A novel method involving quantitative analysis of FRET (Förster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.
Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the “native” proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 ± 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.
1. Plasmid Constructs
2. Protein Expression and Purification
3. Quantitative FRET Spectrum Analysis
where FL530/414 is the total fluorescence emission at 530 nm when excited at 414 nm, FLFRET is the absolute FRET signal, FLCyPet(cont) is the CyPet direct emission when excited at 414 nm, and FLYPet (cont) is the YPet direct emission when excited at 414 nm. The subscript of (cont) stands for contribution.
where FL’530/414 is the total fluorescence emission at 530 nm after digestion when excited at 414 nm, FL’FRET is the remaining absolute FRET signal, FL’CyPet(475/414)is the CyPet emission at 475 nm after digestion when excited at 414 nm (here the CyPet emission is from two parts: undigested CyPet-(pre-SUMO1)-YPet and digested CyPet-SUMO1), and FLYPet (530/475) is the YPet emission when excited at 475 nm, which is constant whether CyPet-(pre-SUMO1)-YPet is digested or not.
where C is the total concentration of CyPet-(pre-SUMO1)-YPet and x is the concentration of digested CyPet-(pre-SUMO1)-YPet.
4. FRET-based Protease Assay for Enzyme Kinetic Study
5. Representative Results
Maturation of pre-SUMO1 by SENP1 can be determined by monitoring the changes in the fluorescence signal at 475 and 530 nm during the process. The result showed that the velocity of pre-SUMO1 digestion by SENP1 in a substrate-dose dependent manner (Figure 4). This suggests that the catalytic domain of SENP1 exhibits excellent activity for pre-SUMO1’s maturation. The initial reaction velocities were calculated by the above analysis with different substrate concentrations (Table 1).
The kcat/KM ratio is generally used to compare the efficiencies of different enzymes with one substrate or a particular enzyme with different substrates. KM and Vmax can be obtained from the Michaelis-Menten equation by plotting the various initial velocities, corresponding to the different concentrations of CyPet-(pre-SUMO1)-YPet (Figure 5). kcat was obtained as:
According to the above analysis, the calculated KM was 0.21 ± 0.04 μM, the kcat was 6.90 ± 0.28 s-1, and the kcat/KM ratio was (3.2 ± 0.55) x107 M-1s-1.
Figure 1. Graph of FRET-based protease assay for SENP’s pre-SUMOs maturation.
Figure 2. Quantitative analysis of fluorescent signal as contributions by the donor, acceptor and FRET. Dissection of emission spectra from CyPet-(pre-SUMO1)-YPet under excitation at 414 nm. FLCyPet(530/414) is CyPet’s emission at 530 nm under excitation of 414 nm, FLFRET is the FRET-induced YPet emission at 530 nm under excitation of 414 nm, and FLYPet(530/414) is YPet’s emission at 530 nm under excitation of 414 nm.
Figure 3. Calculation of direction emission factor α and β.
Figure 4. Quantitative analysis of CyPet-(pre-SUMO1)-YPet digested by different ratios of the catalytic domain of SENP1. Reactions were monitored within the first 5 min.
Figure 5. Michaelis-Menten graphical analysis of CyPet-(pre-SUMO1)-YPet’s digestion by SENP1. Data were plotted and analyzed by GraphPad Prism V and nonlinear regression.
[S](μM) | V0(μM/s) |
0.115 | 0.0023±0.00005 |
0.214 | 0.0028±0.00004 |
0.407 | 0.0033±0.00007 |
0.594 | 0.0037±0.00013 |
0.725 | 0.0043±0.00012 |
1.471 | 0.0051±0.00036 |
1.899 | 0.0050±0.00031 |
2.300 | 0.0050±0.00062 |
Table 1. Initial velocities determination of pre-SUMO1’s maturation by SENP1. In each substrate concentration, four samples were used to measure the digestion. The standard deviation came from the variations of these four samples.
KM(μM) | kcat(s-1) | kcat/kM(M-1•s-1) |
0.21±0.04 | 6.90±0.28 | 3.2±0.55×107 |
Table 2. Kinetic parameters of pre-SUMO1’s maturation by SENP1 by quantitative FRET analysis. The standard deviation came from the four samples in each substrate concentration.
FRET technology has been used to study pre-SUMO1’s maturation by SENP19. CFP-YFP was used as the FRET pair and ratiometric analysis, which is the ratio of acceptor to donor emissions, was used to characterize the kinetic properties. However, there is no consideration of donor and acceptor self-fluorescence in the traditional ratiometric FRET analysis. The ratio does not directly correlate with the amount of digested substrate.
Here we report a developed highly sensitive FRET-based protease assay to study the kinetic of pre-SUMO1’s maturation by SENP1. In contrast to the previous ratiometric approach, we fundamentally improved the method with a new theory of FRET signal for kinetic analysis and an experimental procedure to derive kinetic parameters by determining the quantitative contributions of self-fluorescence from donor and acceptor, and the real FRET-induced acceptor’s emission. Ratiometric analysis cannot do this. The ignorance of self-fluorescent emissions of donor and acceptor may lead to an overestimation of the FRET signal and the donor’s emission. The overestimations might not greatly affect the final kcat/KM ratio (3.81 x107 M-1s-1 for the ratiometric analysis, 3.2 x107 M-1s-1 by our quantitative FRET analysis), but the effect is more obvious when studying the individual parameters, KM (0.098 vs 0.21 μM) and kcat (3.43 s-1 vs 6.90 s-1), which are important in determining the rate-limiting step and inhibitor potency of enzymes.
The method we report here is a one-step assay of protease kinetics parameters and requires only molecular cloning and protein expression without radioactive labeling or expensive instruments. The one-step procedure not only simplifies the experimental procedure but also limits a lot of variations. The fluorescent-tagged proteins are in the aqueous phase, which is typically similar to their natural environment in cells. Fluorescence intensity can be determined by general fluorescence spectroscopy or fluorescence plate readers, which are widely available. Compared with the traditional “gel-based” method, our FRET-based protease assay offers several advantages, including increased sensitivity, real-time measurement, and less time and labor needed. Furthermore, the methodology and procedure of protease kinetics parameter determinations are environmentally friendly and non-hazardous materials, such as radioisotopes or harsh chemicals. In addition, the highly sensitive FRET-based assay can be used in high-throughput biological assays, such as protease inhibitor screenings. The kinetic study can also be used to characterize the properties of the inhibitors (e.g. Ki, IC50).
Therefore, the highly sensitive quantitative FRET-based protease assays could be a powerful approach in developing genome-wide protease-substrate profiling and inhibitor screenings.
The authors have nothing to disclose.
We are very grateful to Victor G.J. Rodgers for valuable advice. We thank all of the members of the Liao group for very close collaborative work and for help with this study. This study was supported by the National Institutes of Health (Grant AI076504 to J.L.).
Name of the reagent | Company | Catalogue number |
PCR II TOPO Kit | Invitrogen | K466040 |
2xYT | Research Products Internationa.l Corp. | X15600 |
Ni-NTA Agrose | Thermo Scientific | 88222 |
Coomassie plus (Bradford) Assay Regent | Thermo Scientific | 23238 |
384-well plate (glass bottom) | Greiner | 781892 |
FlexStation II 384 plate reader | Molecular Device |