The single exponential lifetime of ECFP was measured as 2.49 ns, which is in close agreement with the published value 2.53 ns [121]. Consistently with our FRET measurements, during which the lifetime decreases to 2.01 ns, this value is 1.95 ns for an ECFP-EYFP fusion protein. Literature values for other GFP mutants range from 2.7 to 3.8 ns [86, 122, 123, 124].
The binding constants from the time-resolved FRET assay presented here are very close to the results from the steady-state assay presented and validated in chapter 4. Indeed these results serve as an independent validation of the steady-state analysis based on the 530 nm emission peak.
Time-resolved FRET detection is a promising assay for the quantitative study of protein-protein binding. A major benet is the fact that FRET, measured as the decrease in donor lifetime, is purely a measure of binding and is hence only a function of the relative protein concentrations and their binding anity. This is validated experimentally by adding more ECFP-SUMO1 at the end of the titration and recovering the lifetimes previously measured at that ECFP-SUMO1/EYFP-Ubc9 ratio. Direct excitation and dilution during the titration do not obscure the FRET signal, greatly simplifying the experiments. The sensitivity of this measurement depends on the collection time for each lifetime trace which in turn impacts on the precision of the tting procedure. However, specic equipment is required to perform measurements at this short timescale at photon-counting sensitivity. Several systems are available commercially, however these are not likely to be as prolic in bioscience labo- ratories as steady-state uorimeters.
5.4. CONCLUSION
5.4 Conclusion
In this time-resolved FRET assay to quantify protein-protein binding we mea- sure the uorescence lifetime of a xed amount of donor ECFP-tagged protein solution using a TSCPC setup. The concentration of the EYFP-tagged binding partner in this solution is gradually increased in a titration, and the resulting energy transfer measured as a decrease in the ECFP lifetime at 475 nm. Life- times computed from deconvolution and both single and double exponential ts are used as binding data. Fitting with hyperbolas gives binding constants that compare well with the results obtained in steady state measurements in chapter 4. Together with the comparison to ITC results this validates the use of FRET as a quantitative binding signal, and its use for determining the equilibrium binding constant of a protein pair.
6
Multi-Protein Interactions Analysed by
FRET
A major advantage of monitoring binding by means of FRET is that the eect of third molecules on the binding of two selected proteins can be measured. This provides a convenient method for screening drug candidates which may modify protein interactions and a powerful tool for the study of biochemical cascades within which several proteins bind competitively.
To illustrate this we investigate binding and competition of proteins dur- ing the SUMOylation of substrates, and present a method of quantifying com- petitive binding by determining the inhibition constant Ki.
6.1. MULTI-PROTEIN EXPERIMENTS
It requires the concerted eorts of multiple proteins to catalyse the for- mation of a covalent bond between the small ubiquitin-like modier SUMO1 and a variety of dierent protein targets as discussed in chapter 2.5. The nal step of this conjugation involves the binding of RanBP2 to a covalently linked Ubc9-SUMO1 complex, and the formation of a trimeric bound complex. The covalent Ubc9-SUMO1 link is essential for SUMOylation to occur, although in its absence, a bound trimeric complex could still be formed due to mutual binding sites on the three proteins. The exact mechanism of this nal stage is unknown, but studying the mutual binding anity of the three proteins by FRET can shed light on the cascade of interactions.
Materials used here were ECFP-SUMO1, EYFP-Ubc9, ECFP-RanBP2(l), EYFP-RanBP2(l), SUMO1, RanBP2(l) and RanBP2(s).
6.1 Multi-Protein Experiments
Multi-protein binding was quantied by tagging the two proteins to be mea- sured with a donor ECFP and an acceptor EYFP respectively and making up an equimolar solution of the two. The eect of a third, untagged molecule is measured during a titration into this solution, while spectra are recorded as in chapter 4.
Firstly, experiments were performed to determine the eect of two RanBP2 fragments on the bound SUMO1-Ubc9 complex. The longer RanBP2(l) binds Ubc9 but its SUMO1 binding site is concealed [125]. The shorter RanBP2(s) was shown to bind both SUMO1 and Ubc9 individually in GST pulldown ex- periments. The following titrations in 3.5 ml quartz cuvettes were monitored in a Cary Eclipse Spectrophotometer:
• RanBP2(s) was titrated into 1 ml of 1µM ECFP-SUMO1 + 1µM EYFP-
Ubc9, in 17 x 5 µl additions up to 3.1 µM nal concentration
• RanBP2(l) was titrated into 1 ml of 1 µM ECFP-SUMO1 + 1µM EYFP-
Ubc9
Next, controls were performed with untagged SUMO1 to contrast the competition of SUMO1 for its tagged counterpart with that of the RanBP2 fragments. This reveals the relative binding anity of the proteins for Ubc9.
• SUMO1 was titrated into 1 ml of 1 µM ECFP-SUMO1 + 1 µM EYFP-
Ubc9 in 20 x 5 µl additions up to 3 µM nal concentration (control for
substitution of ECFP-SUMO1 with SUMO1)
Furthermore, the eect of Ubc9 titrated into ECFP-SUMO1 + EYFP- RanBP2(l) and that of SUMO1 on the ECFP-RanBP2(l) + EYFP-Ubc9 com- plex were monitored during the following titrations:
• Ubc9 was titrated into 1 ml of 1 µM ECFP-SUMO1 + 1 µM EYFP-
RanBP2 in 20 x 5 µl additions up to 3 µM nal concentration
• SUMO1 was titrated into 1 ml of 1 µM ECFP-SUMO1 + 1 µM EYFP-
Ubc9 in 20 x 5 µl additions up to 3 µM nal concentration
• The last titration was repeated with more SUMO1, in 5 µl additions in
6.2. THE EFFECT OF RANBP2 ON SUMO1 AND UBC9 BINDING