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NanoBRET—A Novel BRET Platform for the Analysis of Protein–Protein Interactions

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† Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States
‡ Promega Biosciences Incorporated, 277 Granada Drive, San Luis Obispo, California 93401, United States
*E-mail: [email protected]
Cite this: ACS Chem. Biol. 2015, 10, 8, 1797–1804
Publication Date (Web):May 25, 2015
https://doi.org/10.1021/acschembio.5b00143

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Abstract

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Dynamic interactions between proteins comprise a key mechanism for temporal control of cellular function and thus hold promise for development of novel drug therapies. It remains technically challenging, however, to quantitatively characterize these interactions within the biologically relevant context of living cells. Although, bioluminescence resonance energy transfer (BRET) has often been used for this purpose, its general applicability has been hindered by limited sensitivity and dynamic range. We have addressed this by combining an extremely bright luciferase (Nanoluc) with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The small size (19 kDa), high emission intensity, and relatively narrow spectrum (460 nm peak intensity) make Nanoluc luciferase well suited as an energy donor. By selecting an efficient red-emitting fluorophore (635 nm peak intensity) for attachment onto the HaloTag, an overall spectral separation exceeding 175 nm was achieved. This combination of greater light intensity with improved spectral resolution results in substantially increased detection sensitivity and dynamic range over current BRET technologies. Enhanced performance is demonstrated using several established model systems, as well as the ability to image BRET in individual cells. The capabilities are further exhibited in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells.

Dynamic behaviors in cells are mechanistically entwined with a vast interplay of transient interactions occurring between intracellular proteins. Protein–protein interactions (PPI) are thus fundamental to understanding cellular physiology and are attractive as pharmaceutical targets. Although numerous methods are available for the discovery of protein interactions, most notably through analysis by mass spectrometry, it remains challenging to study the dynamics of these interactions in a cellular context. Resonance energy transfer has been an attractive approach for this purpose, where excited-state energy is transferred from one fluorophore to another when brought into close proximity. (1) The rigorous distance constraint of energy transfer restricts proximity detection to roughly the physical span of a typical protein (∼5 nm), making this approach well suited for the crowded molecular space within cells.
Energy transfer is most often achieved using autofluorescent proteins (e.g., YFP), which can be linked to the interacting proteins through genetic fusions. The method allows visualization of subcellular location and interaction dynamics by microscopy imaging but often requires relatively high levels of expression for reliable measurements. This can make analysis at physiological concentrations challenging within the cells and may lead to artifacts of protein behavior. The intensity of the illuminating light source also causes problems with phototoxicity, photobleaching of the fluorophores, direct excitation of the acceptor fluorophore, and background fluorescence from the sample. These issues make sensitive and reliable measurements difficult, particularly for kinetic processes.
Although FRET is generally recognized as requiring an external light source, such as a laser, to generate the excited state of the donor, the energy can be supplied instead by a chemical reaction. For BRET (bioluminescence resonance energy transfer), using a luciferase as the donor sidesteps many of the deficiencies associated with direct illumination of the sample. (2-4) Furthermore, quantitative measurements even at very low expression levels in cells should be possible due to the extraordinary sensitivity typical of luciferase assays. Yet despite these predictable advantages, BRET has not been widely adopted largely due to limited sensitivity and dynamic range of the presently available methods.
Current methods routinely utilize Renilla luciferase (Rluc) as the energy donor, more recently relying on the Rluc8 mutant to provide higher light intensity. (5) In the most commonly used BRET format, BRET1, Rluc is typically matched with a yellow fluorescent protein (YFP) to achieve efficient energy transfer by significant overlap of its excitation spectrum with the bioluminescence. However, the spectral proximity of Rluc and YFP (∼55 nm peak separation) produces a substantial measurement background by the extension of donor signal into the acceptor channel. The high background increases assay noise, which diminished dynamic range and sensitivity.
An alternative BRET, referred to as BRET2, was developed to increase spectral separation between the luciferase and the fluorescent acceptor. (6) The system utilizes a substrate analog, bisdeoxycoelenterazine, to shift the luminescence to much shorter wavelengths (400 nm peak from 480 nm), and GFP2 or GFP10, which exhibit excitation and emission maxima of 395 and 510 nm, respectively. Although the large Stokes shift of GFP2/GFP10 improves spectral resolution (115 nm), this advantage is largely negated by the very low quantum yield and poor luminescence stability. (7) The recent adoption of Rluc8 (8) for BRET2 has improved performance, but light intensities are still far below other BRET configurations. (9)
The recent development of the Nanoluc luciferase (Nluc) offers an opportunity to further improve the performance of BRET assays. (10) Nluc is an engineered protein (19 kDa), which uses a novel coelenterazine derivative (furimazine) as its substrate. Its small size and high physical stability are desirable for use as a fusion tag, imparting minimal influence when tethered onto other proteins. Although it is only about half the size of Rluc8 (36 kDa), it produces sustained luminescence with much greater intensity, allowing very small quantities to be accurately quantified. Moreover, its bioluminescence spectrum is narrower than Rluc8, (10) permitting better spectral discrimination with acceptor fluorophores. To provide distinction from the other configurations of BRET, we refer to energy transfer using Nluc as the donor as NanoBRET.
With Nluc available as a more effective BRET donor, we focused on matching this with an optimal fluorescent acceptor. The HaloTag fusion tag allowed us to rapidly evaluate a range of fluorophores for this purpose. HaloTag (HT) is a two component system consisting of a stable protein (36 kDa) which can be genetically tethered onto other proteins and a chloroalkane ligand that can be chemically coupled onto fluorophores or other molecules of interest. (11) The chloroalkane irreversibly links to the HaloTag through rapid formation of a covalent bond. By using this modular system, we found that long-wavelength fluorophores were preferred for BRET measurements by providing better spectral separation from the luciferase donor. Best performance was achieved with a chloroalkane derivative of nonchloro TOM (NCT) dye, which provides peak light emission at 635 nm. Thus, peak emission from the donor and acceptor were spectrally separated by 175 nm, significantly greater than provided even by the BRET2 system.
The high luminescence intensity of NanoBRET allows detection of protein interactions at low levels comparable to endogenous physiological conditions. The assay exhibits a linear dynamic range extending over several orders of magnitude and provides greater sensitivity than Rluc8/YFP by typically more than an order of magnitude for analogous molecular configurations. The strong performance of NanoBRET was utilized to develop the first assays for quantifying the binding dynamics of bromodomain protein with chromatin in living cells. Furthermore, the high intensity of NanoBRET also raises the potential for quantifying energy transfer in individual cells by microscopy imaging.

