Dynamics of Protein Assemblies by Analytical Ultracentrifugation

本报告所述期间，我们在AUC方面的主要工作重点是进一步改进荧光检测沉降速度（FDS-SV）。为了在单一激发波长下实现多组分分辨率，我们利用了可逆光开关荧光蛋白（rsfp）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性的，这样的时间单一变化可以折叠到沉积过程中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在多信号沉降速度中可以起到与谱域等效的作用。因此，在SEDPHAT软件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ATDs的竞争性同源和异交结合，进行了区分不同蛋白质成分的原理应用证明。

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.