A New Dimension of Detection in Analytical Ultracentrifugation with Fluorescence Detection using Photoswitchable FPs as Time Domain Probes

Abstract

Multi-component protein complex formation is of great interest in physiological and biochemical studies as it is ubiquitous in biological systems. Analytical ultracentrifugation (AUC) provides powerful methods for studying such systems. It offers information on size, shape and binding energies for interacting systems from the analysis of sedimentation profiles of molecular mixtures in free solution. By virtue of the superb hydrodynamic resolution achieved in sedimentation velocity, co-existing complexes can be identified, even in the presence of impurities and aggregates. A recently introduced fluorescence optical detection system (FDS) for AUC substantially extends capabilities for studying high-affinity protein interactions because of the selectivity of fluorescence high sensitivity, allowing studies at picomolar concentrations. To overcome limitations posed by a single excitation wavelength, in the current study, we employed photoswitchable fluorescent proteins (FP) as probes in FDS-AUC. Taking advantage of their characteristic time-dependent fluorescent signal change due to photoswitching during FDS-AUC, a new dimension of detection is created that allows resolving specific FPs in multi-component mixtures. We have developed a computational framework for the analysis of sedimentation data exploiting the new temporal dimension. Experimentally we demonstrated this approach using mixtures of FPs commonly used in imaging, which could be readily distinguished from experimental FDS data. This approach extends FDS-AUC to more complicated protein system with multiple components and allows for quantitative characterization of protein complexes with regard to the binding mechanism and stoichiometry under very low macromolecular concentrations.