Results and Discussion

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Optimization of the Fluorescent Acceptor

Genetically fusing HaloTag directly onto Nluc (Nluc-HT) offered a convenient means for providing a fixed geometry between the donor and acceptor. Fifty-seven different chloroalkane–fluorophore conjugates were synthesized and screened in HEK293 cells transiently expressing the Nluc-HT fusion. By using a cell-based assay, we were able to evaluate the fluorophores for their ability to permeate cellular membranes and produce a BRET signal within the intracellular environment. Each fluorophore was evaluated using a set of four long-pass filters (cut-on 570, 590, 610, and 630 nm) for the acceptor emission, where the BRET response was determined by comparing measurements made both in the presence and absence of the fluorophore. The HaloTag TMR Ligand (590 nm emission peak) was used as a positive control, having been previously shown to bind efficiently and stoichiometrically to HaloTag in living cells. From these experiments, a nonchloro TOM (NCT) ligand was identified as providing the greatest signal over the background. The NCT ligand has an excitation maximum of 595 nm with an emission maximum of 635 nm when bound to HaloTag (Supporting Information Figure S1a). Labeling of HaloTag fusion proteins in living cells is rapid with saturation occurring in less than 10 min after the addition of 250 nM HaloTag NCT ligand (Supporting Information Figure S1b). No obvious cytotoxicity was exhibited by the NanoBRET expression constructs in combination of NCT ligand and furimazine (the principal reagents required for NanoBRET) for periods of up to 24 h (Supporting Information Figure S2).
The Förster equation predicts that the greatest energy transfer will occur with maximal overlap between the emission spectrum of the donor and excitation spectrum of the acceptor. The HaloTag Oregon Green Ligand (512 nm peak emission) has greater overlap, and the NCT ligand has less overlap, with Nluc than does the HaloTag TMR Ligand (Supporting Information Figure S1). Accordingly, in the Nluc-HT fusion, more energy is transferred from Nluc to the Oregon Green Ligand, and less to the NCT ligand, compared to the TMR Ligand (Figure 1A). However, since the decline in energy transfer efficiency is substantially smaller compared to the decrease in Nluc light intensity across the same spectral range, the signal to background ratio increases substantially. Thus, the highest signal to background was achieved with the NCT ligand, even though it does not support the most efficient energy transfer (Supporting Information Figure S3). Moreover, the modest reduction in transfer efficiency is more than compensated by the increased light intensity of Nluc. The specific activity of Nluc is about 150-fold greater than Rluc, and 30-fold greater than Rluc8. Signal decay kinetics are comparable between Nluc and Rluc8, with NanoLuc showing a slightly improved decay profile in biological buffers (Supporting Information Figure S4a–c).

Figure 1

Figure 1. Comparison of different acceptors for NanoBRET. (a) BRET spectra for Nluc-HT (black) and Nluc-HT conjugated to HaloTag Oregon Green (green), HaloTag TMR (red), or HaloTag NCT (blue) ligands. (b) Correlation of BRET with fractional occupancy. Differing degrees of occupancy were created by combining Nluc-HT-NCT (fully acceptor occupied) with Nluc-HT-PEG-biotin (unoccupied donor) in defined ratios at a constant donor concentration. Fractional occupancy (%) indicates the amount of acceptor-occupied Nluc-HT relative to the total amount of Nluc-HT in the sample. Shown are the means ± SD (n = 7) of a representative experiment. The results were plotted as mBRET units.

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In BRET measurements of molecular interactions, since energy transfer occurs when the donor is occupied by an acceptor, assay sensitivity can be specified as the lowest detectable degree of occupancy. This can be modeled using the Nluc-HT fusion, where fully bound to a fluorophore represents 100% occupancy and fully bound with a nonfluorophore represents 0% occupancy. Because the covalently bound HaloTag ligands cannot be exchanged once attached, different ratios of the “occupied” and “unoccupied” fusion protein can be combined to create fractional occupancy. Thus, a series of stable compositions can be readily generated that hold the total amount of Nluc constant while accurately varying the portion occupied by an acceptor. By this method, NanoBRET exhibits linearity with donor occupancy extending beyond 3 orders of magnitude (Figure 1B; see Supporting Information Figure S5 for uncorrected acceptor/donor channel emission ratios). The smallest degree of measurable occupancy can be determined using a statistical metric of discrimination from the unoccupied donor. A commonly used metric is Z′-factor, where quantitatively reliable discrimination is attained for values above 0.5. An evaluation of Z′-factor values relative to fractional occupancy indicates that the NCT ligand provides about 20-fold greater sensitivity than the Oregon Green Ligand and about 4-fold over the TMR Ligand (Table 1).
Table 1. Sensitivity Limits for NanoBRET Acceptors
Z′-factora
 acceptor fluorophore
fractional occupancy (%)NCTTMROGYFPRFP
0.050.46<0<0<0<0
0.100.65<0<0<0<0
0.250.780.57<0<0<0
0.500.850.71<0<0<0
1.00.920.810.046<0<0
2.50.950.900.78<0<0
5.00.960.930.880.230.21
100.980.950.900.750.65
250.980.970.960.890.82
500.980.980.980.920.91
1000.990.990.970.970.93
a

The Z′-factor was determined for each value of % occupancy for Nluc-HT-OG, Nluc-HT-TMR, and Nluc-HT-NCT and for Nluc-turboYFP and Nluc-TagRFP (using Nluc-HT-PEG-biotin as the unoccupied donor).

