Accounting for Photophysical Processes and Specific Signal Intensity Changes in Fluorescence-Detected Sedimentation Velocity

Fluorescence detected sedimentation velocity (FDS-SV) has emerged as a powerful technique for the study of high-affinity protein interactions, with hydrodynamic resolution exceeding that of diffusion-based techniques, and with sufficient sensitivity for binding studies at low picomolar concentrations. For the detailed quantitative analysis of the observed sedimentation boundaries, it is necessary to adjust the conventional sedimentation models to the FDS data structure. 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 radial boundary migration and plateau signal. We show how this new time-domain can be exploited to study molecules exhibiting photobleaching and photoactivation. This e...