Analysis of high affinity self-association by fluorescence optical sedimentation velocity analytical ultracentrifugation of labeled proteins: opportunities and limitations.

Sedimentation velocity analytical ultracentrifugation (SV) is a powerful first-principle technique for the study of protein interactions, and allows a rigorous characterization of binding stoichiometry and affinities. A recently introduced commercial fluorescence optical detection system (FDS) permits analysis of high-affinity interactions by SV. However, for most proteins the attachment of an extrinsic fluorophore is an essential prerequisite for analysis by FDS-SV. Using the glutamate receptor GluA2 amino terminal domain as a model system for high-affinity homo-dimerization, we demonstrate how the experimental design and choice of fluorescent label can impact both the observed binding constants as well as the derived hydrodynamic parameter estimates for the monomer and dimer 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 dimer dissociation studied using a bench-top fluorescence spectrometer and Förster Resonance Energy Transfer. By contrast, Dylight488 labeled GluA2, as well as GluA2 expressed as an EGFP fusion protein, yielded results consistent with estimates for unlabeled GluA2. 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.