Since autofluorescent proteins are most commonly used as the acceptor in BRET assays, their performance was also evaluated in NanoBRET. TurboYFP is a fast maturing YFP variant derived from Phialidium sp., with brightness comparable to Venus and spectral properties similar to the Oregon Green Ligand. TagRFP is derived from Entacmaea quadricolor and has spectral properties similar to the TMR Ligand. (12-14) These were directly fused genetically to Nluc, and fractional occupancy was established as before, using Nluc-HT as an unoccupied donor. Despite the spectral similarity with the fluorescent HaloTag ligands, assay sensitivity was substantially diminished (Table 1). This may be explained in part by having lower quantum yields relative to the HaloTag ligands, inefficient chromophore maturation as well as differences in geometries of these fusions relative to Nluc-HT. (15) In addition, the chromophore in autofluorescent proteins may also be influenced by intracellular conditions, as suggested by an apparent linkage we observed between energy transfer and expression levels for TurboYFP and TagRFP (Supporting Information Figure S6). Both exhibited decreased energy transfer with lower expression while HaloTag provided nearly constant energy transfer. BRET is expected to be independent of concentration, so long as the orientation between the donor and acceptor remains fixed, since the ratiometric values are normalized to donor intensity. This was the case for these fusions when measured as purified proteins (Supporting Information Figure S7).

Performance of NanoBRET for Quantifying Protein Interactions

Having established HaloTag-NCT as an optimal energy acceptor for Nluc, this combination should enhance the performance of BRET for analyzing intracellular protein–protein interactions (PPI). Furthermore, labeling HaloTag with NCT as the energy acceptor is straightforward, requiring only the addition of the tagged fluorophore to the culture medium prior to making measurements (Figure 2). We evaluated this by benchmarking performance to the commonly used pairing of Rluc8 with YFP. For this purpose, we selected the rapamycin-induced interaction between FKBP12 and Frb (Rapamycin-binding domain of mTOR) as the biological model. The Frb/FKBP interaction model has been frequently employed for the evaluation of new PPI analytical methods due to its simplicity and robustness. (16)

Figure 2

Figure 2. Schematic of NanoBRET for detecting protein–protein interactions.

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Because the efficiency of energy transfer can be affected by geometric orientation, it is important to examine all eight possible configurations of donor and acceptor fused onto the interacting proteins. Specifically, these are the donor and acceptor fused to each of the proteins at either terminus of the polypeptide. For all configurations of Nluc/HT-NCT and Rluc8/YFP fused to FKBP and Frb, induction of PPI by rapamycin led to an increase in energy transfer. As expected, the different configurations of the fusions show considerable variation in their relative response to rapamycin (Figure 3a, see Supporting Information Figure S8a,b for uncorrected acceptor/donor channel emission ratios). For all configurations of Rluc8/YFP, the response was lower than for the corresponding configuration of Nluc/HT-NCT and varied by 4 to 70 fold between analogously oriented pairs. The most efficient configurations of Nluc/HT-NCT and Rluc8/YFP (Figure 3a, bars with hatch pattern), respectively, exhibited a 7-fold difference in response to rapamycin.

Figure 3

Figure 3. Comparative analysis of BRET1 and NanoBRET using the Frb/FKBP model system. (a) Optimization of donor and acceptor orientation for the Frb/FKBP interaction. BRET ratios were determined on HEK293 cells transiently transfected with the indicated Frb and FKBP fusion proteins at a donor/acceptor ratio of 1:4, each measured using untreated and rapamycin treated (1 μM, 15 min) samples. The optimal configuration for either BRET1 or NanoBRET is indicated by the hatch pattern. (b) HEK293 cells were transiently transfected with either Frb-Nluc/FKBP-HT or Frb-RLuc8/FKBP-turboYFP at a donor/acceptor ratio of 1:4. The cells were plated in a 384-well plate and left untreated (n = 3) or treated with the indicated concentration of rapamycin (n = 3) for 15 min before determining the BRET ratio.

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The donors and acceptors used in these comparisons are structurally distinct, possibly resulting in some differences in their relative geometry when fused to FKBP and Frb. Nonetheless, it is likely that NanoBRET consistently exhibits greater response dynamics due to its greater spectral separation between the donor and acceptor, narrower emission spectrum of Nluc, and higher quantum yield of NCT ligand. By either choice of donor and acceptor, the EC50 values were essentially unchanged and in agreement with previously published data (Figure 3b, see Supporting Information Figure 9A–D for raw RLU values and uncorrected acceptor/donor channel emission ratios), indicating that the pharmacology of rapamycin was not influenced by the reporter fusions. (16) In addition, NanoBRET measurements of this stable interaction remained relatively constant for 60 min both in the presence and in the absence of rapamycin, indicating the ability to continuously monitor intracellular PPIs over an extended period of time (Supporting Information Figure S10).
As molecular interactions are subject to concentration, having appropriate expression levels of the donor and acceptor is important for achieving biologically relevant data. Because regulatory proteins in particular tend to be present at low levels in cells, energy transfer must be measurable at low expression levels. Furthermore, unphysiologically high expression can lead to biological artifacts such as ligand independent signaling, improper recruitment of other cellular components and mis-folding or mis-localization of the proteins. To explore the capacity for detecting PPI at physiologically relevant levels, we measured rapamycin-induced BRET over a titrated range of donor and acceptor expression plasmids, combined in equal amounts and serially diluted with carrier DNA. For both Nluc/HT-NCT and Rluc8/YFP, a negative correlation between expression level and rapamycin response was evident, possibly due to competition between the introduced fusions and their endogenous counterparts at lower transfection amounts. Western blots for detection of FKBP showed that fusions to either acceptor were present in the sample at amounts comparable to the endogenous protein near the middle of the titration range (Supporting Information Figure S11A,B). Consistency in expression of FKBP fused to either acceptor, over the titrated range of DNA, was also confirmed by flow cytometry analysis of FKBP-HaloTag and FKBP-turboYFP expression (Supporting Information Figure 12A,B).
However, there was a marked difference in signal separation between the BRET pairs, especially at low levels of fusion protein. The NanoBRET pair exhibited complete data separation between rapamycin treated and untreated samples for the entire range of DNA dilutions (Figure 4a, Supplemental Figure 12c), whereas the Rluc8/YFP pair showed convergence to a single value at much higher analyte levels (Figure 4b, Supplemental Figure 12d). A Z′-factor analysis was used to estimate the capacity for reliably distinguishing a rapamycin-induced interaction from an untreated control. (17) The NanoBRET based assay maintained good sensitivity (Z′ > 0.65) even at concentrations of FKBP-HT well below the endogenous FKBP. In comparison, the Rluc8/YFP pair exhibited decreased assay sensitivity even for analyte amounts well above endogenous levels. These results show that NanoBRET can provide better assay sensitivity and response dynamics even at analyte levels comparable to or below physiological concentrations within cells. The ability to perform such measurements at low concentrations of reporter may be particularly significant for technically challenging cell types, such as stem cells and primary cells, which are often difficult to transfect or have limited capacity for expressing transgenes.

