Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Summary

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Abstract

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Introduction

Both classical linkage and next-generation sequencing analyses of human disorders are revealing the clinical relevance of proteins involved in a range of biological pathways. It is often the case that, prior to their identification in such studies, there has been little or no investigation of the biological role of these proteins. One fruitful avenue to begin exploring the biological function of a protein of interest is to identify which other proteins it interacts with in its physiological context. Characterizing molecular networks in this fashion provides insights into the biological pathways underlying the human phenotype.

The most frequently used large-scale screening approaches for identifying candidate interaction partners for proteins of interest are yeast two-hybrid screening¹ and mass spectrometry-based proteomics². These methods can be very successful in suggesting potential interacting proteins, but are vulnerable to false positive results. Therefore, confirmation of an interaction identified by yeast two-hybrid or mass spectrometry screening requires validation of the interaction using a second technique. Typically a co-immunoprecipitation or pull-down assay is used for this purpose³. One disadvantage of using such techniques for validation is the requirement for cell lysis, which destroys the intracellular conditions that may be essential for maintaining certain protein interactions. A second disadvantage is that weak or transient protein interactions may be disrupted during washing steps. Furthermore, these assays demand significant hands-on time, are limited in the number of samples that can be processed simultaneously, and often require time-consuming optimization of reagents and protocols.

To overcome some of the problems associated with co-immunoprecipitation experiments, several assays have been developed based on fluorescent and bioluminescent proteins that can be used in live cells. The first such assays were based on Fluorescence (or Förster) Resonance Energy Transfer (FRET), the non-radiative transfer of energy between two fluorescent proteins with overlapping emission and excitation spectra⁴. The efficiency of energy transfer is strongly distance-dependent, therefore observation of the FRET phenomenon requires that the donor and acceptor fluorophores be in close proximity. To test for an interaction between two proteins of interest, one protein is expressed as a fusion with the donor fluorophore (commonly cyan fluorescent protein; CFP) and the second as a fusion with the acceptor fluorophore (commonly yellow fluorescent protein; YFP). An interaction between the two proteins of interest may bring the donor and acceptor fluorophores sufficiently close for energy transfer to occur, which will result in a measurable increase in the emission of light from the YFP acceptor relative to the CFP donor. FRET has been successful in detecting protein-protein interactions in live cells⁴. The main drawback of using FRET for detecting protein-protein interactions is the requirement for external illumination for excitation of the donor fluorophore. External illumination results in high background in the emission signal, unwanted excitation of the acceptor, and photobleaching of both donor and acceptor fluorophores. These effects reduce the sensitivity of the assay for detecting protein-protein interactions.

A modification of the FRET assay which overcomes the problem of high background from external illumination is the Bioluminescence Resonance Energy Transfer (BRET) assay^5,6. In the BRET system the donor fluorophore is replaced by a luciferase enzyme. Thus the energy for the excitation of the acceptor fluorophore is generated within the system by the oxidation of a luciferase substrate, rendering external illumination unnecessary. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is YFP (for a discussion of alternative donor and acceptor proteins see Pfleger et al.⁵). Accordingly, in this system, a protein of interest is fused to luciferase and a potentially-interacting protein to YFP, or vice versa. The BRET assay requires the addition of coelenterazine as a substrate for luciferase. Because coelenterazine is cell-permeable, it is possible to perform BRET assays in live cells. However, native coelenterazine is unstable in aqueous solution, and the enzyme-independent breakdown of coelenterazine both reduces the concentration of substrate available for the assay and generates autoluminescence, which reduces the sensitivity of measurements of luciferase activity. The use of BRET in live cells has been facilitated by the development of protected coelenterazines, which are stable in aqueous solution but are cleaved by cytosolic esterases after diffusion across the cell membrane to generate active coelenterazine inside the cell⁷.

Following addition of substrate to cells expressing luciferase- and YFP-fusion proteins, energy transfer resulting from protein-protein interactions is quantified by monitoring emission from luciferase and YFP. Because protein interactions can be monitored directly in live cells in multi-well plates, the BRET assay constitutes a simple, scalable method for validating putative interactions that is cost- and time-efficient.

In addition to validating putative interactors identified in proteomic screening studies, the BRET system can also be used to test candidate interactors arising from prior biochemical and structural studies on the protein of interest. Once the existence of a protein-protein interaction has been established (either by using the BRET assay or by other techniques), there is potential for the BRET assay to be employed further to characterize the interaction. For example, the interacting regions can be mapped by generating truncated versions of the proteins, and the involvement of specific residues in the interaction can be demonstrated by creating point mutations. Furthermore, the modulatory effect of posttranslational modifications or small molecules (such as drugs or ligands) on protein-protein interactions can be investigated^8-10.

The BRET assay also has great potential for investigating mutations identified in patient DNA. In cases where a causative role for a mutation has been established, studying the effect of the mutation on protein-protein interactions using BRET may reveal more about the molecular etiology of the phenotype¹¹. Since the advent of next-generation sequencing methodologies, it is increasingly common for several potentially-damaging mutations to be identified within an individual, in which case it is unclear which are relevant to the phenotype¹². In this situation the BRET assay may be valuable in evaluating the impact of mutations on protein function and hence their relevance to the disorder.

