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仪用于分析性超离心机方面取得了重大进展。荧光检测分析超离心（FD-AUC）是一种相对较新的技术，提供亚纳摩尔检测灵敏度。为了探索这种探测器的特性，我们进行了一系列全面的实验。我们发现对标准沉降分析模型进行修正是必要的。在检测器光学设计的激励下，我们开发了可以解释信号放大的空间梯度、激光强度的时间漂移、荧光团的光漂白以及靠近溶液柱末端的检测锥的遮挡的模型。在考虑了这些影响后，发现所得数据质量可与最佳常规检测相媲美或超过，从而首次对FD-AUC数据进行了一致可靠和详细的解释。这些工具在我们的分析软件SEDFIT中实施和传播。

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.