Dynamics of Protein Assemblies by Analytical Ultracentrifugation

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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.