MICROFLUIDIC PLATFORMS FOR LAUE CRYSTALLOGRAPHY

用于劳厄晶体学的微流控平台

• 批准号: 8363681
• 负责人: Paul J. A. Kenis
• 金额: $ 3.04万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363681
• 关键词: Area; Chemicals; Coupling; Crystallization; Crystallography; Data; Data Collection; Devices; Diffuse; Environment; Funding; Grant; Imagery; In Situ; Kinetics; Ligands; Liquid substance; Methods; Microfluidics; Modeling; National Center for Research Resources; Noise; Phase; Principal Investigator; Proteins; Radiation; Research; Research Infrastructure; Resources; Roentgen Rays; Sampling; Signal Transduction; Source; Structure; Techniques; Technology; Time; United States National Institutes of Health; cost; interest; research study; structural biology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. While efforts in microfluidics for protein crystallization have developed dramatically in the past ten years[1-9] the idea of coupling microfluidic technology with advanced crystallographic techniques for in situ analysis is an area where significant advances can be made. Thus far efforts have focused mainly on either simple devices where monochromatic X-rays can be used to interrogate the sample.[2 4 5 10-26] However this approach has been of limited utility because of the need to rotate samples in order to fully sample phase space. An alternative to the more traditional monochromatic X-ray analysis is a polychromatic method termed Laue crystallography.[27] In this method the use of a range of X-ray wavelengths allows for significantly faster data collection enabling the analysis of tiny or fragile crystals that are susceptible to radiation damage[28 29] as well as kinetic experiments.[30] Because of fast data collection it is possible to perform time-resolved experiments by matching the X-ray exposure time to the timescale of chemical and structural changes in the protein. Thus Laue crystallography provides a very elegant platform for performing extremely meaningful kinetic experiments that directly probe changes that occur during enzymatic function. Microfluidic platforms have the benefit of not only enabling experiments at very small volumes but also by creating an environment free of inertial or convective effects and allowing for exquisite control over local conditions and gradients. Here we propose coupling microfluidic platforms for protein crystallization with in situ Laue crystallographic analysis. By combining the control and throughput of microfluidic platforms with this powerful structural analysis method we hope to further enable the field of dynamic crystallographic analysis. Experimentally we need to first validate our platforms for use with Laue analyses in terms of signal to noise rotational requirements visualization and sample handling. A second proof-of-concept effort would involve the structure determination of a model protein either from a single crystal or from an array of microcrystals. Subsequent efforts will involve creating an array of crystals that have been exposed to a range of conditions. For instance the array of crystals could be exposed to a range of ligand molecule concentrations or pH values. More advanced studies will involve coupling real-time fluid handling with in situ analysis for dynamic crystallization experiments. For example a chamber containing a crystal could be controllably exposed to a second chamber containing a chemical of interest. Time resolved structural data could be collected as the chemical of interest diffuses into the crystal.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 虽然蛋白质结晶的微流体技术在过去十年中取得了巨大的发展[1-9]，但将微流体技术与先进的晶体学技术相结合进行原位分析的想法是一个可以取得重大进展的领域。 迄今为止，工作主要集中在可使用单色 X 射线来询问样品的简单设备上。[2 4 5 10-26] 然而，这种方法的实用性有限，因为需要旋转样品才能充分采样相空间。 更传统的单色 X 射线分析的替代方法是称为劳厄晶体学的多色方法。 [27] 在这种方法中，使用一系列 X 射线波长可以显着加快数据收集速度，从而能够分析易受辐射损伤的微小或易碎晶体[28 29]以及动力学实验。[30] 由于数据收集速度快，因此可以通过将 X 射线照射时间与蛋白质化学和结构变化的时间尺度相匹配来执行时间分辨实验。 因此，劳厄晶体学提供了一个非常优雅的平台，用于进行极其有意义的动力学实验，直接探测酶功能过程中发生的变化。 微流控平台的优点是不仅可以进行非常小体积的实验，而且还可以创建一个没有惯性或对流效应的环境，并允许对局部条件和梯度进行精确控制。 在这里，我们建议将用于蛋白质结晶的微流体平台与原位劳厄晶体分析相结合。 通过将微流体平台的控制和吞吐量与这种强大的结构分析方法相结合，我们希望进一步推动动态晶体分析领域的发展。 在实验上，我们需要首先在信噪比旋转要求可视化和样本处理方面验证我们的平台是否可与劳厄分析一起使用。 第二个概念验证工作将涉及从单晶体或微晶体阵列中确定模型蛋白质的结构。 随后的工作将涉及创建一系列暴露在一系列条件下的晶体。 例如，晶体阵列可以暴露于一定范围的配体分子浓度或pH值。更先进的研究将涉及将实时流体处理与动态结晶实验的原位分析相结合。 例如，包含晶体的室可以可控地暴露于包含感兴趣的化学物质的第二室。 当感兴趣的化学物质扩散到晶体中时，可以收集时间分辨的结构数据。