串行X射线液相色谱：用于探索微克量生物分子结构动力学的多维分析框架-动脉网

Serial X-ray liquidography: multi-dimensional assay framework for exploring biomolecular structural dynamics with microgram quantities

Nature 等信源发布 2024-07-26 06:04

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AbstractUnderstanding protein structure and kinetics under physiological conditions is crucial for elucidating complex biological processes. While time-resolved (TR) techniques have advanced to track molecular actions, their practical application in biological reactions is often confined to reversible photoreactions within limited experimental parameters due to inefficient sample utilization and inflexibility of experimental setups.

摘要了解生理条件下的蛋白质结构和动力学对于阐明复杂的生物过程至关重要。虽然时间分辨（TR）技术已经发展到跟踪分子作用，但由于样品利用率低和实验装置不灵活，它们在生物反应中的实际应用通常局限于有限实验参数内的可逆光反应。

Here, we introduce serial X-ray liquidography (SXL), a technique that combines time-resolved X-ray liquidography with a fixed target of serially arranged microchambers. SXL breaks through the previously mentioned barriers, enabling microgram-scale TR studies of both irreversible and reversible reactions of even a non-photoactive protein.

在这里，我们介绍了串行X射线液体成像（SXL），这是一种将时间分辨X射线液体成像与串行排列的微室的固定目标相结合的技术。SXL突破了前面提到的障碍，使微克级TR研究甚至是非光活性蛋白的不可逆和可逆反应成为可能。

We demonstrate its versatility in studying a wide range of biological reactions, highlighting its potential as a flexible and multi-dimensional assay framework for kinetic and structural characterization. Leveraging X-ray free-electron lasers and micro-focused X-ray pulses promises further enhancements in both temporal resolution and minimizing sample quantity.

我们证明了它在研究广泛的生物反应方面的多功能性，突出了它作为动力学和结构表征的灵活和多维分析框架的潜力。利用X射线自由电子激光器和微聚焦X射线脉冲有望进一步提高时间分辨率并最大限度地减少样品量。

SXL offers unprecedented insights into the structural and kinetic landscapes of molecular actions, paving the way for a deeper understanding of complex biological processes..

SXL对分子作用的结构和动力学景观提供了前所未有的见解，为深入了解复杂的生物过程铺平了道路。。

IntroductionProteins play vital roles, orchestrating life’s fundamental functions including enzyme catalysis, signal transduction, and immune response. A wealth of techniques, from site-directed mutagenesis to high-throughput screening in diverse chemical libraries has enriched our understanding of protein functions, revealing regulatory pathways, unveiling protein-protein interactions, and identifying promising drug targets1,2,3,4,5.

简介蛋白质起着至关重要的作用，协调生命的基本功能，包括酶催化，信号转导和免疫反应。。

In addition, progress in structural biology using X-ray crystallography, nuclear magnetic resonance, and electron microscopy, has propelled our comprehension of protein structure and function, revealing intricate details about protein folding, conformational changes, and molecular interactions6,7,8.

此外，使用X射线晶体学，核磁共振和电子显微镜的结构生物学进展推动了我们对蛋白质结构和功能的理解，揭示了蛋白质折叠，构象变化和分子相互作用的复杂细节6,7,8。

Yet, proteins are dynamic entities continuously shifting their shapes and interactions with other cellular components, as they perform their specific functions. To grasp the essence of the dynamic nature of proteins, understanding how their structures evolve over time is crucial. Despite employing various time-resolved (TR) approaches, capturing these fleeting changes during biological reactions remains time-consuming, resource-intensive, and laborious.Unraveling the complexity of molecular interactions and orchestrated movements during biological reactions demands the ability to initiate reactions with exquisite precision and track their progression with the utmost temporal acuity.

然而，蛋白质是动态的实体，在执行其特定功能时，不断改变其形状并与其他细胞成分相互作用。为了掌握蛋白质动态性质的本质，了解其结构如何随时间演变至关重要。尽管采用了各种时间分辨（TR）方法，但在生物反应过程中捕获这些短暂的变化仍然是耗时，资源密集且费力的。解开生物反应过程中分子相互作用和协调运动的复杂性需要能够以精确的精度启动反应，并以最大的时间敏锐度跟踪其进展。

Traditional mixing methods offer a universal approach to trigger molecular reactions, but they typically demand a substantial sample quantity while offering only a modest temporal resolution9,10,11. On the other hand, laser-based techniques, particularly those using the pump-probe scheme, where a pump (optical) pulse initiates a reaction and a time-delayed probe (op.

