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: 8743775
• 负责人: PETER SCHUCK
• 金额: $ 28.05万
• 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/8743775
• 关键词: Accounting; Affinity; Antibodies; Behavior; Binding; Biological Models; Calibration; Chemicals; Chimeric Proteins; Computer software; Data; Data Quality; Detection; Devices; Equilibrium; Exhibits; Experimental Designs; Fluorescence; Foundations; Goals; Intention; Investigation; Label; Laboratories; Lasers; Left; Literature; Macromolecular Complexes; Manufacturer Name; Measurement; Measures; Modeling; Modification; Mono-S; Optics; Procedures; Property; Protein Dynamics; Proteins; Radial; Reaction; Reporting; Retinal Cone; Sedimentation process; Series; Signal Transduction; Solid; Solutions; Structure; System; Techniques; Temperature; Testing; Time; Writing; analytical ultracentrifugation; data acquisition; data integrity; design; detector; expectation; fluorophore; improved; instrument; macromolecular assembly; optical imaging; research study; sedimentation coefficient; sedimentation velocity; technique development; tool

Significant progress during the reporting period was made in the use of the newly purchased fluorescence detector for the analytical ultracentrifuge. Fluorescence-detected analytical ultracentrifugation (FD-AUC) is a relatively new technique that provides sub-nanomolar detection sensitivity. We have carried out comprehensive series of experiments aimed at exploring the properties of this detector. We found that modifications of the standard sedimentation analysis models are necessary. Motivated by the optical design of the detector, we developed models that can account for spatial gradients of signal magnification, temporal drifts of laser intensity, photo-bleaching of the fluorophore, and the obscurement of the detection cone close to the end of the solution column. After accounting for these effects, the resulting data quality was found to be comparable or exceeding that of the best conventional detection, allowing for the first time a consistently reliable and detailed interpretation of the FD-AUC data. These tools were implemented and disseminated in our analysis software SEDFIT. Next, we explored the effect of different labelling and experimental design strategies for FD-AUC in the study of high-affinity protein self-association and hetero-association. Using GluA2 ATD as a model system, we found the fluorescent label, FAM could create multiple classes of molecules with different binding properties, whereas the more polar Dylight label appears to leave the binding properties of GluA2 unaffected, indistinguishable from unlabelled molecules as well as GFP fusion proteins. The broad isotherm caused by the polydispersity of FAM-GluA2 explains previously observed discrepancies of FAM-labeled GluA2 ATD apparent hydrodynamic behaviour with the hydrodynamic expectation. This removes previously noted concerns about the reliability of sedimentation coefficients measured in FD-AUC. Finally, we developed an approach to improve the detection limits of FD-AUC by 1 to 2 orders of magnitude, which now brings interacting systems with picomolar binding constants into the dynamic range of AUC. As an example, we revisited an GFP antibody previously reported in the literature to be of too high affinity for FD-AUC, and to show (unexpectedly) only mono-valent binding. With our new tools, we were able to determine a 10 pM binding constant with bi-valent binding, consistent with the antibody structure. Thus, taken together, these results set FD-AUC on a solid foundation for the quantitative study of high-affinity assembly reactions, extending the corresponding capabilities of conventional AUC by three orders of magnitude. In a different line of investigation we tested existing and developed new calibration procedures for analytical ultracentrifugation (conventional and FD-AUC). The need for this arose from the observation of inconsistent sedimentation coefficients measured in different instruments. We discovered that the current manufacturer data acquisition software misreported experimental times by 10%. We wrote and disseminated software to restore data integrity. Further, we discovered similarly serious deficiencies in the temperature and radial calibration. We developed a new device for temperature measurement in the spinning rotor, and for the accurate determination of the radial magnification of the imaging optics. By combination of these three external calibrations, we were able to restore accuracy of the sedimentation parameters of a test protein across eleven different instruments. Our intention is to expand this study to many laboratories, in order to assure a uniform quality standard for hydrodynamic measurements.

在报告期间，在分析超级离心设备中使用新购买的荧光检测器时取得了重大进展。 荧光检测的分析超速离心（FD-AUC）是一种相对较新的技术，可提供亚纳摩尔检测敏感性。 我们进行了一系列旨在探索该检测器的特性的全面实验。 我们发现，必须对标准沉积分析模型进行修改。 通过检测器的光学设计，我们开发了模型，这些模型可以解释信号放大的空间梯度，激光强度的时间漂移​​，荧光团的光泽以及接近溶液柱末端的检测锥的晦涩。 在考虑了这些效果之后，发现所得的数据质量是可比或超过最佳常规检测的效果，这首先允许对FD-AUC数据进行一致可靠且详细的解释。 这些工具已在我们的分析软件Sedfit中实施和传播。 接下来，我们探讨了FD-AUC在高亲和力蛋白质自我关联和异质关联研究中的不同标记和实验设计策略的影响。 使用GLUA2 ATD作为模型系统，我们发现荧光标签可以创建具有不同结合特性的多种分子，而更极性Dylight标签似乎使GLUA2的结合特性不受影响，与未分配的分子以及GFP PPP融合蛋白无法区别。由FAM-GLUA2的多分散性引起的广泛等温线解释了先前观察到的FAM标记的GLUA2 ATD明显的流体动力行为的差异，并具有流体动力学的预期。这消除了以前对FD-AUC测量沉积系数的可靠性的担忧。 最后，我们开发了一种方法，将FD-AUC的检测限制提高了1至2个数量级，现在将具有皮摩尔结合常数的相互作用系统带入了AUC的动态范围。 例如，我们重新审视了先前在文献中报道的GFP抗体对FD-AUC的亲和力过高，并且仅显示（意外）单价结合。 借助我们的新工具，我们能够通过双价结合确定10 pm的结合常数，与抗体结构一致。 因此，这些结果共同将FD-AUC设定在固体基础上，以进行高亲和力组装反应的定量研究，从而将常规AUC的相应能力扩展到三个数量级。 在不同的调查中，我们测试了现有的分析超速离心（常规和FD-AUC）的新校准程序。 对此的需求来自观察到不同仪器中测得的不一致的沉积系数。 我们发现当前的制造商数据采集软件将实验时间误导了10％。我们编写并传播软件以恢复数据完整性。 此外，我们在温度和径向校准中发现了类似的严重缺陷。 我们开发了一种新的设备，用于旋转转子中的温度测量，并准确地确定成像光学元件的径向放大倍率。 通过这三个外部校准的结合，我们能够恢复11种不同仪器中测试蛋白的沉积参数的准确性。 我们的目的是将这项研究扩展到许多实验室，以确保用于流体动力学测量的统一质量标准。