Protocol

1. Creation of Plasmids

1. Subclone the cDNAs for each protein of interest into both the pLuc and pYFP vectors, using standard molecular biology techniques (Figure 1). For detailed protocols see Green et al¹³.
2. Sequence all constructs to verify that the proteins of interest are in frame with the Luc/YFP sequence with no intervening stop codons.
3. Perform functional assays as desired to confirm that the fusion proteins retain biological activity. In the case presented here the subcellular localization of YFP fusion proteins was ascertained by fluorescence microscopy.
4. Make expression plasmids for appropriate Luc and YFP control proteins by engineering the relevant targeting signals into the pLuc and pYFP expression vectors. In the case presented here, generate nuclear-targeted Luc and YFP constructs by inserting a nuclear localization signal into the pLuc and pYFP vectors.
5. Make a positive control construct in which Luc and YFP are fused into a single polypeptide. In the case presented here, generate a positive control in which the YFP coding sequence is inserted into the pLuc vector.
6. Select a neutral filler plasmid to be used to equalize the mass of DNA in transfection mixes. Use a plasmid that has no eukaryotic promoter and will therefore be transcriptionally inactive in mammalian cells, such as a bacterial cloning vector.

2. Preparation of DNA Mixes

1. Estimate the concentration of all the plasmids described in Section 1 based on absorbance at 260 nm (1 absorbance unit is equivalent to 50 µg/ml of DNA). Determine the molecular mass of each plasmid by multiplying the number of base pairs by 650 Da. Use the concentration and molecular mass to calculate the molar concentration of each plasmid preparation. Dilute the plasmid DNA preparations to a concentration of 36 nM. These will be the working stocks that will be used to prepare the DNA mixes for transfection.
2. Prepare a control DNA mix containing 1,800 ng of filler plasmid in a final volume of 20 µl of water.
3. Prepare a control DNA mix containing 5 µl of the pLuc-control construct. Add filler plasmid to bring the total DNA mass to 1,800 ng. Add water to bring the final volume to 20 µl.
4. Prepare a control DNA mix containing 5 µl of pLuc-control construct and 5 µl of pYFP-control construct. Add filler plasmid to bring the total DNA mass to 1,800 ng. Add water to bring the final volume to 20 µl.
5. Prepare a control DNA mix containing 5 µl of positive control construct. Add filler plasmid to bring the total DNA mass to 1,800 ng. Add water to bring the final volume to 20 µl.
6. Prepare the following DNA mixes to test for homodimerization of a protein of interest, X. For each DNA mix combine 5 µl of the relevant pLuc construct and 5 µl of the pYFP construct. Add filler plasmid to bring the total DNA mass to 1,800 ng. Add water to bring the final volume to 20 µl.
   A) pLuc-control and pYFP-X
   B) pLuc-X and pYFP-control
   C) pLuc-X and pYFP-X
7. Prepare the following DNA mixes to test for an interaction between a pair of proteins of interest, X and Y. For each DNA mix combine 5 µl of the relevant pLuc construct and 5 µl of the pYFP construct. Add filler plasmid to bring the total DNA mass to 1,800 ng. Add water to bring the final volume to 20 µl.
   A) pLuc-control and pYFP-X
   B) pLuc-X and pYFP-control
   C) pLuc-control and pYFP-Y
   D) pLuc-Y and pYFP-control
   E) pLuc-X and pYFP-Y
   F) pLuc-Y and pYFP-X

3. Transfection

1. Harvest subconfluent HEK293 cells from a 75 cm² flask. Dilute 10% of the total cells into 13 ml of culture medium. Dispense 130 µl of cell suspension into each well of a white clear bottomed 96-well tissue culture plate. Culture cells for 24 hr.
2. Calculate the number of wells to be transfected by multiplying the number of DNA mixes by 3. Bring serum-free culture medium to room temperature. Prepare a master mix containing 6.3 µl of serum-free medium and 0.18 µl transfection reagent per well. Mix by vortexing and incubate at room temperature for 5 min.
3. Prepare transfection mixes by adding 2 µl of DNA mix to 20 µl of serum-free medium/transfection reagent master mix. Do not vortex. Incubate at room temperature for 10 min.
4. Transfect three wells with each transfection mix dispensing 6.5 µl of transfection mix per well. Culture the cells for a further 36-48 hr.

