ray diffraction analysis; base; beamline; biophysical analysis; chemical reaction; crystallinity; detector; equipment acquisition; flexibility; improved; insight; macromolecule; molecular modeling; novel; oxygen transport; protein structure; radiation adverse effect; temperature jump

Enzymatic reactions exhibit remarkable selectivity and efficiency, the likes of which are rarely achieved in bench-top chemical reactions. While it is clear that the biochemical prowess of an enzyme arises from its highly-ordered structure, the detailed mechanism by which it functions has proven elusive. This is because enzymes are not simply static macromolecules that host an active site, as depicted by their crystal structure; rather, they are dynamic molecules whose choreographed motions can gate the transport of substrate to and from the active site and can modulate over time the activity of that site. To develop a mechanistic understanding into how enzymes function, it is essential to study this choreography of life with structurally-sensitive methods capable of ultrafast time resolution. In this report, we focus on time-resolved X-ray studies that employ the pump-probe method. Briefly, a laser pulse (pump) photoactivates or thermally excites a biomolecule, after which a suitably delayed X-ray pulse (probe) passes through the sample and records a diffraction or scattering pattern on a 2D detector. Thanks to significant capital equipment investments made by the NIH (over $1.2M since 2006), we have developed the ability to pursue studies of biomolecules on the BioCARS 14-IDB beamline via both time-resolved Laue crystallography and time-resolved SAXS/WAXS. Time-resolved Laue crystallography takes advantage of a polychromatic X-ray pulse, which can produce thousands of reflections in a single shot, and boosts substantially the rate at which time-resolved diffraction data can be acquired. The information needed to determine the protein's structure is encoded in the relative intensities of the diffraction spots observed. Since the structural information contained in a single Laue diffraction image is incomplete, repeated measurements at multiple crystal orientations are required to produce a complete set of data. Prior studies have generally required a small number of very large, homogeneous crystals, which has limited the application of this methodology to a handful of proteins. The impact of time-resolved Laue crystallography would be boosted significantly if we were to develop methods capable of acquiring high S/N diffraction images from a large number of relatively small crystals (30-35 microns), rather than small number of large crystals. To that end, we continue our efforts to develop novel microfluidic methods for growing crystals and delivering them in a fashion that can be automated. Briefly, we have developed an alternating drop microfluidic mixer that has been used to grow more than 1000 uniformly sized lysozyme crystals ( 30-35 microns) in a 1-m long glass capillary. A microfluidic crystal delivery system based on a home-built multi-axis syringe-pump tower is being developed with an aim to automate crystal delivery to the BioCARS 14-IDB beamline. This effort also includes the development of a high-speed diffractometer capable of rapidly and precisely positioning crystals at the intersection of the laser and X-ray beams. While much progress has been made, more work remains to be done. Our aim to automate the