Figure 4

Figure 4. Effect of expression level on assay sensitivity for detecting the interaction of FKBP with Frb. HeLa cells were transfected with a serial dilution of expression constructs for (a) Frb-Nluc and FKBP-HT or (b) Frb-Rluc8 and FKBP-TurboYFP (1:1 ratio) and plated in thre 96-well plates. BRET ratios were determined for untreated or rapamycin treated samples (1 μM rapamycin, 15 min). Shown is a representative experiment with all individual data points (n = 12) as well as mean ± SD plotted against DNA dilution used for transfection.

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Analysis of Bromodomain Interactions

The role of PPI in many disease relevant signaling pathways has motivated interest in compounds that target these interactions for therapeutic purposes. (18) NanoBRET provides a means for exploring the action of these compounds within the intracellular environment. This is exemplified by characterizing compounds able to inhibit binding of bromodomain proteins to chromatin, which reveals the importance of the cellular context and how biochemical studies can sometimes be misleading. The binding of bromodomain proteins to acetylated histones is one of the key mechanisms for epigenetic regulation of transcriptional activity. The ability to block binding by specific bromodomain proteins is an emerging area of drug therapy for a variety of cancers, which has been most elegantly shown for the BET family members, BRD2, BRD3, and BRD4. (19-21)
We show that the interaction between BRD4 and chromatin can be monitored with NanoBRET by expressing Nluc-BRD4 in mammalian cells with human histone H3.3-HT fusions (Figure 5A). Treatment of the cells with increasing doses of known inhibitor I-BET151, which competes for binding to acetyl lysine, showed a dose dependent decrease of BRET signal with an IC50 close to published values (Figures 5A, see Supporting Information Figure S13A for uncorrected acceptor/donor channel emission ratios). (22) In similar studies with a non-BET family bromodomain protein, CBP, we do not see any displacement from chromatin with I-BET151, rather a slight increase we believe is due to increased occupancy of CBP after BET family displacement (Figures 5B, see Supporting Information Figure S13B for uncorrected acceptor/donor channel emission ratios). Similar results in both systems were obtained using histone H4-HT as the acceptor partner in place of histone H3.3-HT (Supporting Information Figure S13C and D). These results demonstrate that the action of I-BET151 can be observed by NanoBRET for specific pairs of bromodomain proteins and histones in living cells.

Figure 5

Figure 5. NanoBRET assays for bromodomain protein interactions with chromatin. NanoBRET assays were developed to measure the binding of Nluc-BRD4 (a) or Nluc-CBP (b) to Histone H3.3-HT. HEK293 cells were transfected with the indicated combination of constructs. The BRET ratio for each sample was determined following treatment for 18 h with the indicated concentrations of I-BET151. Shown are the means ± SEM of three independent experiments performed in quadruplicate.

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NanoBRET for Cellular Imaging

The ability to image BRET within individual cells has previously been restricted by the relatively low photon flux provided by bioluminescence. However, the bright luminescence of Nluc combined with recent advances in cellular imaging technologies is beginning to change this situation. We recently demonstrated that Nluc enables visualization of subcellular translocation events within living cells with subsecond temporal resolution. (10) In this study, the well-established ligand-induced recruitment of β-arrestin 2 (ARRB2) to vasopressin receptor 2 (AVPR2) was used as a model for visualizing protein interactions. (23)
In the absence of a ligand, Nluc-ARRB2 showed the anticipated uniform distribution in cells (Figure 6A). A weak signal was also observed in the acceptor channel, which can be attributed to “bleed-through” of the Nluc luminescence. The addition of arginine vasopressin (AVP) induced a substantial signal increase in the acceptor channel, accompanied by the appearance of a vesicular pattern in both detection channels, which indicates association of Nluc-ARRB2 with AVPR2-HT, followed by trafficking into clathrin-coated pits (Figure 6A, Supporting Information Figure S14A). (24) The sequential imaging with short exposure times allowed the recording of arrestin recruitment kinetics in individual cells. Although transient transfection leads to variable expression levels between cells, the relative response kinetics were found to be similar and corresponded well with data obtained for a cell population in a 96-well plate format (Figure 6B, see Supporting Information Figure S14B and C for uncorrected acceptor/donor channel emission ratios).