4. Measurement of BRET Signal

1. Dissolve live-cell luciferase substrate at 34 mg/ml in DMSO by vortexing.
2. Dilute reconstituted live-cell luciferase substrate at 1:1,000 in substrate dilution medium pre-warmed to 37 °C. Allow 50 µl of substrate dilution medium per well. Vortex to mix. A precipitate may form, but will not interfere with the assay.
3. Aspirate the culture medium from the 96-well plate. Dispense 50 µl of diluted live-cell luciferase substrate into each well. Culture cells for at least 2 hr (up to 24 hr).
4. Remove the lid from the 96-well plate and incubate the plate for 10 min at room temperature inside the luminometer.
5. Measure emission from Luc and YFP one well at a time. Measure emission from Luc using a filter blocking wavelengths longer than 470 nm. Measure emission from YFP using a 500-600 nm band-pass filter. Integrate emission signals over 10 sec. 

5. Data Analysis

1. Average the Luc emission readings from the 3 wells that received only the filler plasmid. Subtract this value from all other Luc emission readings to produce the background-subtracted Luc emission values.
2. Average the YFP emission readings from the 3 wells that received only the filler plasmid. Subtract this value from all other YFP emission readings to produce the background-subtracted YFP emission values.
3. For each well, divide the background-subtracted YFP emission value by the background-subtracted Luc emission value to give the uncorrected BRET ratio.
4. Average the uncorrected BRET ratios from the three wells that were transfected with only the pLuc-control plasmid. Subtract this value from all the other uncorrected BRET ratios to give the corrected BRET ratios.
5. For each of the remaining sets of corrected BRET ratios, average the values from the three transfected wells to obtain a final BRET ratio.

Results

The principle of the BRET assay is illustrated in Figure 2. The assay setup used throughout the experiments presented here is depicted in Figure 3. The detection of a strong BRET signal from cells transfected with a luciferase-YFP fusion protein confirmed that energy transfer was observable in this experimental setup (Figure 4).

Our research focuses on the role of the FOXP family of transcriptional repressors in brain development. Heterozygous...

Discussion

The design of the fusion protein expression constructs is a critical step in setting up the BRET assay. In the experiments presented here, the proteins of interest were fused to the C-terminus of luciferase or YFP. It is also possible, and may be necessary, to fuse proteins to the N-terminus of luciferase/YFP. For some proteins, fusions may only be accepted at either the N- or C-terminus in order to avoid disruption of protein structure and function. Furthermore, for transmembrane proteins in which the N and C termini re...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Nanodrop 8000	Nanodrop		Any spectrophotometer capable of reading absorbances at 260 nm will be suitable. Determining the molecular mass of the plasmid is crucial for calculating DNA quantities to be used in transfection mixes.
96-microwell plates, flat bottom, white	Greiner Bio One	655098	White plates reduce the crosstalk between wells and maximize the sensitivity of luminescence detection. Clear-bottomed wells allow monitoring of cell density. Plates must be suitable for cell culture. If using a top-reading luminometer the plate lid should be taken off.
Infinite F200Pro plate reader with control software	TECAN		Use the 'Blue 1' and 'Green 1' filters for luminescence measurement and the filter sets and dichoic mirror for GFP for fluorescence measurement. Any top-reading plate reader with capability of measuring dual-color luminescence and fluorescence is suitable.
pLuc, pYFP, positive control plasmid	N/A	N/A	Plasmids available from the authors upon request.
pGEM-3Zf(+)	Promega	P2271	Filler plasmid for equilization of DNA mass in transfection mixes. Any plasmid lacking a eukaryotic promoter would be suitable.
HEK293 cells	ECACC	85120602	Other cell lines that transfect with reasonable efficiency may be suitable.
DMEM, high glucose, with phenol red	Gibco	41966	This is the medium used for culturing HEK293 cells. Warm in 37 °C waterbath before use. If using a different cell line, replace the growth medium described here with cell-line specific medium.
DMEM, high glucose, no phenol red (substrate dilution medium)	Gibco	21063	This is the substrate dilution medium used for dilution of the luciferase substrate (EnduRen) as it does not contain phenol red, which reduces the sensitivity of the assay. Contains HEPES to maintain correct pH during luminescence measurements while cells are out of the CO2 incubator. Warm in 37 °C waterbath before use.
OptiMEM	Gibco	31985	OptiMEM is used for dilution of GeneJuice transfection reagent. Other serum-free media would also be suitable. Warm to room temperature before use.
Fetal bovine serum	Gibco	10270	For supplementation of cell culture media at a concentration of 10% v/v.
GeneJuice transfection reagent	Novagen	70967	If using a cell line other than HEK293, it may be necessary to adjust the ratio of Genejuice transfection reagent to DNA in the transfection mixes. Other transfection reagents may be used. If using an alternative transfection reagent, it may be necessary to optimize the amount of DNA used in the transfection mixes based on manufacturer's instructions.
DMSO	Sigma	D2650	Use sterile DMSO that is suitable for tissue culture.
EnduRen live-cell substrate	Promega	E6481	Reconstitute EnduRen at 34 mg/ml in DMSO. Upon dilution of EnduRen in culture medium a precipitate may form. This will not interfere with the assay. Store reconstituted EnduRen at -20 °C, and avoid multiple freeze-thaw cycles. Ensure that reconstituted EnduRen is completely thawed before diluting it in culture medium.