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 optimum excitation wavelength to use in future FRET experiments with scaffolded mCerulean3 and mVenus.
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 emitted when excited. Every fluorophore has a range of wavelengths at which it can be excited, i.e. absorb electromagnetic energy, known as its absorption spectrum. After being excited, a fluorophore then emits energy at a range of wavelengths, known as its emission spectrum. The excitation wavelength is of higher energy (shorter wavelength) than the emission wavelength. The concept behind FRET is the excitation of one fluorophore which requires a high energy wavelength for excitation and another which requires a comparatively lower energy wavelength. The intensity of the second fluorophore's emission will depend on the distance that the emitted light from the first fluorophore has travelled (Figure 1). Therefore, intensity of the second fluorophore's emission represents fluorophore proximity. It is important to note that when selecting appropriate fluorophores, a large amount of overlap between the emission of the first fluorophore, in our case, mCerulean3, and the excitation of the second fluorophore, mVenus, is preferred.
As FRET has the ability to measure distances between fluorophores that are between 1 and 10 nm apart1, by attaching mCerulean3 and mVenus to our Assemblase scaffold 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.
Figure 1: Animation showing how fluorophores do not interact when too far apart, yet are able to transfer electromagnetic energy when brought close together.
Aim
Our aims were two-fold:
- To perform FRET with unscaffolded mCerulean3 and mVenus in order to obtain their emission spectra
- To perform FRET with mCerulean3 and mVenus attached to our Assemblase scaffold to measure the distance between the molecules
Due to time constraints, we were only able to meet our first aim of this experiment.
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 2 (below) was used as a starting point for determining the optimum excitation and emission wavelengths for the fluorescent proteins.
Figure 2: Excitation and emission spectra reported in the literature for mCerulean3 (A) and mVenus (B).
From this information from the literature, the following data (Table 1) was extrapolated as ideal values and ranges for mCerulean3 and mVenus excitation and emission.
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 the excitation and emission spectra of mCerulean3 and mVenus 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 maximise the excitation of mCerulean3 whilst minimising the excitation of mVenus.
Once this ideal value was obtained, mCerulean3 and mVenus were mixed in a 1:1 ratio, producing a solution containing each fluorescent protein at 0.5 mg/mL. Controls were also prepared using each fluorescent protein individually, 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 with scaffolded mCerulean3 and mVenus.
The protocols used in this experimentation can be found here.
| 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.
Results
After scanning for the ideal wavelengths for mCerulean3 and mVenus, the resulting excitation and emission spectras were obtained (Figure 4):
Figure 4: The excitation and emission spectra generated for Cerulean and Venus, similar to those reported in the literature, 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 451/478 nm for mCerulean3 and 519/539 for mVenus. The excitation value of mCerulean3 (451 nm) was used to excite the combined mixtures of mCerulean3 and mVenus for FRET, producing the graph shown in Figure 5 below. The emission observed from the wells containing only mCerulean3 is shown in blue, the wells containing only mVenus in yellow, and those containing both fluorescent proteins in green. Three replicates were performed for each condition.
Figure 5: FRET emission curves after excitation at 451 nm. The triplicate wells containing mCerulean3, mVenus or both are represented by the blue, yellow and green curves, respectively.
Discussion
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 5. The peak in the green curves (mCerulean3 and mVenus mixture) is not FRET occurring, but rather it is a result of superposing the two curves of mCerulean3 and mVenus on their own. Fluorescence is observed from these fluorophores as the range scanned includes the tail end of the emission spectrum of mCerulean3, while the excitation wavelength, although carefully selected, is still just within the excitation range of mVenus.
Due to delays in the cloning and protein expression, mCerulean3 and mVenus were not expressed as fusions to Snoop and Spy Tag. This means that they could not be attached to our Assemblase scaffold and FRET was not performed on scaffolded fluorescent proteins. Instead, mCerulean3 and mVenus were purified after obtaining the expression plasmids from Dr Dominic Glover's laboratory on campus at UNSW. Despite being unable to perform the FRET that was planned for our scaffold, we achieved the first aim of our FRET experiments, generating data on the negative controls for our future FRET experiments with scaffolded mCerulean3 and mVenus, as well as the optimum excitation and emission wavelengths of the fluorescent proteins.
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.