Analysis of High-Affinity Protein Interactions by Fluorescence Optical Analytical Ultracentrifugation

Abstract

Analytical ultracentrifugation (AUC) offers substantial advantages for studying protein complex formation, providing information on size, shape and binding energies for reversible systems from analysis of the sedimentation profiles of molecular mixtures in free solution. By virtue of the hydrodynamic resolution achieved in sedimentation velocity experiments, multiple co-existing complexes can be identified, even in the presence of impurities and aggregates, but until recently the limited sensitivity of AUC has prevented accurate analysis of high affinity interactions. A recently introduced fluorescence optical detection system (FDS) for AUC offers specific advantages for studying high-affinity protein interactions due to the high sensitivity of fluorescence, but the unique characteristics of the optical system in this instrument require new analytical tools. We have developed computational approaches for analysis of FDS data that provide excellent fits to the observed sedimentation boundaries, correcting for effects intrinsic to the use of confocal laser optics. With these tools we have explored the detection limit of FDS, and have now recorded sedimentation signals with hydrodynamic resolution at low picomolar concentrations. This allows us to analyze sedimentation isotherms for interacting systems with Kds in the pM to nM range. Using different systems with high-affinity interaction we will demonstrate how the FDS-AUC system can be reliably applied to study high-affinity protein self- and hetero-associations at orders of magnitude lower concentrations and with much higher accuracy than previously possible.