In previous years we have detected significant calibration problems with the time and radial dimensions in the analytical ultracentrifuge (AUC), along with large temperature errors. Since these parameters are critical for all quantitative thermodynamic and hydrodynamic applications of AUC, we have previously developed suitable external calibration devices, and organized a large benchmark study with > 70 participating laboratories worldwide. In the reporting period we have concluded and published this study. The results show that the external calibration tools are both highly effective and essential for quantitative AUC. 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. Another focus of our AUC work in the reporting period was the further development of fluorescence-detected sedimentation velocity. With the goal to develop multi-wavelength fluorescence detection capabilities, we have continued to work on the modification of one of our analytical ultracentrifuges. After the equipment of the rotor chamber with fiber optical and electrical feedthroughs, we have now installed an optical stage in the rotor chamber, and proceeded to the design of data acquisition electronics in collaboration with Dr. Thomas Pohida. We have also explored the application of fluorescence detected AUC to the study of unpurified proteins directly in diluted cell extracts. We have established detection limits for endogenously expressed EGFP in bacterial and mammalian cell extracts, and were able to demonstrate the detection of interactions between EGFP or EFFP-fused proteins with different binding partners. Departing from tradition in AUC, we have developed strategies that exploit time-varying centrifugal fields. We have first implemented algorithms for the solution of the master equation of sedimentation and diffusion in the presence of arbitrarily time-varying fields for the case of diffusion-dominated processes. Embedded in an optimization program for the centrifugal field changes, we have shown that it is possible to achieve sedimentation equilibrium at a fraction of the time required for conventional experiments. This is an important experimental consideration, as the common time-requirement for sedimentation equilibrium studies was on the order of days and therefore prohibitive for many samples. This is now reduced to several hours. We have further extended the implementation of time-varying fields to sedimentation-dominated processes in sedimentation velocity AUC. We have developed computational and experimental tools for the gravitational sweep sedimentation, where a steadily increasing rotor speed allows one to explore a very large size range of particles quasi-simultaneously. We expect this to be highly useful for the study of very large macromolecular assemblies (such as virions, fibrils, or reversibly formed multi-protein complexes) simultaneously with much smaller building blocks.
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