Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Biological activities are ultimately carried out by proteins, most of which interact with others for proper biological functions. Using a computational approach, the total amount of protein-protein interactions in human is estimated to be ~650,000¹, and disruption of these interactions often leads to diseases². Due to their essential roles in controlling cellular and organismal processes, numerous methods have been developed to study protein-protein interactions, such as yeast-two-hybrid, bimolecular fluorescence complementation, split-luciferase complementation, and co-immunoprecipitation assay³. While these methods are good at discovering and confirming protein-protein interactions, they are usually non-quantitative and thus provide limited information about the affinity between the interacting protein partners. Quantitative pull-downs can be used to measure the binding affinity (e.g., the dissociation constant K_d), but it does not measure the kinetics of the binding, nor can it be applied when the K_d is very low due to an inadequate signal-to-noise ratio⁴. Surface plasmon resonance (SPR) spectroscopy quantifies the binding kinetics, but it requires a specific surface and immobilization of one reactant on the surface, which can potentially change the binding property of the reactant⁵. Moreover, it is difficult for SPR to measure fast association and dissociation rates⁵, and it is not appropriate to use SPR to characterize the event of exchanging protein subunits in a protein complex. Here, we describe a method that allows measuring rates of protein complex assembly and disassembly at a millisecond time scale. This method was essential for determining the role of Cullin-associated-Nedd8-dissociated protein 1 (Cand1) as the F-box protein exchange factor^6,7.

Cand1 regulates the dynamics of Skp1•Cul1•F-box protein (SCF) E3 ligases, which belong to the large family of Cullin-RING ubiquitin ligases. SCFs consist of the cullin Cul1, which binds the RING domain protein Rbx1, and an interchangeable F-box protein, which recruits substrates and binds Cul1 through the adaptor protein Skp1⁸. As an E3 ligase, SCF catalyzes the conjugation of ubiquitin to its substrate, and it is activated when the substrate is recruited by the F-box protein, and when Cul1 is modified by the ubiquitin-like protein Nedd8⁹. Cand1 binds unmodified Cul1, and upon binding, it disrupts both the association of Skp1•F-box protein with Cul1 and the conjugation of Nedd8 to Cul1^10,11,12,13. As a result, Cand1 appeared to be an inhibitor of SCF activity in vitro, but Cand1 deficiency in organisms caused defects that suggests a positive role of Cand1 in regulating SCF activities in vivo^14,15,16,17. This paradox was finally explained by a quantitative study that revealed the dynamic interactions among Cul1, Cand1, and Skp1•F-box protein. Using fluorescence resonance energy transfer (FRET) assays that detect the formation of the SCF and Cul1•Cand1 complexes, the association and dissociation rate constants (k_on and k_off, respectively) were measured individually. The measurements revealed that both Cand1 and Skp1•F-box protein form extremely tight complex with Cul1, but the k_off of SCF is dramatically increased by Cand1 and the k_off of Cul1•Cand1 is dramatically increased by Skp1•F-box protein^6,7. These results provide the initial and critical support for defining the role of Cand1 as a protein exchange factor, which catalyzes the formation of new SCF complexes through recycling Cul1 from the old SCF complexes.

Here, we present the procedure of developing and using the FRET assay to study the dynamics of the Cul1•Cand1 complex⁷, and the same principle can be applied to study the dynamics of various biomolecules. FRET occurs when a donor is excited with the appropriate wavelength, and an acceptor with excitation spectrum overlapping the donor emission spectrum is present within a distance of 10-100 Å. The excited state is transferred to the acceptor, thereby decreasing the donor intensity and increasing the acceptor intensity¹⁸. The efficiency of FRET (E) depends on both the Förster radius (R₀) and the distance between the donor and acceptor fluorophores (r), and is defined by: E = R₀⁶/(R₀⁶ + r⁶). The Förster radius (R₀) depends on a few factors, including the dipole angular orientation, the spectral overlap of the donor-acceptor pair, and the solution used¹⁹. To apply the FRET assay on a stopped-flow fluorimeter, which monitors the change of the donor emission in real-time and enables measurements of fast k_on and k_off, it is necessary to establish efficient FRET that results in a significant reduction of donor emission. Therefore, designing efficient FRET by choosing the appropriate pair of fluorescent dyes and sites on the target proteins to attach the dyes is important and will be discussed in this protocol.