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Overview
Below you can find a description about the approaches that we used to proof that we indeed have made two constructs that will aggregate and form a large structure, similar to a gel.
We have investigated the following conditions, in triplo:
- Scaffold Construct (14-3-3 with GFP) + Binding Partner (CT33 with Strep-tag®II and mCherry) + Inducer + Strep-Tactin®XT
- Scaffold Construct (14-3-3 with GFP) + Binding Partner (CT33 with Strep-tag®II and mCherry) + Inducer
- Scaffold Construct (14-3-3 with GFP) + Binding Partner (CT33 with Strep-tag®II and mCherry) + Strep-Tactin®XT
Table 1: Concentration of our constructs used to obtain the results
| Construct |
Proteins |
Concentration (µM) |
| Scaffold |
14-3-3 with GFP |
66 |
| Center Point |
Strep-Tactin®XT |
49.5 |
| Binding Partner |
CT33 with mCherry and Strep-tag®II |
198 |
| Inducer |
Fusicoccin |
330 |
FRET Measurements
The first approach was executed with a plate reader measurement. The first step is to excite the fluorophore GFP, which will then emit light with a higher wavelength. A part of this emitted light has a wavelength that can excite the fluorophore mCherry, which will then also emit light with an even higher wavelength. This principle is called Förster Resonance Energy Transfer (FRET)[1] and mostly used to indicate a close proximity between two proteins. Therefore we expect to see the effect of the fusicoccin stabilizer, which brings together the 14-3-3 and CT33 proteins.
In Figure 1, fluorescence measurements are shown after 1 h and after 24 h incubation times at room temperature. Looking at the results after 1 h, the FRET signal for the conditions with fusicoccin is elevated compared to the condition where fusicoccin is left out. The results after 24 h also show that the FRET signal is relatively low for the condition without fusicoccin, compared to the other two conditions, indicating the effect of fusicoccin to the 14-3-3/CT33 interaction.
Microscopy
To further assess the proximity between the two constructs fluorescence miscroscopy was used. Using microscopy, two images were made: one to visualize GFP and one to visualize mCherry. Those two images have been overlayed to see if the two fluorescent signals are present in the same construct.
In Figure 2, fluorescence microscopy images are shown after 48 h of incubation at room temperature. As can be seen from the overlay spectra, the presence of fusicoccin as well as the presence of Strep-Tactin®XT is beneficial for network formation. Without fusicoccin, formation of larger clusters is negligible compared to conditions where fusicoccin is present. Without the presence of Strep-Tactin®XT, mCherry and GFP signals are seen in small speckles, but it is unclear whether network formation has occured. Looking at the condition where all components are present larger structures and a lower signal to noise ratio are present, compared to the other two conditions. This indicates that our system works as expected: a network is formed in presence of an inducer, and multivalency plays a key role in network formation.
Conclusion
The microscopy results show a clear difference between the different conditions. All constructs together lead to clusters with sizes of approxixmately 10-20 µm. The absence of Strep-Tactin®XT results in very small clusters, probably presenting single scaffold constructs bound to binding partners. This interaction is due to the presence of fusicoccin. If fusicoccin is absent, no network formation is visible, as can be seen in the overlay picture (Figure 2i). FRET measurements also indicate that no networks are formed without fusicoccin present. Thus, our system works as expected: a network is formed in presence of an inducer, and multivalency plays a key role in network formation.
[1] B.T. Bajar, E.S. Wang, S. Zhang, M.Z. Lin and J. Chu, "A Guide to Fluorescent Protein FRET Pairs", Sensors (Basel), vol 16, no. 9, pp 1488, (2016)
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