A major focus in our work on analytical ultracentrifugation (AUC) technology in the reporting period was the further improvement of fluorescence-detected sedimentation velocity (FDS-SV). To this end, we have embarked on the development of an improved detector, which we expect to have higher sensitivity through a different optical design, to allow recording faster sedimentation processes, and to increasing the dynamic range by simultaneously acquiring absorbance data. We have also examined in more detail the sedimentation process of rapidly reacting protein mixtures where transient binding events lead to enhancement or quenching of the fluorescence signals. Such binding-induced modulations of fluorescence signals are commonly observed in many protein interactions. For such systems, we have discovered anomalies in the concentration-dependence of the average sedimentation coefficients, which can be explained and modeled using effective particle theory (EPT). EPT describes the formation of reaction boundaries and, more generally, the physical principles of the coupled sedimentation boundary patterns in solutions where dissolved particles exhibit rapidly reversible binding. Previously EPT was known only for simple two-component systems, and we have now extended EPT to the general case of mixtures with an arbitrarily high number of components and complexes. This can serve as a quantitative framework for the analysis of complicated interacting mixtures. To improve experimental precision, 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 Part II and III of a book series that comprehensively describes data analysis for interacting and non-interacting systems, respectively, extending Part I on experimental procedures previously published in 2015. We have also written a detailed step-by-step protocol to better enable colleagues to adopt the technique of FDS-SV in studies of high-affinity interactions.
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