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Method Article
Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique.
Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.
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 Kd), but it does not measure the kinetics of the binding, nor can it be applied when the Kd 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 factor6,7.
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 Cul110,11,12,13. 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 vivo14,15,16,17. 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 (kon and koff, respectively) were measured individually. The measurements revealed that both Cand1 and Skp1•F-box protein form extremely tight complex with Cul1, but the koff of SCF is dramatically increased by Cand1 and the koff of Cul1•Cand1 is dramatically increased by Skp1•F-box protein6,7. 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 kon and koff, 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.
1. Design the FRET assay.
Figure 1: The crystal structure of Cul1•Cand1 and measurement of the distance between potential labeling sites. The crystal structure file was downloaded from Protein Data Bank (File 1U6G), and viewed in PyMOL. Measurements between selected atoms were done by PyMOL. Please click here to view a larger version of this figure.
Figure 2: The excitation and emission spectra of the fluorescent dyes for FRET. Spectra of AMC (7-amino-4-methylcoumarin) and FlAsH are shown. Dashed lines indicate excitation spectra, and solid lines indicate emission spectra. The image was originally generated by the Fluorescence SpectraViewer and was modified for better clarity. Please click here to view a larger version of this figure.
2. Preparation of Cul1AMC•Rbx1, the FRET donor protein
3. Preparation of FlAsHCand1, the FRET acceptor protein
NOTE: Most of steps in this part are the same as Step 2. Conditions that differ are described in detail below.
4. Preparation of Cand1, the FRET chase protein
NOTE: The protein preparation protocol is similar to Step 3, with the following modifications.
5. Test and confirm the FRET assay
6. Measure the association rate constant (kon) of Cul1•Cand1
NOTE: Details of operating a stopped-flow fluorimeter has been described in a previous report26.
7. Measure the dissociation rate constant (koff) of Cul1•Cand1 in the presence of Skp1•F-box protein.
NOTE: This step is similar to Step 6, with the following modifications.
To test the FRET between Cul1AMC and FlAsHCand1, we first determined the emission intensity of 70 nM Cul1AMC (the donor) and 70 nM FlAsHCand1 (the acceptor), respectively (Figure 3A-C, blue lines). In each analysis, only one emission peak was present, and the emission of FlAsHCand1 (the acceptor) was low. When 70 nM each of Cul1AMC and FlAsHCand1 were mixed to genera...
FRET is a physical phenomenon that is of great interest for studying and understanding biological systems19. Here, we present a protocol for testing and using FRET to study the binding kinetics of two interacting proteins. When designing FRET, we considered three major factors: the spectral overlap between donor emission and acceptor excitation, the distance between the two fluorophores, and the dipole orientation of the fluorophores28. To choose the fluorophores for FRET, ...
The authors have nothing to disclose.
We thank Shu-Ou Shan (California Institute of Technology) for insightful discussion on the development of the FRET assay. M.G., Y.Z., and X.L. were funded by startup funds from Purdue University to Y.Z. and X.L.This work was supported in part by a seed grant from Purdue University Center for Plant Biology.
Name | Company | Catalog Number | Comments |
Anion exchange chromatography column | GE Healthcare | 17505301 | HiTrap Q FF anion exchange chromatography column |
Benchtop refrigerated centrifuge | Eppendorf | 2231000511 | |
BL21 (DE3) Competent Cells | ThermoFisher Scientific | C600003 | |
Calcium Chloride | Fisher Scientific | C78-500 | |
Cation exchange chromatography column | GE Healthcare | 17505401 | HiTrap SP Sepharose FF |
Desalting Column | GE Healthcare | 17085101 | |
Floor model centrifuge (high speed) | Beckman Coulter | J2-MC | |
Floor model centrifuge (low speed) | Beckman Coulter | J6-MI | |
Fluorescence SpectraViewer | ThermoFisher Scientific | https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html | |
FluoroMax fluorimeter | HORIBA | FluoroMax-3 | |
FPLC | GE Healthcare | 29018224 | |
GGGGAMC peptide | New England Peptide | custom synthesis | |
Glutathione beads | GE Healthcare | 17075605 | |
Glycerol | Fisher Scientific | G33-500 | |
HEPES | Fisher Scientific | BP310-100 | |
Isopropyl-β-D-thiogalactoside (IPTG) | Fisher Scientific | 15-529-019 | |
LB Broth | Fisher Scientific | BP1426-500 | |
Ni-NTA agarose | Qiagen | 30210 | |
Ovalbumin | MilliporeSigma | A2512 | |
pGEX-4T-2 vector | GE Healthcare | 28954550 | |
Protease inhibitor cocktail | MilliporeSigma | 4693132001 | |
Reduced glutathione | Fisher Scientific | BP25211 | |
Refrigerated shaker | Eppendorf | M1282-0004 | |
Rosetta Competent Cells | MilliporeSigma | 70953-3 | |
Size exclusion chromatography column | GE Healthcare | 28990944 | Superdex 200 10/300 GL column |
Sodium Chloride (NaCl) | Fisher Scientific | S271-500 | |
Stopped-flow fluorimeter | Hi-Tech Scientific | SF-61 DX2 | |
TCEP·HCl | Fisher Scientific | PI20490 | |
Thrombin | MilliporeSigma | T4648 | |
Tris Base | Fisher Scientific | BP152-500 | |
Ultrafiltration membrane | MilliporeSigma | UFC903008 | Amicon Ultra-15 Centrifugal Filter Units, Ultra-15, 30,000 NMWL |
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