Investigating High Affinity Protein Self-Association by Fluorescence Optical Sedimentation Velocity Analytical Ultracentrifugation

Sedimentation velocity analytical ultracentrifugation (SV) is a powerful first-principle technique for the study of protein interactions and the rigorous characterization of binding stoichiometry and affinities in free solution. With a recently introduced commercial fluorescence optical detection system (FDS) it has gained the potential for applications to be extended to high-affinity interactions. However, for most proteins the attachment of an extrinsic fluorophore is an essential prerequisite for the study by FDS-SV. Fluorescent labeling induced artifacts present one key difficulty in the study of the homo-oligomerization of proteins by FDS-SV. Using the high-affinity homo-dimerization of the glutamate receptor GluA2 amino terminal domain as a model system, we demonstrate how the choice of fluorescent label can impact both the observed binding constants as well as the derived estimates of the hydrodynamic parameters of protein species. Specifically, FAM (5,6-carboxyfluorescein) was found to create different populations of artificially high-affinity and low-affinity dimers, as indicated by both FDS-SV and the kinetics of Forster Resonance Energy Transfer observed in bench-top spectro-fluorometry. By contrast, Dylight488 labeled protein as well as protein expressed as an EGFP fusion, yielded results consistent with estimates from unlabeled protein. Our study suggests considerations for the choice of labeling strategies, and highlights experimental designs that exploit specific opportunities of FDS-SV for improving the reliability of the binding isotherm analysis of interacting systems.