Dynamics of Protein Assemblies by Analytical Ultracentrifugation

在本报告所述期间，在使用新购买的荧光检测器进行超痕量分析方面取得了重大进展。 荧光检测的分析超滤（FD-AUC）是一种相对较新的技术，可提供亚纳摩尔的检测灵敏度。 我们已经进行了一系列旨在探索这种探测器的性能的实验。 我们发现，标准的沉降分析模型的修改是必要的。 出于检测器的光学设计，我们开发的模型，可以占信号放大率的空间梯度，激光强度的时间漂移，荧光团的光漂白，以及接近溶液柱末端的检测锥的模糊。 考虑到这些影响后，发现所得数据质量与最佳常规检测相当或超过最佳常规检测，首次对FD-AUC数据进行了一致可靠和详细的解释。 这些工具已在我们的分析软件SEDFIT中实施和传播。 接下来，我们探索了不同标记和实验设计策略对FD-AUC在高亲和力蛋白质自缔合和异缔合研究中的影响。 使用GluA 2 ATD作为模型系统，我们发现荧光标记，FAM可以产生具有不同结合特性的多种类型的分子，而极性更大的Dylight标记似乎不影响GluA 2的结合特性，与未标记的分子以及GFP融合蛋白难以区分。由FAM-GluA 2的多分散性引起的宽等温线解释了先前观察到的FAM标记的GluA 2 ATD表观流体动力学行为与流体动力学预期的差异。这消除了先前注意到的关于FD-AUC中测量的沉降系数的可靠性的问题。 最后，我们开发了一种方法，以提高检测限的FD-AUC的1至2个数量级，现在带来的相互作用系统的皮摩尔结合常数的AUC的动态范围。 作为一个例子，我们重新审视了先前在文献中报道的GFP抗体，其对FD-AUC具有太高的亲和力，并且（出乎意料地）仅显示单价结合。 使用我们的新工具，我们能够确定与二价结合的10 pM结合常数，与抗体结构一致。 因此，总的来说，这些结果为高亲和力组装反应的定量研究奠定了坚实的基础，将常规AUC的相应能力扩展了三个数量级。 在不同的调查路线中，我们测试了现有的和开发的新的分析超离心校准程序（常规和FD-AUC）。 之所以需要这样做，是因为观察到不同仪器测量的沉降系数不一致。 我们发现，当前制造商的数据采集软件误报了10%的实验时间。我们编写并分发了恢复数据完整性的软件。 此外，我们发现在温度和径向校准方面存在类似的严重缺陷。 我们开发了一种新的装置，用于测量旋转转子中的温度，并用于精确确定成像光学系统的径向放大率。 通过组合这三种外部校准，我们能够在11种不同仪器上恢复测试蛋白质的沉降参数的准确性。 我们的目的是将这项研究扩展到许多实验室，以确保流体动力学测量的统一质量标准。

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.