Figure 6

Figure 6. Real time imaging of ligand induced interaction of AVPR2-HT with Nluc-β-arrestin2 interaction. HeLa cells were transfected with expression constructs for AVPR2-HT and Nluc-β-arrestin2. Sequential acquisition of images was initiated under unstimulated conditions. After establishing a baseline for seven acquisition cycles, 1 μM arginine vasopressin was added to the sample by injection followed by acquisition cycles (13.4 s per cycle) covering approximately 20 min. (a) The panel shows pseudocolored images of the donor (cyan) and acceptor (yellow) channel at indicated time points following treatment with AVP. The images are representative of the results obtained in five independent experiments (scale bar = 10 μm). (b) Kinetic of Nluc-β-arrestin2 recruitment following stimulation with AVP measured by imaging or in plate reader. The imaging data represent five individual regions of interest (ROI), each representing a single cell. The data shown for the plate based assay format are the results of a representative experiment performed in triplicate. In order to compare results obtained from different analytical platforms, the data were plotted as relative change in BRET normalized to the maximum BRET ratio obtained for each data set.

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In addition to the vasopressin receptor model, NanoBRET imaging was successfully applied to the rapamycin-induced interaction of FKBP with Frb, and the EGF-induced recruitment of grb2 to the EGF receptor (Supporting Information Figure S15A and B). The sustained high luminescence output of Nluc allows continuous monitoring of protein–protein interactions for up to 2 h and also improves spatial and temporal resolution. The enhanced feasibility for imaging of PPI in living cells afforded by NanoBRET may be of particular use for signaling events that are confined to specific subcellular compartments, especially if only a small subset of the donor-tagged protein is engaged in the interaction of interest.

Conclusions

BRET has been used for nearly 15 years to analyze protein–protein interaction in living cells. Benefiting from the capacity of luminescent chemistries to elucidate intracellular events, BRET assays are further advantaged by their ratiometric format for enhancing data precision and robustness. By representing acceptor proximity in relation to an available donor, the ratiometric normalization provides automatic correction for variable cell numbers or gene expression, inconsistent reagent potency, inadvertent luciferase inhibitors, and so forth. Yet, despite these advantages, usage of this technology has remained largely confined to relatively specific areas of investigation, most notably the analysis of cell surface receptor interactions. The main reason may be the comparatively limited sensitivity and dynamic range provided by current BRET systems.
With the recent development of Nluc, which emits bright, stable, and spectrally narrow luminescence, we saw an opportunity to design a BRET platform with substantially improved capabilities. This was achieved by pairing Nluc with a spectrally well separated acceptor, thus effectively reducing the background caused by “bleed through” of the donor signal into the acceptor channel. Our results indicate that NanoBRET (Figure 2) provides a reliable platform for analyzing protein–protein interactions at low expression levels in living cells, which is important for maintaining appropriate interaction dynamics. This applies not only to the tagged proteins subject to measurement but also to the many untagged endogenous proteins whose contributing influence comprises the physiological environment. The increased sensitivity of NanoBRET should be useful in combination with genomic editing technologies such as TALEN or CRISPR, allowing protein expression to be directed by the corresponding endogenous gene locus. (25)
Due to the delicate energy relationships associated with transient protein interactions, the influence of synthetic molecules on these interactions may be particularly reliant on the intracellular environment. Drug development founded on biochemical approaches may not adequately encompass the essential elements necessary to recapitulate a living molecular process. We have demonstrated that NanoBRET can deliver precise quantitative measurements of dynamic interactions as they occur within cells. Moreover, the technology delivers sufficient sensitivity to reveal these interactions occurring even to relatively small extents (i.e., at low fractional occupancies). The high emission intensity of NanoBRET also opens possibilities for imaging protein interactions within individual cells, and we speculate that the red-shifted emission of the acceptor may be suitable for imaging in living animals. In addition, linking fluorophores onto other types of molecules may prove useful for revealing a broader range of intracellular interactions.
The technical challenge of working with isolated full-length proteins also promotes the common usage of protein fragments as surrogates. Such simplified assays may not capture the multidomain and multifunctional nature of intracellular proteins and the many cellular factors which regulate their interactions. This is clearly demonstrated in our examples using NanoBRET to monitor interactions with chromatin. These interactions are highly modulated and dictated by the chromatin environment, including nucleosome positioning, presence of other transcription factors and transcription machinery, and the array of modifications present at any given time on the various histones. This environment cannot be replicated in a biochemical setting and thus highlights the necessity for monitoring such interactions within cells. NanoBRET configured with histones as energy acceptors should provide the ability to monitor interactions not only of bromodomains, as demonstrated here, but other chromatin interacting proteins as well.

Material and Methods

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Fractional Occupancy Assay

A 400 pM dilution of recombinant Nluc-HT, Nluc-turboYFP, and Nluc-tagRFP protein was prepared in PBS + 0.1% BSA (Promega). The Nluc-HT solution was split in separate samples and labeled for 180 min at RT with 5 μM of either the indicated fluorescent HaloTag ligands (Oregon Green, TMR, and NCT) or the nonfluorescent HaloTag PEG-biotin ligand. To prepare solutions representing different degrees of occupancy, the stock solutions of Nluc-HT-ligand, Nluc-TurboYFP, and Nluc-tagRFP were combined with Nluc-HT-PEG-biotin (as unoccupied donor) at defined ratios and a total concentration of 2 nM in PBS + 0.1% BSA. The percentage of acceptor occupied donor relative to total donor is referred to as fractional occupancy. BRET measurements were performed in white 96-well plates by combining 50 μL of the donor/acceptor mixtures with 50 μL of 20 μM furimazine in PBS. All measurements were performed at RT on a Thermo Scientific Varioskan Flash plate reader equipped with a 460/80 nm band-pass filter (donor) and 530 nm (HaloTag Oregon Green Ligand/TurboYFP), 590 nm (HaloTag TMR Ligand/Tag RFP), or 610 nm (HaloTag NCT Ligand) long-pass filters (acceptor) using an integration time of 0.5 s.

