Analytical Ultracentrifugation with Fluorescence Detection System Using Photoswitchable GFPs as Time Domain Probes

Authors

• H Zhao
• Y Fu
• C Glasser
• E Andrade
• M Mayer
• G Patterson
• P Schuck

Abstract

Multi-component protein complex formation is of great interest in physiological and biochemical studies as it is ubiquitous in numerous biological systems. Analytical ultracentrifugation (AUC) provides powerful methods for studying such systems. It offers information on size, shape and binding energies for reversible systems from analysis of sedimentation profiles of molecular mixtures in free solution. By virtue of the superb hydrodynamic resolution achieved in sedimentation velocity, multiple co-existing complexes can be identified, even in the presence of impurities and aggregates. A recently introduced fluorescence optical detection system (FDS) for AUC offers specific advantages for studying high-affinity protein interactions because of the high sensitivity and selectivity of fluorescence. In the current study, we employed photoswitchable GFP molecules as fluorescent probes in FDS-AUC and took the advantage of their time-dependent fluorescent signal change due to photoswitching as a new dimension of detection. We have developed computational approaches to account for such unique time-dependent signal so the size and shape of the species carrying the photoswitchable molecule can be resolved in the time domain spectrum. The photoswitchable molecules with different switching kinetics were examined with FDS and the different time domain spectra were demonstrated to be distinguishable for the model system under the current study. This time domain detection adds a new dimension of detection in FDS-AUC and will expand AUC application to more complicated protein system with multiple components and allow for quantitative and reliable analysis of the binding mechanism and stoichiometry under very low macromolecular concentrations.

Scientific Focus Area: Biomedical Engineering and Biophysics

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