Fluorescence Detected Sedimentation Velocity Analytical Ultracentrifugation for Investigating Affinity and Stoichiometry of Protein Interactions

Abstract

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is monitoring the migration of particles in free solution with real-time optical detection. It provides powerful methods for studying multi-component systems of biomacromolecules such as protein and DNA. It offers exquisite resolution regarding the size and shape of the components and population of co-existing complexes, and therefore binding energies and stoichiometries for reversible interacting systems can be resolved. A recently introduced fluorescence optical detection system (FDS) for AUC offers specific advantages for studying molecules at low concentration because of the high sensitivity and selectivity of fluorescence. In order to make FDS-SV a robust and quantitative method for studying macromolecules, we have previously developed computational tools to specifically account for the unique data structure posed by the confocal design and other geometric factors. Such developments allow FDS-SV to be applied to high-affinity binding systems at concentrations well into the picomolar regime. Recently we extended FDS-SV to multi-dimensional signal detection by employing photoswitchable fluorescent proteins and taking advantage of their characteristic time-dependent fluorescent signal change due to photoswitching. Thus, FDS-SV is an excellent tool for the study of high-affinity protein interactions. We will show applications to protein-protein and protein-nucleic acid interactions with multiple states, quantitatively determining the binding affinity, stoichiometry and cooperativity.