Fixed-Target Platforms for Time-Resolved Crystallography

用于时间分辨晶体学的固定目标平台

• 批准号: 10634328
• 负责人: SARAH LOUISE PERRY
• 金额: $ 31.51万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10634328
• 关键词: 3-hydroxy-3-methylglutaryl-coenzyme A; Address; Affect; Attention; Biological; Biology; Chemicals; Coenzyme M; Collaborations; Collection; Communities; Consumption; Coupled; Crystallography; Data; Data Collection; Dehydration; Democracy; Development; Devices; Electron Transport; Electronics; Environment; Future; Generations; Goals; Heat shock proteins; Human; Image; In Situ; Influenza Hemagglutinin; Knowledge; Laboratories; Lasers; Light; Location; Massachusetts; Measurement; Metals; Methods; Microfluidic Microchips; Microfluidics; Mitochondria; Molecular; Motion; Noise; Optics; Oxidoreductase; Oxygen; Performance; Phosphoric Monoester Hydrolases; Photons; Polymers; Protein Dynamics; Proteins; Qualifying; Reaction; Reaction Time; Recording of previous events; Reporting; Research; Research Design; Research Personnel; Robotics; Roentgen Rays; Sampling; Science; Signal Transduction; Source; Spectrum Analysis; Stream; Structure; Structure-Activity Relationship; Synchrotrons; System; Technology; Testing; Time; Translations; United States National Institutes of Health; Universities; Validation; Work; X ray diffraction analysis; X-Ray Crystallography; base; beamline; biomacromolecule; cost; design; electric field; experimental study; fabrication; graphene; hydrodynamic flow; improved; new technology; next generation; novel; prevent; protein structure; sensor; structural biology; temperature jump; voltage; x-ray free-electron laser

PROJECT SUMMARY/ABSTRACT Next generation structural biology experiments look to move beyond static observations of structure to a dynamic, time-resolved understanding of function. These next-level experiments are enabled by serial crystallography at X-ray free-electron lasers (XFELs) and microfocus synchrotron beamlines but are limited by issues of sample consumption. The long-term goal of this project is to democratize studies of protein structural dynamics by developing robust fixed-target mounting strategies for light-, chemical-, and electrically-triggered time-resolved protein crystallography experiments while maximizing sample utilization. This effort leverages the expertise of the SLAC National Accelerator Laboratory and the BioCARS facility at the Advanced Photon Source for testing and validation of new mounting strategies. These relationships will also enable rapid translation of developed technologies to the larger structural biology community. We have four initial aims to do this: Aim 1: Develop fixed-target strategies to facilitate the study of macromolecular crystallography targets while maintaining biological activity. We will design and fabricate X-ray compatible sample holders that maintain the stability of protein crystals over time while also allowing for easy sample loading, robotic handling, and in situ spectroscopy. A particular focus will include enabling data collection in a fully anaerobic environment, and the technologies developed here will serve as a basis for efforts in subsequent Aims. Aim 2: Develop fixed-target platforms for photo-triggering of reactions for analysis via time-resolved serial crystallography. We will develop X-ray compatible sample holders that take advantage of hydrodynamic forces to create large-scale arrays of crystals for use in serial crystallography experiments. The polymer-based fabrication strategy will enable fast, low-cost device fabrication, as well as allowing for direct imaging of samples, in situ spectroscopy, and laser-based triggering of reactions for time-resolved X-ray crystallography studies. Aim 3: Develop fixed-target platforms for chemical triggering of reactions for time-resolved serial crystallography analysis. We will develop strategies to enable the controlled addition of a chemical species (i.e., substrate, pH change) to enable the chemical triggering of reactions in crystals. Aim 4: Utilize graphene-based devices to study structural dynamics based on high voltage triggering. We will integrate graphene-based circuitry into our microfluidic devices to enable the use of a voltage-jump as both a general strategy for triggering protein motions and to control electron transfer reactions. The team is well qualified, merging expertise in microfluidics, X-ray science, and structural biology, with an established history of developing new technology to address challenges within the structural biology community. The impact will be improvements in the ability to perform time-resolved studies of protein structural dynamics that will immediately enhance the research capabilities of a large base of NIH-supported and other researchers.

项目摘要/摘要 下一代结构生物学实验着眼于超越对结构的静态观察，而是 对功能的动态、时间分辨的理解。这些下一层次的实验是通过串口实现的 X射线自由电子激光(XFELs)和微聚焦同步光束线的结晶学，但受限于 样品消耗的问题。这个项目的长期目标是使蛋白质结构的研究民主化。 通过为光、化学和电触发开发强大的固定目标安装策略来实现动态 时间分辨蛋白质结晶学实验，同时最大化样品利用率。这一努力利用了 SLAC国家加速器实验室和先进光子源的BioCARS设施的专业知识 用于测试和验证新的安装策略。这些关系还将使快速翻译 为更大的结构生物界开发技术。为此，我们有四个初步目标： 目标1：制定固定靶标策略，以促进大分子晶体靶标的研究 同时保持生物活性。我们将设计和制造与X射线兼容的样品支架 随着时间的推移保持蛋白质晶体的稳定性，同时还允许轻松的样品加载、机器人操作、 和原位光谱分析。一个特别的重点将包括在完全厌氧环境中实现数据收集， 这里开发的技术将作为后续目标努力的基础。 目标2：开发用于光触发反应的固定靶标平台，用于通过时间分辨进行分析 系列结晶学。我们将开发与X射线兼容的样品支架，利用流体动力 产生大规模晶体阵列的力量，用于系列结晶学实验。以聚合物为基础的 制造策略将实现快速、低成本的器件制造，以及允许样品的直接成像， 用于时间分辨X射线结晶学研究的原位光谱分析和基于激光的反应触发。 目标3：开发用于时间分辨序列的化学触发反应的固定靶标平台 晶体分析。我们将制定策略，控制一种化学物质的添加 (即底物、pH变化)，以实现晶体中化学反应的触发。 目的4：利用基于石墨烯的器件研究基于高压触发的结构动力学。 我们将把基于石墨烯的电路集成到我们的微流体设备中，以便能够使用电压跳跃作为 这既是触发蛋白质运动和控制电子转移反应的一般策略。 该团队资历深厚，融合了微流体、X射线科学和结构生物学方面的专业知识，并 建立了开发新技术以应对结构生物界挑战的历史。 其影响将是对蛋白质结构动力学进行时间分辨研究的能力的提高 这将立即增强NIH支持的大量研究人员和其他研究人员的研究能力。