Team:UNSW Australia/Lab/FRET
FRET
Abstract
By performing FRET, we aimed to investigate the distance between two fluorescent proteins, mCerulean3 and mVenus, attached to our Assemblase scaffold. This would allow us to gain an understanding of the proximity of enzymes in our Assemblase system and therefore help us perform more accurate modelling of reaction kinetics. We started by obtaining purified mCerulean3 and mVenus and performing FRET using these unscaffolded proteins. Due to time constraints, we did not have the opportunity to perform this experiment using scaffolded mCerulean3 and mVenus. Nevertheless, we have established FRET protocols to use in the future, and have obtained the absorption and emission spectra of these fluorescent proteins.
Figure 1: Animation showing how fluorophores do not interact when too far apart, yet are able to transfer electromagnetic energy when brought close together.
Introduction
Förster Resonance Energy Transfer (FRET) is a method whereby the distance between two fluorophores can be calculated based on the amount of fluorescence signal given off when excited. Every fluorophore has a range of wavelengths at which it can be excited, that is, take up electromagnetic energy, and a range of wavelengths at which it will release that energy, known as its emission spectrum. The idea behind FRET is that you can excite one fluorophore at a higher energy wavelength, which it will subsequently emit at a lower energy wavelength. This lower energy wavelength then travels to the second fluorophore to excite it, followed again by emission at an even lower energy wavelength. This final emission can be measured on a plate reader, the strength of which allows us to calculate the distance between the fluorophores. When selecting appropriate fluorophores, you want the largest amount of overlap between the emission of the first fluorophore, in our case, cerulean, and the excitation of the second fluorophore, venus.
FRET has the ability to measure distances between fluorophores that are between 1 and 10 nm apart1, and hence by scaffolding our two selected proteins, we hoped that FRET would occur between the scaffolded molecules. This would allow us to gain experimental data on the distance between the molecules that we scaffold, and hence more accurately calculate the diffusion patterns that would occur between enzymes that were scaffolded to our system.
Aim
To measure the distance between scaffolded proteins using FRET.
Methods
Solutions of mCerulean3 and mVenus were made up to 1 mg/mL and a serial dilution of each made with Phosphate Buffered Saline (PBS) from 1:1 through to 1:1000. The data presented by Markwardt et al.2 and Jonáš et al.3 in the figure below was used as a starting point for determining the optimum excitation and emission wavelengths for the fluorophores.
Figure 2: Excitation and emission spectra of Cerulean reported by Markwardt et al.2 in A) and Venus reported by Jonáš et al.3 in B). Excitation in A) is shown by a dotted line while emission is a solid line.
From this, the following data was extrapolated,
Table 1: Ideal values and ranges of excitation and emission extracted from Figure 2.
| Excitation | Emission | |
|---|---|---|
| mCerulean3 | 433 (400-465) | 475 (465-525) |
| mVenus | 515 (500-525) | 528 (520-550) |
These wavelengths were used to investigate each of the excitation and emission spectra of the fluorophores to determine the ideal wavelengths specific to our expressed proteins. Based on the resulting output graphs generated, curve smoothing was used before selecting an ideal excitation value that would sufficiently excite the Cerulean fluorophore, yet was low on the Venus excitation spectrum.
Once this value was obtained, Cerulean and Venus were mixed in a 1:1 ratio, producing a solution containing 0.5 mg/mL Cerulean and 0.5 mg/mL Venus. Controls were made using only one fluorophore made up to the same concentration with PBS. Using the ideal excitation value for cerulean determined previously, these wells were scanned to create the negative controls for future FRET experiments, the results of which are shown in Figure X.