传统的混合方法提供了一种通用的方法来触发分子反应，但它们通常需要大量的样品，同时只能提供适度的时间分辨率9,10,11。另一方面，基于激光的技术，特别是那些使用泵浦探针方案的技术，其中泵浦（光学）脉冲引发反应和延时探针（op）。

Data availability

数据可用性

The data supporting the feasibility of the SXL method for Figs. 2, 3 are provided in source data format. In-depth analysis of the data for Figs. 4–6, which demonstrate the general applicability of the SXL method but have not yet been fully analyzed at the structural level, is planned for a separate publication.

支持图2、3的SXL方法可行性的数据以源数据格式提供。图4-6的数据的深入分析证明了SXL方法的普遍适用性，但尚未在结构层面进行充分分析，计划单独发布。

To avoid redundancy and focus on key findings, we are not releasing the raw data publicly in source data format at this time. Instead, we are prepared to present processed graphical representations of the raw scattering profiles at various stages of reaction in the Supplementary Information. The datasets intended for the forthcoming publication, along with the complete original 2D scattering images, which are too extensive to include as source data, can be obtained from the corresponding author upon request. Source data are provided with this paper..

为了避免冗余并专注于关键发现，我们目前不以源数据格式公开发布原始数据。。即将出版的出版物的数据集，以及完整的原始2D散射图像，这些图像太广泛而不能作为源数据，可以根据要求从相应的作者那里获得。本文提供了源数据。。

Code availability

代码可用性

In this study, we conducted a brief kinetic analysis of each reaction using built-in plugin functions in OriginLab (for exponential fitting) and MATLAB (for SVD analysis). These functions can be utilized with the manufacturer’s manuals. The KCA codes used in this study are available from the corresponding author upon request..

在这项研究中，我们使用OriginLab（用于指数拟合）和MATLAB（用于SVD分析）中的内置插件函数对每个反应进行了简短的动力学分析。这些功能可与制造商手册一起使用。本研究中使用的KCA代码可应要求从通讯作者处获得。。

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Download referencesAcknowledgementsWe thank the 4 C beamline staff at Pohang Accelerator Laboratory (PAL, Korea) for assistance with SAXS data collection. This work was supported by the Institute for Basic Science (IBS-R033). The use of BioCARS Sector 14 was supported by the National Institute of General Medical Sciences of the National Institutes of Health under grant number P41 GM118217.

下载参考文献致谢我们感谢浦项加速器实验室（PAL，韩国）的4 C光束线工作人员对SAXS数据收集的帮助。这项工作得到了基础科学研究所（IBS-R033）的支持。生物炭部门14的使用得到了美国国立卫生研究院国家普通医学科学研究所的支持，资助号为P41 GM118217。

Time-resolved set-up at Sector 14 was funded in part through a collaboration with Philip Anfinrud (NIH/NIDDK). We also thank Prof. M. Schmidt (University of Wisconsin-Milwaukee) for supporting the nanosecond laser system for time-resolved experiments at the BioCARS beamline.Author informationAuthors and AffiliationsCenter for Advanced Reactions Dynamics (CARD), Institute for Basic Science (IBS), Daejeon, 34141, Republic of KoreaSeong Ok Kim, So Ri Yun, Hyosub Lee, Junbeom Jo, Doo-Sik Ahn, Doyeong Kim, Jungmin Kim, Changin Kim, Seyoung You, Hanui Kim, Sang Jin Lee & Hyotcherl IheeDepartment of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, Republic of KoreaSeong Ok Kim, So Ri Yun, Hyosub Lee, Junbeom Jo, Doo-Sik Ahn, Doyeong Kim, Jungmin Kim, Changin Kim, Seyoung You, Hanui Kim, Sang Jin Lee & Hyotcherl IheeCenter for Advanced Radiation Sources, The University of Chicago, 9700 South Cass Avenue, Argonne, IL, 60439, USAIrina Kosheleva & Robert HenningAuthorsSeong Ok KimView author publicationsYou can also search for this author in.

