Team:TU-Eindhoven/Results/Functionality

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Functionality

• Heterodimer
  • Introduction
  • Functionality
  • Orthogonality
  • Conclusion
• Tetramer
• NanoBit curve
• Proof of concept
• Interlab Study
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Functionality

In order to get an understanding of the functionality of our heterodimers, NanoLuc assays were conducted. The relative intensity of each scaffold was measured, and the concentration of scaffold was varied get information about the functionality of the scaffold. All data was visualized by integrating the measured bioluminescence over a certain period of time. This time period was the time in which the bioluminescence decreased in a linear pattern. This is visualized in bar graphs. An example of a measured bioluminescence curve and the integration over time can be found

here.

Relative intensity

The relative intensity was depicted in order to check the functionality of each set of mutations. On the y-axis the bioluminescence is presented for each set of mutations in a bar plot. The heterodimers and a homodimer are depicted next to their negative controls.

Figure 1: T14-3-3 functionality measurement representing the relative intensities from each mutation set. The scaffold concentrations used are 1 µM. 50 µM fusicoccin was used for all measurements. CT52-LargeBiT and CT52-SmallBiT were used with a concentration of 200 nM, CT52-LargeBiT contained the mutation. The substrate used was 1250x diluted from stock.

The first interesting thing is the activity when a wildtype homodimer is used, it is significantly lower than three of the four heterodimers (figure 1). The NanoBiTs are two non-identical fragments and when a homodimer is used there is no control over which NanoBiT binds where. Thus resulting in scaffolds with two LargeBiTs or two SmallBiTs. This might partially explain the difference in bioluminescence.

The second interesting thing that is observed, is the activity from the heterodimer with the W237R mutation (figure 1). The bioluminescence measured is close to the background activity, suggesting no significant increase in active NanoLuc could be created by the scaffold. So apparently the mutation in the T14-3-3 or the complementary mutation in CT52 disrupts the binding interaction, assuming both proteins were successfully expressed.

The last notable thing are the intensities observed from the other three heterodimers (figure 1), the S71L, S71L&I72V and E19R mutation sets. They all give significant higher bioluminescence values compared to their background, even when their standard deviation has been taken into account. This suggests that functional NanoLuc complexes have been formed on the scaffolds. The E19R mutation set has already been used before and has been known to work [1]. The S71L and S71L&I72V mutation sets both have comparable bioluminescence to the E19R mutation set, this suggests that both sets are functional.

Varied scaffold concentrations

The concentrations of the scaffolds were varied in order to check if the scaffold functions as expected. When higher concentrations of scaffold are used, we expect the amount of scaffold bound NanoBiTs to increase as well. When the number of bound NanoBiTs increases, the measured bioluminescence will increase as well. Of course this is not infinitely true, eventually the concentration of CT52 fused NanoBiTs will become a limiting factor leading to a biphasic effect, to read more about this phenomenon visit our modelling page. We varied the concentration of scaffold from 0 to 1 µM and the following results were obtained. On the y-axis the bioluminescence is presented, and each bar plot represents a different scaffold concentration, with 0 on the left and 1µM on the right.

Figure 2: T14-3-3 functionality measurement with varying scaffold concentrations from 0 nM to 1 µM T14-3-3(S71L)-T14-3-3. 50 µM fusicoccin was used for all measurements. Mutant forms CT52 (I947H)-LargeBiT and CT52(wildtype)-SmallBiT were used with a concentration of 200 nM. The substrate used was 1250x diluted from stock.

In this graph (figure 2) a rising trend can be observed, a higher concentration scaffold clearly has an effect on the measured luminescence. The higher the scaffold concentration, the higher the level of luminescence. The relative intensity graph suggested that the S71L scaffold and complementary CT52 form a functional system, and these measurements strongly suggest the same.

Figure 3: T14-3-3 functionality measurement with varying scaffold concentrations from 0 nM to 1 µM T14-3-3(S71L&I72V)-T14-3-3. 50 µM fusicoccin was used for all measurements. Mutant forms CT52 (I947F)-LargeBiT and CT52(wildtype)-SmallBiT were used with a concentration of 200 nM. The substrate used was 1250x diluted from stock.

In this graph (figure 3) a rising line can be observed, however it is not as steep and smooth as the one observed in the graph from the S71L scaffold. This graph suggests that the S71L&I72V mutation works.

Figure 4: T14-3-3 functionality measurement with varying scaffold concentrations from 0 nM to 1 µM T14-3-3(W237R)-T14-3-3. 50 µM fusicoccin was used for all measurements. Mutant forms CT52 (I953K)-LargeBiT and CT52(wildtype)-SmallBiT were used with a concentration of 200 nM. The substrate used was 1250x diluted from stock.

The trend observed in the graph from the heterodimer with a W237R mutation (figure 4) is more or less a horizontal line. Meaning the concentration of scaffold has no influence on the measured luminescence. Another matter to take into account are the intensities, these are very low. So even though some observed differences might seem substantial, they are not significant. So it seems like this set of mutations results in a none functional system. This was expected, because the relative intensity graph suggested that the W237R scaffold or complementary mutated CT52 was not functional, and these measurements would support that suggestion.

Figure 5: T14-3-3 functionality measurement with varying scaffold concentrations from 0 nM to 1 µM T14-3-3(E19R)-T14-3-3. 50 µM fusicoccin was used for all measurements. Mutant forms CT52 (K943D)-LargeBiT and CT52(wildtype)-SmallBiT were used with a concentration of 200 nM. The substrate used was 1250x diluted from stock.

The graph (figure 5) does not show an increasing trend as smooth as we would expect, also some of the standard deviations are rather large. Another interesting matter is the background activity, where no scaffold is present, which is rather high. So the ‘relative intensity’ graph suggests that the E19R mutation in T14-3-3 and the complementary mutated CT52 resulted in a functional system. However, this graph shows that the scaffold shows not expected behaviour, which is propably caused by an inaccuracy in measurement.

References

• [1] References: Skwarczynska, M., Molzan, M., & Ottmann, C. (2013). Activation of NF-κB signalling by fusicoccin-induced dimerization. Proceedings of the National Academy of Sciences, 110(5), E377-E386.

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