Live Cell Labeling with HaloTag NCT Ligand

For labeling of cells with the HaloTag NCT ligand, we developed two alternative protocols based on the needs of experimental PPI models (protocol use is also indicated in the description of the PPI models). Rapid labeling protocol: Cells were labeled by replacing the growth medium with labeling medium (Opti-MEM supplemented with 250 nM HaloTag NCT ligand) followed by incubation for 60–90 min at 37 °C. Overnight labeling protocol: Cells were labeled by replacing the growth medium with labeling medium (Opti-MEM supplemented with 4% FBS and 100 nM HaloTag NCT ligand) followed by incubation for 12–18 h at 37 °C. All HaloTag NCT ligand dilutions were performed using a stock solution of 5 mM HaloTag NCT ligand in DMSO. Subsequent treatment and processing of samples was performed as outlined in assay specific experimental procedures. Removal of excess ligand is not required since no unspecific bystander BRET was observed between the HaloTag NCT ligand and Nluc in biochemical or cell based assays for HaloTag NCT ligand concentrations of up to 500 nM.

Frb/FKBP Interaction Assays

HEK293 cells were transiently transfected with expression constructs for the indicated Frb and FKBP fusions and plated into a white 96-well tissue culture plate (Corning Costar #3917) at 2 × 104 cells/well in 100 μL volume. After 24 h of incubation, the growth medium was replaced by 50 μL Opti-MEM (Life Technologies). HaloTag containing samples were labeled by the addition of 50 μL of Opti-MEM containing HaloTag NCT Ligand at a concentration of 250 nM followed by incubation for 90 min at 37 °C. Interaction of Frb and FKBP was induced by the addition of rapamycin at the indicated concentration. After 15 min of incubation at 37 °C, furimazine (NanoBRET) or Coelenterazine h (BRET1) was added to each sample to a final concentration of 10 or 20 μM, respectively. All measurements were taken at RT using a Thermo Scientific Varioskan Flash plate reader using the filter setting as outlined above.

Supporting Information

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Detailed experimental protocols, supplementary figures, and tables. This material is available free of charge at The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.5b00143.

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Author Information

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  • Corresponding Author
    • Thomas Machleidt - Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States;  Email: [email protected]
  • Authors
    • Carolyn C. Woodroofe - Promega Biosciences Incorporated, 277 Granada Drive, San Luis Obispo, California 93401, United States
    • Marie K. Schwinn - Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States
    • Jacqui Méndez - Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States
    • Matthew B. Robers - Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States
    • Kris Zimmerman - Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States
    • Paul Otto - Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States
    • Danette L. Daniels - Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States
    • Thomas A. Kirkland - Promega Biosciences Incorporated, 277 Granada Drive, San Luis Obispo, California 93401, United States
    • Keith V. Wood - Promega Corporation, 2800 Woods Hollow, Madison, Wisconsin 53711, United States
  • Notes
    The authors declare no competing financial interest.

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This article references 25 other publications.

