Dynamics of Protein Assemblies by Analytical Ultracentrifugation

分析超速离心的蛋白质组装动力学

• 批准号: 9361484
• 负责人: PETER SCHUCK
• 金额: $ 57.32万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 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/9361484
• 关键词: 3D Print; Blinking; Books; Calibration; Characteristics; Chemicals; Collaborations; Computer software; Data Analyses; Development; Discrimination; Educational workshop; Equilibrium; Exhibits; Fluorescence; Foundations; Glutamate Receptor; Goals; Homo; Institutes; Knowledge; Light; Lighting; Macromolecular Complexes; Masks; Methodology; Methods; Modification; Morphologic artifacts; Optics; Play; Process; Protein Dynamics; Proteins; Publishing; Radial; Reaction; Reference Standards; Reporting; Research; Resolution; Role; Sampling; Sapphire; Sedimentation process; Signal Transduction; Source; Technology; Time; Work; analytical ultracentrifugation; improved; macromolecular assembly; nanoparticle; quantum; research study; sedimentation coefficient; sedimentation velocity; technique development

A major focus in our work on AUC in the reporting period was the further improvement of fluorescence-detected sedimentation velocity (FDS-SV). In order to achieve multi-component resolution despite the single excitation wavelength available, we have exploited the characteristic temporal change in fluorescence quantum yield of reversibly photoswitchable fluorescent proteins (rsFPs). We found these to be highly quantitative and reproducible, such that the temporal single change can be folded into the analysis of spatio-temporal concentration changes during sedimentation. We have shown that this new temporal signal domain can play an equivalent role as the spectral domain in multi-signal sedimentation velocity. Thus, a monochromatic multi-component sedimentation coefficient distribution analysis was embedded in the software SEDPHAT. A proof of principle application that different protein components can be distinguished was carried out using the competitive homo- and hetero-association of glutamate receptor ATDs of GluA2 and GluA3. Exploring further the new signal modulation capabilities provided by rsFPs, we took advantage of our customized analytical ultracentrifuge that allows us to illuminate the spinning rotor with light from high-powered LEDs to periodically restore the rsFPs state after photoswitching. The resulting blinking signal from rsFPs-tagged molecules can improve the discrimination of different components, especially for slowly-sedimenting molecules. To continue this research direction and examine different modes of illumination, we have installed a newly fabricated mock centrifuge on an optical table. Another opportunity to create new modes of analytical ultracentrifugation experiments is the modification of the sample container. To this end, we have developed 3d printing methodology for centerpieces and ancillary accessories. We found them to be cheap, reliable, and to perform surprisingly well. This will offer a versatile platform for further developments. For the purpose of studying nanoparticles, we have recently extended the implementation of time-varying fields to sedimentation-dominated processes in sedimentation velocity AUC. In order to clarify discrepancies obtained with different data analysis methods, we have studied their mathematical foundation and found new relationships between the time-derivative of the boundary and the sedimentation coefficient distribution, which led to a clarification of the source of significant artifacts in a time-difference method that was historically used. We have continued our collaboration with the National Institutes of Standards and Technology (Dr. Jeffrey Fagan and Dr. Thomas LeBrun) to develop a lithographic mask on sapphire substrate as a standard reference material for radial calibration in AUC. Finally, to better disseminate knowledge of analytical ultracentrifugation, in addition to organizing workshops, we have published a book that comprehensively describes its physical foundation and experimental practice.

报告期内我们对AUC工作的一个主要重点是进一步提高荧光检测沉降速度（FDS-SV）。 为了实现多组分分辨率，尽管单一的激发波长可用，我们已经利用了可逆的光开关荧光蛋白（rsFPs）的荧光量子产率的特征时间变化。 我们发现这些是高度定量和可重复的，这样的时间单一的变化可以被折叠到沉降过程中的时空浓度变化的分析。 我们已经表明，这个新的时间信号域可以发挥相当于多信号沉积速度的谱域的作用。 因此，单色多组分沉降系数分布分析被嵌入到软件SEDPHAT中。 使用GluA 2和GluA 3的谷氨酸受体ATDs的竞争性同源和异源缔合进行了可以区分不同蛋白质组分的原理应用的证明。 进一步探索rsFP提供的新信号调制功能，我们利用了我们定制的分析超级电容器，使我们能够用来自高功率LED的光照射旋转转子，以在光开关后定期恢复rsFP状态。 从rsFP标记的分子产生的闪烁信号可以提高对不同组分的区分，特别是对于缓慢沉降的分子。 为了继续这一研究方向并检查不同的照明模式，我们在光学工作台上安装了一个新制造的模拟离心机。 创建分析超离心实验的新模式的另一个机会是样品容器的修改。 为此，我们开发了用于中心装饰品和辅助配件的3D打印方法。 我们发现它们便宜、可靠，而且性能出奇地好。 这将为进一步发展提供一个多功能平台。 为了研究纳米粒子的目的，我们最近扩展了随时间变化的领域的实施，以沉降为主的过程中的沉降速度AUC。 为了澄清不同的数据分析方法得到的差异，我们研究了它们的数学基础，并发现了边界的时间导数和沉降系数分布之间的新关系，从而澄清了历史上使用的时间差方法中的重要工件的来源。 我们继续与美国国家标准与技术研究院（Jeffrey Fagan博士和托马斯LeBrun博士）合作，在蓝宝石衬底上开发光刻掩模，作为AUC径向校准的标准参考材料。 最后，为了更好地传播分析超离心的知识，除了组织研讨会外，我们还出版了一本书，全面介绍了它的物理基础和实验实践。