第14部门的时间解决设置部分是通过与Philip Anfinrud（NIH/NIDDK）的合作获得资金的。我们还要感谢M.Schmidt教授（威斯康星大学密尔沃基分校）支持纳秒激光系统在BioCARS光束线上进行时间分辨实验。作者信息作者和附属机构大田基础科学研究所（IBS）高级反应动力学研究所（CARD），34141，大韩民国Ok Kim，So Ri Yun，Hyosb Lee，Junbeom Jo，Doo Sik Ahn，Doyeong Kim，Jungmin Kim，Changin Kim，Seyoung You，Hanui Kim，Sang Jin Lee＆Hyotcherl IheedDepartment of Chemistry，韩国高级科学技术研究所（KAIST），大田，34141，大韩民国Ok Kim，So Ri Yun，Hyosb Lee，Junbeom Jo，Doo Sik Ahn，Doyeong Kim，Jungjing金敏（Min Kim）、金昌宁（Changin Kim）、尤赛英（Seyoung You）、金哈妮（Hanui Kim）、李桑进（Sang Jin Lee）和李海哲（Hyotcherl Iheeecenter for Advanced Radiation Sources），芝加哥大学，伊利诺伊州阿贡市南卡斯大道9700号，邮编60439，USAIrina Kosheleva&Robert HenningAuthorsSeong Ok KimView Author Publications，您也可以在中搜索这位作者。

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PubMed Google ScholarContributionsS.O.K., D.A. and H.I. conceived the idea and developed the method. S.O.K., J.J. and D.A. designed and fabricated the S.X.L. chip. S.O.K. screened and selected the target samples, and S.O.K., S.R.Y. prepared the samples. I.K., R.H. and S.O.K. set up the beamline and developed the data collection schemes.

PubMed谷歌学术贡献。O、 K.，D.A.和H.I.构思了这个想法并开发了这个方法。S、 O.K.，J.J.和D.A.设计并制造了S.X.L.芯片。S、 O.K.筛选并选择了目标样品，S.O.K.，S.R.Y.制备了样品。一、 K.，R.H.和S.O.K.建立了光束线并制定了数据收集方案。

S.O.K., J.J., S.R.Y., H.L., D.K., D.A., C.K., S.Y., H.K. and S.L. participated in the data collection. S.O.K., S.R.Y., H.L., J.K., and D.K. processed data. S.O.K., S.R.Y., H.L. and D.K. analyzed the data. S.O.K. and H.I. wrote and edited the original drafts. HI supervised the whole project. All authors read and commented on the manuscript.Corresponding authorCorrespondence to.

S、 O.K.，J.J.，S.R.Y.，H.L.，D.K.，D.A.，C.K.，S.Y.，H.K.和S.L.参与了数据收集。S、 O.K.，S.R.Y.，H.L.，J.K。和D.K.处理了数据。S、 O.K.，S.R.Y.，H.L.和D.K.分析了数据。S、 O.K.和H.I.撰写并编辑了原始草稿。他监督了整个项目。所有作者都阅读并评论了手稿。对应作者对应。

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Reprints and permissionsAbout this articleCite this articleKim, S.O., Yun, S.R., Lee, H. et al. Serial X-ray liquidography: multi-dimensional assay framework for exploring biomolecular structural dynamics with microgram quantities.

转载和许可本文引用本文Kim，S.O.，Yun，S.R.，Lee，H。等人。系列X射线液相色谱：用于探索微克量生物分子结构动力学的多维测定框架。

Nat Commun 15, 6287 (2024). https://doi.org/10.1038/s41467-024-50696-0Download citationReceived: 08 February 2024Accepted: 17 July 2024Published: 26 July 2024DOI: https://doi.org/10.1038/s41467-024-50696-0Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

《国家公社》156287（2024）。https://doi.org/10.1038/s41467-024-50696-0Download引文收到日期：2024年2月8日接受日期：2024年7月17日发布日期：2024年7月26日OI：https://doi.org/10.1038/s41467-024-50696-0Share。复制到剪贴板。

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Enzyme mechanismsKineticsMolecular conformationSAXS

酶机制皮肤分子构象SAXS

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