Dynamics of Protein Assemblies by Analytical Ultracentrifugation

在本报告所述期间，我们在AUC方面工作的一个主要重点是进一步提高荧光检测沉积速度(FDS-SV)。为了在单一激发波长下实现多组分分辨，我们利用了可逆光开关荧光蛋白(RsFP)的荧光量子产率随时间变化的特性。我们发现这些都是高度定量和可重复性的，因此时间上的单一变化可以合并到沉积过程中的时空浓度变化的分析中。我们已经证明，这种新的时间信号域可以在多信号沉积速度中起到与谱域相同的作用。因此，在软件SEDPHAT中嵌入了单色多组分沉降系数分布分析。利用GluA2和GluA3的谷氨酸受体ATD的竞争性同源和异源结合，进行了区分不同蛋白质组分的原理应用的证明。 我们进一步探索了rsfps提供的新信号调制功能，利用我们定制的分析超离心机，我们可以用高功率LED发出的光照射旋转的转子，从而在光开关后定期恢复rsfps的状态。Rsfps标记的分子产生的闪烁信号可以改善对不同成分的区分，特别是对缓慢沉淀的分子。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台新制造的模拟离心机。 另一个创造分析超速离心法实验新模式的机会是样品容器的改装。为此，我们开发了用于中心件和辅助附件的3D打印方法。我们发现它们便宜、可靠，而且表现得出人意料地好。这将为进一步的发展提供一个通用的平台。 为了研究纳米粒子，我们最近将时变场的实现扩展到沉降速度AUC中的沉降主导过程。为了澄清不同数据分析方法得到的差异，我们研究了它们的数学基础，发现了边界的时间导数与沉积系数分布之间的新关系，从而澄清了历史上使用的时差方法中重要伪迹的来源。 我们继续与美国国家标准与技术研究院(Jeffrey Fagan博士和Thomas LeBrun博士)合作，在蓝宝石衬底上开发一种光刻掩模，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速离心法的知识，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。

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.