| PBS | Cerulean 1 in 1000 | Cerulean 1 in 100 | Cerulean 1 in 10 | Cerulean 1 in 1 | ||||
| PBS | Venus 1 in 1000 | Cerulean 1 in 100 | Venus 1 in 10 | Venus 1 in 1 | ||||
| PBS | ||||||||
| PBS | 0.5 mg/mL Cerulean | 0.5 mg/mL Venus | 0.5 mg/mL Cerulean, 0.5 mg/mL Venus | |||||
| PBS | ||||||||
| PBS | 0.5 mg/ml Cerulean | 0.5 mg/mL Venus | 0.5 mg/mL Cerulean, 0.5 mg/mL Venus | |||||
| PBS | ||||||||
| PBS | 0.5 mg/ml Cerulean | 0.5 mg/mL Venus | 0.5 mg/mL Cerulean, 0.5 mg/mL Venus |
Figure 3: Picture of the plate used for FRET analysis of the negative controls and the contents within each well. A space was left between wells wherever possible to reduce fluorescent signal from nearby wells due to the lack of a black sided plate.
The protocols used in this experimentation can be found here.
Results
After scanning for the ideal wavelengths for the proteins we used, the resulting graphs were as follows,
###Insert the 4 white graphs from Drive - just one after another cause they're kinda long and the text is small so don't want to make them too small by doing side by side.###Figure 4: The excitation and emission spectra generated for Cerulean and Venus, similar to those of Markwardt et al.2 and Jonáš et al.3 in Figure 2, yet ensured to be suitable for the specific versions of the proteins that we have expressed.
After curve smoothing of the maximum fluorescence curves, the ideal excitation/emission wavelengths were determined to be Cerulean (451/478) and Venus (519/539). The excitation value of cerulean (451 nm) was used to excite the fluorophores for FRET, producing the graph shown in Figure Y below. The emission observed from the wells containing only cerulean is shown in blue, the wells containing only venus in yellow, and that containing both in green.
Figure 5: Emission curves observed for the plate analysed in Figure 3 after excitation at 451 nm. The triplicate wells containing only Cerulean are shown in blue, Venus in orange, and the two together as the green curves.
Discussion
Due to delays in the cloning and protein expression side of the experimentation, the two proteins Cerulean and Venus were not expressed as fusions to Snoop and Spy Catcher. This means that they could not be scaffolded and FRET was not performed on the scaffolded molecules. Instead, Cerulean and Venus were expressed from Dr Dominic Glover's laboratory on campus at UNSW from the expression vector pET19b. These versions were only tagged with 6 histidine residues for purification. Despite not being able to perform the FRET that was planned for our scaffold, these two that were successfully expressed and purified allowed us to generate data on the negative controls of the experiment, as well as the optimum excitation and emission wavelengths of the fluorescent molecules.
One of the limitations of FRET is the overlap of curves seen between any two fluorophores. The excitation and emission spectra must be close enough so that the fluorophores interact, yet sufficiently distant to limit signal generated in the second fluorophore from the excitation wavelength meant for the first molecule. A balance between these is sought, yet no pairing of fluorophores is perfect, and hence there will always be some level of fluorescence observed in a solution where FRET is not occurring. This is the case observed in Figure Y. The green curve is not FRET occurring, but rather it is a result of superposing the two curves of Cerulean and Venus on their own. Fluorescence is observed from these fluorophores as the range scanned includes the tail end of the emission spectrum of Cerulean, while the excitation wavelength, although carefully selected, is still within the excitation range of the Venus excitation curve as it tails off towards the higher energy wavelengths.
Figure 6: Falcon tubes containing our expressed and purified proteins. The fluorescence of these proteins can be seen here when placed under a UV light source.
References
- Hevekerl, H., Spielmann, T., Chmyrov, A. & Widengren, J. Förster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores. The Journal of Physical Chemistry B 115, 13360-13370 (2011).
- Markwardt, M. et al. An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching. PLoS ONE 6, e17896 (2011).
- Jonáš, A. et al. In vitro and in vivo biolasing of fluorescent proteins suspended in liquid microdroplet cavities. Lab Chip 14, 3093-3100 (2014).
Supplementary Materials
Download the supplementary data for our experiments here, including exact parameters used.