Fluorescence Optical Sedimentation Velocity With Photoswitchable Proteins

Monday, September 22, 2014 — Poster Session I
12:00 p.m. – 2:00 p.m.	FAES Academic Center	NIBIB	BIOENG-9

Authors

• P Schuck
• J Ma
• G Patterson
• H Zhao

Abstract

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 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. Furthermore, this provides the potential for monochromatic multi-component detection by discrimination of the time-dependent signals of different fluorophores. This should be highly useful in the study of high-affinity heterogeneous protein interactions and multi-protein complex formation.

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