Illuminating Protein: Protein Interactions in Living Cells

May 1, 2015 (Vol. 35, No. 9)

Using NanoBRET Technology to Follow Changes in Intracellular Protein Binding Events

Significant advances in the understanding of the human proteomics network have advanced knowledge of the roles of specific protein:protein interactions, particularly related to disease and disease progression. This has resulted in an increased effort to target particular protein:protein interactions or monitor protein:protein nodules within signalling pathways in numerous areas of drug discovery research.

To successfully do so, technology with a high degree of specificity and sensitivity is needed to detect protein interactions and their dynamic changes within cells. One such technology capable of this is bioluminescence resonance energy transfer, BRET, which measures energy transfer from a luminescent donor to a fluorescent acceptor within a defined distance range between appropriately labelled proteins (Figure 1A).

Current BRET assay systems however lack the necessary sensitivity and are not able to detect subtle changes of protein:protein interactions. The main issues are the lack of donor brightness and the close proximity of donor and acceptor emission spectra that result in poor sensitivity and limited dynamic range. Compounding the issue is the need to express the donor luciferase at high levels to achieve efficient energy transfer to the acceptor, which can in certain cases lead to altered biological response. Here we present a new configuration of BRET, termed NanoBRET™, which addresses these challenges through the use of a NanoLuc® luciferase donor in combination with a fluorescently labelled HaloTag® acceptor (Figures 1A and 1B).

The NanoBRET combination of a bright donor with a spectrally well-separated acceptor results in reduced donor background in the acceptor channel and leads to significant improvements in sensitivity and dynamic range (Figure 1B). NanoLuc luciferase is >100-fold brighter than other luciferases, and therefore expression of the NanoLuc donor protein in the assay can be lowered to levels of the endogenous protein expression. Appropriate expression of the protein (i.e., not highly overexpressed) is important for obtaining a proper physiological response and for improving the overall signal:background compared to other BRET donor:acceptor configurations.

In the NanoBRET method, the two proteins of interest are expressed in a mammalian cell line of choice as either NanoLuc or HaloTag protein fusions. It is important that fusions are created in optimal physiological orientation and spatial proximity in order to achieve optimal energy transfer. This is typically determined experimentally. Similarly, the relative expression of the fusion donor protein to the fusion protein acceptor is important for obtaining the optimal NanoBRET signal, as the amount of unbound donor within the cell should be minimized.

NanoBRET protein:protein interactions can then be measured from live cells in either 96- or 384-well format using an instrument capable of measuring dual filtered signals. NanoBRET, like other BRET assays, is a ratiometric assay with the calculation shown in Figure 1B. Inherent to ratiometric assays and observed with NanoBRET assay are several advantages for screening; a high level of robustness results in high Z factors and low CVs, an independence of cell number, and insulation of data variance due to slight changes in assay setup or plating.

Figure 1. Principle of the NanoBRET assay for detecting intracellular protein:protein interactions. (A) The NanoBRET assay is a proximity based assay measuring energy transfer between a NanoLuc donor fusion protein and a fluorescently labelled HaloTag acceptor fusion protein. (B) Spectral separation of donor and acceptor signals in the NanoBRET assay leads to reduced background within the assay and calculation of the NanoBRET ratio, which is the acceptor signal divided by the donor signal. Indicated are the emission wavelength of the NanoLuc (peak at 450 nM) and the fluorescent HaloTag 618 ligand (peak at 618 nM), as well as the region wherein BRET is measured.

The NanoBRET assay can be used to understand how a specific protein:protein interaction is modulated upon treatment of the cells with different stimuli (Figure 2). We used full-length proteins of human p53 fusion as donor and human MDM2 fusion as acceptor, expressed in HEK293 cells to show the ability to measure the interaction and specific inhibition of the interaction with Nutlin 3A (Figure 2A). These live-cell measurements are used to determine the IC50 and they showed high Z factor values, which are important for screening. One can also perform inhibition studies by using the defined N-terminal region of p53 (amino acids 1-118), which is known to mediate the interaction with MDM2 (Figure 2B).

A similar in-cell IC50 value is obtained when using the p53 N-terminal domain in the NanoBRET assay, compared to full-length proteins, indicating that this terminal region is indeed important for the interaction and response to inhibitor (Figures 2A and 2B).

Since NanoBRET method works in live cells, kinetic and real-time measurements can be performed to understand the dynamic nature of protein interactions. NanoBRET assay optimization of the membrane protein receptor, EGFR, with its adaptor protein Grb2 is shown in Figure 2C. As expected, this interaction is increased upon addition of the growth ligand for EGFR, EGF, with a known EC50 value. Figure 2D demonstrates the ability to monitor the rate of increase of this interaction immediately after addition of EGF, which occurs within seconds inside the cell.

Figure 2. NanoBRET assays showing inhibition, activation, and real-time measurements in living cells. (A) NanoBRET assay of full-length p53 and MDM2 proteins shows inhibition with Nutlin-3A. (B) NanoBRET assay of an N-terminal fragment (amino acids 1-118) and full-length MDM2 showing inhibition with Nutlin-3A. (C) NanoBRET assay of full-length EGFR and Grb-2 showing induction of interaction using the growth factor, EGF. (D) Real-time NanoBRET assay of EGFR and Grb2 showing kinetic induction of interaction upon addition of EGF. All graphs show calculated BRET ratio units*1000 (mBU) on the y-axis and indicated drug treatment on the x-axis (A, B, and C) or the time of treatment (D).

We have developed and optimized over 100 protein:protein interaction NanoBRET assays, which are available from Promega. Table 1 shows a highlighted list of currently available NanoBRET assays, many of which cover important areas of drug targeting in epigenetics, transcription, and receptor biology. A significant number of pre-built NanoBRET assays are for monitoring interactions of bromodomain proteins with chromatin, many of which have been qualified by existing inhibitors.

As most inhibitors to these targets are initially found using the bromodomain fragment alone, several of the assays include bromodomains along with the full-length protein as indicated. For many of these family members, we have observed inhibition from chromatin of the bromodomain alone can be achieved inside the cell, mimicking the in vitro screening conditions, but this does not always translate to inhibition or eviction of the full-length protein by the inhibitor. This is likely due to the complexity of interactions and the multiple points of contact with chromatin that are present within the full-length proteins, which these cellular NanoBRET assays can begin to deconvolute.

These predesigned, full-length protein NanoBRET assays are available for expression in mammalian cells. Bromodomain assays, which are available as bromodomain-alone vectors, are indicated with an asterisk (*).

Danette L. Daniels, Ph.D. ([email protected]), is a group leader, Thomas Machleidt, Ph.D., is head of cell biology in advanced technologies, Jacqui Méndez is a senior research scientist, Kristin Riching, Ph.D., is a postdoctoral fellow, Marie Schwinn, Ph.D., is a senior research scientist, Nancy Murphy is a research scientist, Thomas Kirkland, Ph.D., is head of advanced technology chemistry research, Keith Wood, Ph.D., is head of research, and Marjeta Urh, Ph.D., is director of R&D research at Promega, which supplies assay and reagent materials as well as custom assay services.