This is a beautiful example of the precision, simplicity and versatility of using FRET for quantitative studies. The FRET-based post-translational modi- cation assay is ideal for the detection of further SUMO-modied substrates. As shown in previous experiments, the format is easily adapted to multi-well plate and high-throughput screening formats.
The high temporal sensitivity and the quantitative nature of the detec- tion by FRET can not only lead to new insights into substrate specicity, but also characterise the conjugation process as a function of the concentration of catalysing molecules such as Ubc9 and RanBP2. Buer constituents, ATP con- centration and temperature are further variables of interest. Initial conjugation rates, determined for a range of substrate concentrations form the basis of the analysis of Michaelis-Menten kinetics. Similar to the binding curve hyperbola in chapter 4, this analysis yields the constant Km, the substrate concentration
at which the initial reaction rate is half the maximum rate. Monitoring reac- tion rates by FRET gives a set of data ideal for advanced analysis. This can give valuable input to models of pathways, and enable predictions concerning in vivo interactions.
Following this proof of principle, we planned further work to explore the options discussed above, consolidate the analysis techniques and present a comprehensive method. However these plans were shelved when similar work
7.3. DISCUSSION OF FRET IN POST-TRANSLATIONAL MODIFICATION
was published by Bossis et al [1].
Figure 7.3: Figures 2 A and 2 B from Bossis et al [1]:"(A) Unprocessed emission data. Twenty-ve microliter reaction mixes containing YFP-SUMO1, CFP- GAPtail, Aos1/ Uba2, and Ubc9 were incubated in a 384-well plate at 30C in the absence or presence of 1 mM ATP. Fluorescence after excitation at 430 nm was measured every minute at 485 and 527 nm. (B) Processed data. The rate of conjugation correlates directly with the change in the ratio of emissions (527 nm/485 nm). The value observed in the absence of ATP is due to the uorescence of CFP at 485 and 527 nm (YFP is not excited to signicant levels at 430 nm) and reects 0%modication. After the addition of ATP, the ratio
of emissions increases linearly until it reaches a stable plateau. This reects 100% modication[...]"
In a very similar experiment, presented in well-plate format, they demon- strate the conjugation of YFP-SUMO1 to a version of CFP-RanGAP1. They expand the initial experiment to include varying concentrations of ATP, Aos1/Uba2 (SAE1/2), untagged RanGAP1, Ubc9 mutants and a demonstration of decon- jugation by SenP1. The data is presented in unprocessed form and as the 527 nm / 485 nm ratio, but no advanced analysis such as Michaelis-Menten tting was attempted. Figure 7.3 shows the initial results and data handling and
includes the original gure caption. The rst graph shows the uorescence data collected at 485 and 527 nm (lter bandpass width is not quoted) during a conjugation reaction and a control reaction without ATP. In both cases the uorescence at 485 nm decreases with time - however a decrease due to energy transfer would only be expected in the active experiment with ATP. Indeed, the decrease with ATP appears more prominent. However in the control - in which the 527 nm emission decreases as well - this "is most likely due to pho- tobleaching and/or denaturation of the recombinant proteins" [1]. This is a surprising conclusion, since both uorescent proteins have proven very stable both photophysically and structurally in our measurements. The changes in uorescence presented here are more likely to be a result of increasing tempera- ture and hence vibrational quenching. Since the experiments are performed at elevated temperatures, this may reect the equilibration of the well-plate. Fur- thermore, the resulting wavelength ratio results in a zero gradient line, which reinforces the theory that losses may be due to temperature, as a uniform loss of signal across the spectrum will cancel when dividing the intensity of two wavelengths.
According to Bossis et al, "the change in the emission ratio over time is a direct readout for the rate of the conjugation reaction." This statement is however neither veried nor referenced. While the ratio is a convenient way of eliminating intensity changes across whole spectra (such as due to dilution and temperature changes) and visualising spectral shifts, the division of two ltered readouts by no means represents a direct measure of the number of molecules undergoing FRET and hence a readout of rates. In fact, if denatu- ration and photobleaching were present as stated above this would denitely not be true. In chapter 8 we experimentally validate the use of the acceptor peak as a linearly proportional measure of molecules undergoing FRET. How-
7.3. DISCUSSION OF FRET IN POST-TRANSLATIONAL MODIFICATION
ever dividing this by the decreasing donor emission leads to an overestimation of rates.
Figure 7.4: Emission spectra (excitation 400 nm) of ECFP-SUMO1 and EYFP- RanGAP before (blue) and after (red) conjugation.
A further point to note about the rst graph is that the emission intensity at 527 nm is about half that at 485 nm. Since details of the emission lters are not disclosed, the spectral shape cannot be derived from the information given. Moreover, lter-based well-plate readers allow for the separate adjustment of a gain ("fudge") factor which is multiplied by the emission readout. The authors state that YFP is not excited to signicant levels at 430 nm - it is clear from our results in preceeding chapters that direct excitation indeed has to be born in mind when using FRET data quantitatively. However, since the authors do not attempt a rigorous analysis, this omission is not catastrophic. Figure 7.4 shows corrected spectra for a solution of ECFP-SUMO1 and EYFP-RanGAP1 before (blue) and after (red) conjugation, demonstrating the spectral change due to FRET in this case, and the true relative peak emissions.
7.4 Conclusion
The techniques most commonly used in the kinetic analysis of protein conju- gation, such as the SUMOylation of substrates, require the manual removal of sample from a reaction before fractionation on electrophoresis gels. This is a labour-intensive process and the temporal resolution is clearly limited.
We demonstrated that these limitations can be overcome when using FRET as a conjugation signal. The emission at 530 nm from an ECFP-SUMO1 and EYFP-RanGAP1 solution increases as a result of FRET during the conju- gation of SUMO1 to RanGAP1, and closely follows gel data obtained in paral- lel. A sensitive quantitative kinetic analysis of post-translational modication is achievable by this method. This is readily rolled out to high-throughput screening format for dierent substrates, potential ligases and inhibitors.
8
FRET Detection of Protease Activity
Using Doubly-Tagged SUMO1
The protease SENP1 activates SUMO1 precursors by cleaving peptide bonds during the initial maturation of SUMO1, and is involved again in the nal stage of SUMOylation, where it deconjugates SUMO1 from modied substrates by cleaving isopeptide bonds. As in the conjugation presented in chapter 7, deconjugation is also usually quantied by immunoblotting or autoradiography of samples fractionated by SDS electrophoresis. Reaction rates determined from such experiments give valuable insights into the concentration-dependent timescales of deconjugation and the specicity of a protease for a range of
possible substrates.
In this chapter we present uorescently labelled substrates that enable the kinetic analysis and detailed characterization of both the C-terminal hydro- lase and the isopeptidase activities of SUMO1 proteases by FRET [117], as well as measurement and analysis methods. In particular, SUMO1 doubly-tagged with ECFP and EYFP is examined as a substrate for SenP1 which cleaves a GG-HSTV motif on the C-terminus of SUMO1. This approach opens a vast range of experimental possibilities for the study of proteases interacting with ubiquitin-like proteins, some of which are presented here, and forms the basis of subsequent research [127].
Materials used in this chapter were SUMO1, EYFP-SUMO1-ECFP, ECFP- SUMO1-EYFP, EYFP-SUMO1-RanGAP1-ECFP, SenP1(415−644)(kind gift from
L. Shen, University of Dundee)