Accounting for Photophysical Processes and Specific Signal Intensity Change in Fluorescence-Detected Sedimentation Velocity Analytical Ultracentrifugation

Fluorescence detected sedimentation velocity (FDS-SV) analytical ultracentrifugation has emerged as a powerful technique for the study of macromolecular interactions, particularly high-affinity protein interactions, with hydrodynamic resolution exceeding that of diffusion-based techniques, and with sufficient sensitivity for binding studies at low picomolar concentrations. In order to fit the FDS data structure, in the quantitative analysis it is essential to adjust the conventional sedimentation models for detailed description of the sedimentation boundaries. A key consideration is the change in the macromolecular fluorescence intensity during the course of the experiment, caused by slow drifts of the excitation laser power, and/or by photophysical processes. In the present work we demonstrate that FDS-SV data have inherently a reference for the time-dependent macromolecular signal intensity, resting on a geometric link between boundary migration and plateau signal. We show how this new time-domain can be exploited to study molecules exhibiting photobleaching and photoactivation. This expands the application of FDS-SV to proteins tagged with photoswitchable fluorescent proteins, organic dyes, or nanoparticles, such as those recently introduced for sub-diffraction microscopy. At the same time, we find conventional fluorophores undergo minimal photobleaching under standard illumination in the FDS. These findings support the application of a high laser power density for the detection, which we demonstrate can further increase the data quality.