acquisition of X-ray diffraction images from thousands of crystals without user intervention will hopefully be realized within the coming year. Time-resolved Laue crystallography, as its name implies, can only be performed on crystalline samples. The intermolecular forces that maintain crystalline order constrain large amplitude conformational motion, and this loss of flexibility may perturb or even inhibit the function of a protein. Though Laue crystallography stands alone in its ability to track structural changes in proteins on ultrafast time scales with near-atomic spatial resolution, it is crucial to also study structural dynamics of biomolecules in solution where the full range of conformational motion is permitted. Without external alignment forces, biomolecules in solution are randomly oriented, and the structural information contained in their orientationally-averaged diffuse scattering pattern is one dimensional. Nevertheless, it is well known that the SAXS region of the diffraction pattern reports on the size and shape of the biomolecule, while the WAXS region is sensitive to secondary and tertiary structure. Time-resolved SAXS/WAXS scattering patterns therefore provide 'fingerprints' that can be correlated with protein structure via molecular models, and can assess which models best describe reaction pathways in solution. Our time-resolved SAXS/WAXS diffractometer currently employs a secondary K-B mirror pair to focus the X-ray beam onto the sample capillary with independent control of the the vertical and horizontal dimensions, a very small beamstop (0.51 mm diameter), and a large area (340x340 mm), high-speed (up to 10 Hz) X-ray detector. With the sample-detector distance set at 185.8 mm, scattering data can be acquired over a broad range of q (momentum transfer) spanning 0.02 to 5.4 inverse Angstroms, which corresponds to spatial resolution below 1.2 Angstroms. To mitigate the adverse effects of radiation damage during X-ray exposure, the capillary containing the protein solution is rapidly translated over a 20-mm span using a home-built, high-speed diffractometer that is based on 1-micron resolution linear motor translation stages capable of more than 1-g acceleration. Thanks to a closed-loop circulation system, about 150 microliter of protein solution is sufficient to acquire a high signal-to-noise ratio data set. With our recently improved capillary holder and high-precision temperature controller, we are able to characterize structural changes over a broad range of temperatures spanning from approximately -16 to 120 degrees Celsius. Moreover, thanks to the relatively small spot size that can be generated with the secondary K-B mirror pair, it is possible to focus a 1 mJ infrared pulse down to a dimension small enough to heat samples in a glass capillary by more than 20 degrees Celsius. When setting the sample temperature just below its unfolding temperature, this magnitude T-jump is sufficient to trigger unfolding of the biomolecule, and allows us to investigate the dynamics of protein unfolding with unprecedented spatial resolution. The time-resolution achieved is currently limited by the duration of the infrared laser pulse, which is about 5 ns. Prior time-resolved studies of the R to T structure transition in human hemoglobin unveiled complicated kinetics which seemed to point to heterogeneity in the R state. To explore this possibility, we pursued a time-resolved T-jump study of