One of the techniques with major promise for the study of multi-protein complexes is multi-signal sedimentation velocity, and we have continued the development of this approach. We have devised a novel mass conservation constraint to be used as a form of Bayesian regularization. We have mathematically shown that it can significantly aid in the spectral discrimination of components. In collaboration with Dr. Chad Brautigam, the performance of this approach is now being tested with simulated and experimental data sets. A fundamental quantity derived from sedimentation velocity is the hydrodynamic frictional coefficient of translation, which reports on the protein solution conformation. In particular, this coefficieint is very informative in conjunction with structural predictions. In a collaboration with Drs. Ghirlando, Brautigam, and Aragon, we have set out to compare results from the new high-precision boundary element methods for structure-based prediction of friction coeffcients with experimental data from sedimentation velocity. From initial collection of data, we have identified key experimental factors. All ultracentrifugation experiments are subject to macromolecular buoyancy as a dominating factor of sedimentation behaviour. To complement existing compositional and densimetry approaches for the determination of macromolecular partial-specific volume, we have developed a density contrast sedimentation velocity method. This was successfully tested on several model proteins. Finally, we have updated our SEDFIT software with new tools for sedimentation equilibrium and sedimentation velocity studies of intrinsically polydisperse macromolecules, such as carbohydrates.

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National Institute of Biomedical Imaging and Bioengineering
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LeBrun, Thomas; Schuck, Peter; Wei, Ren et al. (2018) A radial calibration window for analytical ultracentrifugation. PLoS One 13:e0201529
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