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    Fluorescence-based methods in the study of protein-protein interactions in living cells
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    A review. Multiprotein complexes partake in nearly all cell functions, thus the characterization and visualization of protein-protein interactions in living cells constitute an important step in the study of a large array of cellular mechanisms. Recently, noninvasive fluorescence-based methods using resonance energy transfer (RET), namely bioluminescence-RET (BRET) and fluorescence-RET (FRET), and those centered on protein fragment complementation, such as bimol. fluorescence complementation (BiFC), have been successfully used in the study of protein interactions. These new technologies are nowadays the most powerful approaches for visualizing the interactions occurring within protein complexes in living cells, thus enabling the investigation of protein behavior in their normal milieu. Here we address the individual strengths and weaknesses of these methods when applied to the study of protein-protein interactions.
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    We describe a method for assaying protein interactions that offers some attractive advantages over previous assays. This method, called bioluminescence resonance energy transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and a green fluorescent protein mutant fused to another protein of interest. Interactions between the two fusion proteins can bring the luciferase and green fluorescent protein close enough for resonance energy transfer to occur, thus changing the color of the bioluminescent emission. By using proteins encoded by circadian (daily) clock genes from cyanobacteria, we use the BRET technique to demonstrate that the clock protein KaiB interacts to form homodimers. BRET should be particularly useful for testing protein interactions within native cells, esp. with integral membrane proteins or proteins targeted to specific organelles.
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    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXjvV2itw%253D%253D&md5=eb87cabdad3ba1ee84b52692e4ef8f20
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    Pfleger, K. D. and Eidne, K. A. (2006) Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET) Nat. Methods 3, 165– 174
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    Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET)
    Pfleger, Kevin D. G.; Eidne, Karin A.
    Nature Methods (2006), 3 (3), 165-174CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    A review. Bioluminescence resonance energy transfer (BRET) is a straightforward biophys. technique for studying protein-protein interactions. It requires: (1) that proteins of interest and suitable controls be labeled with either a donor or acceptor mol., (2) placement of these labeled proteins in the desired environment for assessing their potential interaction, and (3) use of suitable detection instrumentation to monitor resultant energy transfer. There are now several possible applications, combinations of donor and acceptor mols., potential assay environments and detection system perturbations. Therefore, this review aims to demystify and clarify the important aspects of the BRET methodol. that should be considered when using this technique.
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    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhsFOru7k%253D&md5=bc83e4f90f5ab10ed12286eb20849ccc
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    Bertrand, L., Parent, S., Caron, M., Legault, M., Joly, E., Angers, S., Bouvier, M., Brown, M., Houle, B., and Menard, L. (2002) The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS) J. Recept. Signal Transduction Res. 22, 533– 541
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    The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS)
    Bertrand, Lucie; Parent, Stephane; Caron, Mireille; Legault, Mireille; Joly, Erik; Angers, Stephane; Bouvier, Michel; Brown, Mike; Houle, Benoit; Menard, Luc
    Journal of Receptors and Signal Transduction (2002), 22 (1-4), 533-541CODEN: JRSTCT ISSN:. (Marcel Dekker, Inc.)
    In BRET2 (Bioluminescence Resonance Energy Transfer), a Renilla luciferase (RLuc) is used as the donor protein, while a Green Fluorescent Protein (GFP2) is used as the acceptor protein. In the presence of the cell permeable substrate DeepBlueC, RLuc emits blue light at 395 nm. If the GFP2 is brought into close proximity to RLuc via a specific biomol. interaction, the GFP2 will absorb the blue light energy and reemit green light at 510 nm. BRET2 signals are therefore easily detd. by measuring the ratio of green over blue light (510/395 nm) using appropriate dual channel luminometry instruments (e.g., Fusion Universal Microplate Analyzer, Packard BioScience). Since no light source is required for BRET2 assays, the technol. does not suffer from high fluorescent background or photobleaching, the common problems assocd. with std. FRET-based assays. Using BRET2, the authors developed a generic G Protein-Coupled Receptor (GPCR) assay based on the observation that activation of the majority of GPCRs by agonists leads to the interaction of β-arrestin (a protein that is involved in receptor desensitization and sequestration) with the receptor. The authors established a cell line stably expressing the GFP2:β-arrestin 2 fusion protein, and showed that it can be used to monitor the activation of various transiently expressed GPCRs, in BRET2/arrestin assays. In addn., using the HEK 293/GFP2:β-arrestin 2 cell line as a recipient, the authors generated a double-stable line co-expressing the vasopressin 2 receptor (V2R) fused to RLuc (V2R:RLuc) and used it for the pharmacol. characterization of compds. in BRET2/arrestin assays. This approach yields genuine pharmacol. and supports the BRET2/arrestin assay as a tool that can be used with recombinant cell lines to characterize ligand-GPCR interactions which can be applied to ligand identification for orphan receptors.
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    Hamdan, F. F., Audet, M., Garneau, P., Pelletier, J., and Bouvier, M. (2005) High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assay J. Biomol. Screening 10, 463– 475
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    High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based β-arrestin2 recruitment assay
    Hamdan, Fadi F.; Audet, Martin; Garneau, Philippe; Pelletier, Jerry; Bouvier, Michel
    Journal of Biomolecular Screening (2005), 10 (5), 463-475CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)
    In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amts. of β-arrestin2-Rluc and VENUS fusions of G protein-coupled receptors (GPCRs) belonging to both class A and class B receptors, which include receptors that interact transiently or stably with β-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1-β-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and β-arrestin2-/Renilla luciferase was then used for high-throughput screening (HTS) for antagonists of the chemokine receptor CCR5, the primary co-receptor for HIV. A total of 26,000 compds. were screened for inhibition of the agonist-promoted β-arrestin2 recruitment to CCR5, and 12 compds. were found to specifically inhibit the agonist-induced β-arrestin2 recruitment to CCR5. Three of the potential hits were further tested using other functional assays, and their abilities to inhibit CCR5 agonist-promoted signaling were confirmed. This is the 1st study describing a BRET1-β-arrestin recruitment assay in stable mammalian cells and its successful application in HTS for GPCRs antagonists.
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    Loening, A. M., Fenn, T. D., Wu, A. M., and Gambhir, S. S. (2006) Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output Protein Eng., Des. Sel. 19, 391– 400
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    Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output
    Loening, Andreas Markus; Fenn, Timothy David; Wu, Anna M.; Gambhir, Sanjiv Sam
    Protein Engineering, Design & Selection (2006), 19 (9), 391-400CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)
