Part:BBa_K3945010
BRET: A fluorescence-based measurement system for rare earth elements
Usage and Biology
mCherry is a red fluorescent protein derived from Discosoma sea anemones, well-known for its use in molecular biology. NanoLuc is a patented luciferase enzyme from Promega, derived from Oplophorus gracilirostris [1]. It is unique in the sense that it’s much smaller than traditional firefly luciferase, produces a signal 100 times greater than that of firefly luciferase, and has a broad range of stability throughout differing pH and temperature conditions [1]. When used together, these two proteins undergo the phenomena of BRET (bioluminescence resonance energy transfer), which is a non-radiative energy transfer between a luminophore donor (NanoLuc) and a fluorophore acceptor (mCherry) [2]. BRET only occurs when these proteins are within a certain proximity to each other, making it a good tool to determine protein-protein interactions [2]. As the binding of LanM to lanthanide ions results in its folding, these protein fragments are held in close proximity to each other once folding occurs but held apart from each other when LanM is in an unbound state. As such, this system can be used to determine whether LanM is binding to lanthanide ions, and the intensity of the signal can quantify the ion concentration. As this system uses NanoLuc, a furimazine substrate will be required to ensure the NanoLuc produces light, and a luminometer will be used to measure signal output in the 550-650nm range.
Design
Previous experiments using a LanM-based FRET system were done in the past, where the authors established a certain R0,(Forster distance) at which the system worked [3]. Forster distance refers to the distance at which BRET undergoes 50% of resonance energy transfer [2]. As that paper demonstrated a successful signal, we decided to utilize a BRET system with a similar R0. The mCherry and NanoLuc combination was shown to have the closest R0 out of a number of BRET systems modelled by Weihs et al, which is why it became our chosen system [3].
As physical distance plays such a large role in the success of this system, linkers between LanM and the BRET proteins were modelled. This was done to ensure that the proteins wouldn’t interact when LanM was in an unbound state, but would fall within the minimum distance required when LanM was bound and folded. Furthermore, we needed to ensure that the presence of the BRET proteins would not interfere with the folding and unfolding mechanisms of LanM. This was determined through molecular docking simulations. The linkers used for the BRET system provided the optimized values for reaching the R0, as well as provided the best physical distance between BRET components and LanM.
According to previous studies, the signal output of the BRET system changes depending on the protein-terminus the luminophore and fluorophore are fused to [2]. As such, in order to increase our signal output, the mCherry was fused to the N-terminus of LanM and NanoLuc to the C-terminus.
Experimental Workflow
In order to measure REE concentration using the BRET system, there were several steps we would need to undergo. After successful protein production, we would need to determine background signal emittance. We would do this by measuring the luminescence output of just the protein and the added furimazine. Afterwards, a calibration curve using known REE concentrations will have to be calculated. This will be done by resuspending a constant amount of the protein in differing concentrations of REE solution, adding furimazine, and measuring the output using a luminometer. Once this calibration curve was completed, we would test the REE solution after it had already undergone the metal separation step of our system. This would help us determine the range of detection of BRET, and also determine the viability of it for testing REE concentrations after the Neocycle separation process. This experimental process will be identical to the Lucifer experimental process apart from the wavelength at which the signal output is measured. Future optimization experiments will be conducted to better understand the range of the BRET system.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 811
Illegal NgoMIV site found at 1150 - 1000COMPATIBLE WITH RFC[1000]
References
1. Promega. 2021. NanoLuc Luciferase. Retrieved online from https://www.promega.ca/resources/technologies/nanoluc-luciferase-enzyme/
2. Weihs F, Wang J, Pfleger K.D, Dacres H. 2020. Experimental determination of the bioluminescence resonance energy transfer (BRET) Forster distances of NanoBRET and red-shifted BRET pairs. Anal. Chim. Acta. 6, 100059.
3. Mattocks JA, Ho JV, Contruvo JA. A Selective, Protein-Based Fluorescent Sensor with Picomolar Affinity for Rare Earth Elements. J Am Chem Soc. 2019. 141(7): 2857-2861. https://doi.org/10.1021/jacs.8b12155
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