the R state of human hemoglobin. If more than one R state exists, one would expect their relative population to be temperature dependent, and would lead to a time-dependent change in its X-ray scattering pattern as the relative population of two (or more) states comes into equilibrium at the higher temperature. Indeed, we recently reported in "Dynamics of Quaternary Structure Transitions in R-State Carbonmonoxyhemoglobin Unveiled in Time-Resolved X-ray Scattering Patterns Following a Temperature Jump," J Phys Chem B 122, 11488-11496, that human hemoglobin has two (or more) R states, whose rate of interconversion is approximately 30 microseconds. Armed with this knowledge, we are working to develop a comprehensive model capable of unveiling in mechanistic detail how this allosteric transition enables hemoglobin to efficiently transport oxygen from the lungs to the tissues. As our time-resolved SAXS/WAXS methodology becomes more precise and easier to use, we expect it to become an ever more important complement to time-resolved Laue studies and time-resolved optical spectroscopy studies of biomolecules, and will help provide a structural basis for understanding how biomolecules function.

酶促反应表现出显着的选择性和效率，这样的反应在台式化学反应中很少实现。虽然酶的生化能力显然源自其高度有序的结构，但其发挥作用的详细机制已被证明难以捉摸。这是因为酶不仅仅是具有活性位点的静态大分子，如其晶体结构所示；相反，它们是动态分子，其精心设计的运动可以控制底物进出活性位点的运输，并可以随着时间的推移调节该位点的活性。为了从机制上理解酶的功能，有必要使用能够超快时间分辨率的结构敏感方法来研究这种生命的编排。 在本报告中，我们重点关注采用泵浦探针方法的时间分辨 X 射线研究。简而言之，激光脉冲（泵）光激活或热激发生物分子，然后适当延迟的 X 射线脉冲（探针）穿过样品并在 2D 检测器上记录衍射或散射图案。得益于 NIH 的大量资本设备投资（自 2006 年以来超过 120 万美元），我们已经具备了通过时间分辨劳厄晶体学和时间分辨 SAXS/WAXS 在 BioCARS 14-IDB 光束线上进行生物分子研究的能力。 时间分辨劳厄晶体学利用多色 X 射线脉冲，一次可以产生数千次反射，并大大提高了获取时间分辨衍射数据的速率。确定蛋白质结构所需的信息被编码在观察到的衍射斑点的相对强度中。由于单个劳厄衍射图像中包含的结构信息不完整，因此需要在多个晶体取向上重复测量才能产生完整的数据集。先前的研究通常需要少量非常大的均质晶体，这限制了该方法在少数蛋白质上的应用。如果我们开发出能够从大量相对较小的晶体（30-35 微米）而不是少量的大晶体中获取高信噪比衍射图像的方法，那么时间分辨劳厄晶体学的影响将会显着增强。为此，我们继续努力开发新颖的微流体方法来生长晶体并以自动化的方式提供它们。简而言之，我们开发了一种交替滴微流体混合器，已用于在 1 米长的玻璃毛细管中生长 1000 多个均匀尺寸的溶菌酶晶体（30-35 微米）。正在开发一种基于自制多轴注射泵塔的微流体晶体输送系统，旨在自动将晶体输送到 BioCARS 14-IDB 光束线。这项工作还包括开发高速衍射仪，能够将晶体快速精确地定位在激光束和 X 射线束的交叉点处。尽管已经取得了很大进展，但仍有更多工作要做。我们的目标是在无需用户干预的情况下自动从数千个晶体中获取 X 射线衍射图像，这一目标有望在来年实现。 时间分辨劳厄晶体学，顾名思义，只能在晶体样品上进行。维持晶体秩序的分子间力限制了大幅构象运动，而这种灵活性的丧失可能会扰乱甚至抑制蛋白质的功能。尽管劳厄晶体学以其在超快时间尺度上以近原子空间分辨率追踪蛋白质结构变化的能力而独树一帜，但在允许全方位构象运动的溶液中研究生物分子的结构动力学也至关重要。在没有外部对准力的情况下，溶液中的生物分子是随机定向的，并且其定向平均漫散射图案中包含的结构信息是一维的。然而，众所周知，衍射图样的 SAXS 区域报告生物分子的大小和形状，而 WAXS 区域对二级和三级结构敏感。因此，时间分辨 SAXS/WAXS 散射模式提供了“指纹”，可以通过分子模型与蛋白质结构相关联，并可以评估哪些模型最能描述溶液中的反应途径。 我们的时间分辨 SAXS/WAXS 衍射仪目前采用辅助 K-B 镜对将 X 射线束聚焦到样品毛细管上，并独立控制垂直和水平尺寸、非常小的束光阑（直径 0.51 mm）和大的束光阑。面积 (340x340 mm)、高速（高达 10 Hz）X 射线探测器。当样品-探测器距离设置为 185.8 毫米时，可以在 0.02 至 5.4 反埃的较宽 q（动量传递）范围内获取散射数据，这对应于低于 1.2 埃的空间分辨率。为了减轻 X 射线照射期间辐射损伤的不利影响，使用基于 1 微米分辨率线性电机平移的自制高速衍射仪，将含有蛋白质溶液的毛细管快速平移 20 毫米跨度能够提供超过 1 g 加速度的平台。得益于闭环循环系统，约150微升的蛋白质溶液足以获取高信噪比数据集。借助我们最近改进的毛细管支架和高精度温度控制器，我们能够表征约 -16 至 120 摄氏度的广泛温度范围内的结构变化。此外，由于二级 K-B 镜对可以产生相对较小的光斑尺寸，因此可以将 1 mJ 红外脉冲聚焦到足够小的尺寸，以将玻璃毛细管中的样品加热超过 20 摄氏度。当将样品温度设置为略低于其展开温度时，这种幅度的 T 跃变足以触发生物分子的展开，并使我们能够以前所未有的空间分辨率研究蛋白质展开的动力学。目前所实现的时间分辨率受到红外激光脉冲持续时间的限制，约为 5 ns。 先前对人类血红蛋白中 R 到 T 结构转变的时间分辨研究揭示了复杂的动力学，这似乎表明了 R 状态的异质性。为了探索这种可能性，我们对人类血红蛋白的 R 状态进行了时间分辨 T 跳跃研究。如果存在多个 R 态，人们会期望它们的相对布居与温度相关，并且当两种（或更多）状态的相对布居在温度越高。事实上，我们最近在“温度跳跃后时间分辨 X 射线散射模式中揭示的 R 态碳单氧血红蛋白四级结构转变动力学”中报道，J Phys Chem B 122, 11488-11496，人类血红蛋白有两个（或更多）R状态，其相互转换速率约为30微秒。有了这些知识，我们正在努力开发一个综合模型，能够详细揭示这种变构转变如何使血红蛋白有效地将氧气从肺部输送到组织。 随着我们的时间分辨 SAXS/WAXS 方法变得更加精确和易于使用，我们预计它将成为时间分辨劳厄研究和生物分子时间分辨光学光谱研究的更重要的补充，并将有助于提供结构基础以了解生物分子如何发挥作用。