    Luciferases, which have seen expansive employment as reporter genes in biol. research, could also be used in applications where the protein itself is conjugated to ligands to create probes that are appropriate for use in small animal imaging. As the bioluminescence activity of commonly used luciferases is too labile in serum to permit this application, specific mutations of Renilla luciferase, selected using a consensus sequence driven strategy, were screened for their ability to confer stability of activity in serum as well as their light output. Using this information, a total of eight favorable mutations were combined to generate a mutant Renilla luciferase (RLuc8) that, compared with the parental enzyme, is 200-fold more resistant to inactivation in murine serum and exhibits a 4-fold improvement in light output. Results of the mutational anal. were also used to generate a double mutant optimized for use as a reporter gene. The double mutant had half the resistance to inactivation in serum of the native enzyme while yielding a 5-fold improvement in light output.These variants of Renilla luciferase, which exhibit significantly improved properties compared with the native enzyme, will allow enhanced sensitivity in existing luciferase-based assays as well as enable the development of novel probes labeled with the luciferase protein.
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  9. 9
    Kocan, M., See, H. B., Seeber, R. M., Eidne, K. A., and Pfleger, K. D. (2008) Demonstration of improvements to the bioluminescence resonance energy transfer (BRET) technology for the monitoring of G protein-coupled receptors in live cells J. Biomol. Screening 13, 888– 898
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    Demonstration of improvements to the bioluminescence resonance energy transfer (BRET) technology for the monitoring of G protein-coupled receptors in live cells
    Kocan, Martina; See, Heng B.; Seeber, Ruth M.; Eidne, Karin A.; Pfleger, Kevin D. G.
    Journal of Biomolecular Screening (2008), 13 (9), 888-898CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)
    The bioluminescence resonance energy transfer (BRET) technique has become extremely popular for studying protein-protein interactions in living cells and real time. Of particular interest is the ability to monitor interactions between G protein-coupled receptors, such as the TSH-releasing hormone receptor (TRHR), and proteins crit. for regulating their function, such as β-arrestin. Using TRHR/β-arrestin interactions, we have demonstrated improvements to all 3 generations of BRET (BRET, BRET, and eBRET) by using the novel forms of luciferase, Rluc2 and Rluc8, developed by the Gambhir lab. Furthermore, for the 1st time it was possible to use the BRET2 system to detect ligand-induced G protein-coupled receptor/β-arrestin interactions over prolonged periods (on the scale of hours rather than seconds) with a very stable signal. As demonstrated by our Z'-factor data, these luciferases increase the sensitivity of BRET to such an extent that they substantially increase the potential applicability of this technol. for effective drug discovery high-throughput screening.
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    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXjtVymtr0%253D&md5=52bd4ecaa2dd5f265e7a02deeb407556
  10. 10
    Hall, M. P., Unch, J., Binkowski, B. F., Valley, M. P., Butler, B. L., Wood, M. G., Otto, P., Zimmerman, K., Vidugiris, G., Machleidt, T., Robers, M. B., Benink, H. A., Eggers, C. T., Slater, M. R., Meisenheimer, P. L., Klaubert, D. H., Fan, F., Encell, L. P., and Wood, K. V. (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate ACS Chem. Biol. 7, 1848– 1857
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    Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate
    Hall, Mary P.; Unch, James; Binkowski, Brock F.; Valley, Michael P.; Butler, Braeden L.; Wood, Monika G.; Otto, Paul; Zimmerman, Kristopher; Vidugiris, Gediminas; Machleidt, Thomas; Robers, Matthew B.; Benink, Helene A.; Eggers, Christopher T.; Slater, Michael R.; Meisenheimer, Poncho L.; Klaubert, Dieter H.; Fan, Frank; Encell, Lance P.; Wood, Keith V.
    ACS Chemical Biology (2012), 7 (11), 1848-1857CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochem. and phys. characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high phys. stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degrdn. sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metab. or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.
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  11. 11
    Los, G. V., Encell, L. P., McDougall, M. G., Hartzell, D. D., Karassina, N., Zimprich, C., Wood, M. G., Learish, R., Ohana, R. F., Urh, M., Simpson, D., Mendez, J., Zimmerman, K., Otto, P., Vidugiris, G., Zhu, J., Darzins, A., Klaubert, D. H., Bulleit, R. F., and Wood, K. V. (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis ACS Chem. Biol. 3, 373– 382
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    11
    HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis
    Los, Georgyi V.; Encell, Lance P.; McDougall, Mark G.; Hartzell, Danette D.; Karassina, Natasha; Zimprich, Chad; Wood, Monika G.; Learish, Randy; Ohana, Rachel Friedman; Urh, Marjeta; Simpson, Dan; Mendez, Jacqui; Zimmerman, Kris; Otto, Paul; Vidugiris, Gediminas; Zhu, Ji; Darzins, Aldis; Klaubert, Dieter H.; Bulleit, Robert F.; Wood, Keith V.
    ACS Chemical Biology (2008), 3 (6), 373-382CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in soln., in living cells, or in chem. fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful mols., such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiol. conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple mol. processes assocd. with NF-κB-mediated cellular physiol., including imaging of subcellular protein translocation and capture of protein-protein and protein-DNA complexes.
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    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXmvVGru7k%253D&md5=66e11d2da0992e634150392ea9575f75
  12. 12
    Shagin, D. A., Barsova, E. V., Yanushevich, Y. G., Fradkov, A. F., Lukyanov, K. A., Labas, Y. A., Semenova, T. N., Ugalde, J. A., Meyers, A., Nunez, J. M., Widder, E. A., Lukyanov, S. A., and Matz, M. V. (2004) GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity Mol. Biol. Evol. 21, 841– 850
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    12
    GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity
    Shagin, Dmitry A.; Barsova, Ekaterina V.; Yanushevich, Yurii G.; Fradkov, Arkady F.; Lukyanov, Konstantin A.; Labas, Yulii A.; Semenova, Tatiana N.; Ugalde, Juan A.; Meyers, Ann; Nunez, Jose M.; Widder, Edith A.; Lukyanov, Sergey A.; Matz, Mikhail V.
    Molecular Biology and Evolution (2004), 21 (5), 841-850CODEN: MBEVEO; ISSN:0737-4038. (Oxford University Press)
    Homologs of the green fluorescent protein (GFP), including the recently described GFP-like domains of certain extracellular matrix proteins in Bilaterian organisms, are remarkably similar at the protein structure level, yet they often perform totally unrelated functions, thereby warranting recognition as a superfamily. Here we describe diverse GFP-like proteins from previously undersampled and completely new sources, including hydromedusae and planktonic Copepoda. In hydromedusae, yellow and nonfluorescent purple proteins were found in addn. to greens. Notably, the new yellow protein seems to follow exactly the same structural soln. to achieving the yellow color of fluorescence as YFP, an engineered yellow-emitting mutant variant of GFP. The addn. of these new sequences made it possible to resolve deep-level phylogenetic relationships within the superfamily. Fluorescence (most likely green) must have already existed in the common ancestor of Cnidaria and Bilateria, and therefore GFP-like proteins may be responsible for fluorescence and/or coloration in virtually any animal. At least 15 color diversification events can be inferred following the max. parsimony principle in Cnidaria. Origination of red fluorescence and nonfluorescent purple-blue colors on several independent occasions provides a remarkable example of convergent evolution of complex features at the mol. level.
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  13. 13
    Merzlyak, E. M., Goedhart, J., Shcherbo, D., Bulina, M. E., Shcheglov, A. S., Fradkov, A. F., Gaintzeva, A., Lukyanov, K. A., Lukyanov, S., Gadella, T. W., and Chudakov, D. M. (2007) Bright monomeric red fluorescent protein with an extended fluorescence lifetime Nat. Methods 4, 555– 557
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    13
    Bright monomeric red fluorescent protein with an extended fluorescence lifetime
    Merzlyak, Ekaterina M.; Goedhart, Joachim; Shcherbo, Dmitry; Bulina, Mariya E.; Shcheglov, Aleksandr S.; Fradkov, Arkady F.; Gaintzeva, Anna; Lukyanov, Konstantin A.; Lukyanov, Sergey; Gadella, Theodorus W. J.; Chudakov, Dmitriy M.
    Nature Methods (2007), 4 (7), 555-557CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Fluorescent proteins have become extremely popular tools for in vivo imaging and esp. for the study of localization, motility and interaction of proteins in living cells. Here the authors report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.
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  14. 14
    Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., and Miyawaki, A. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications Nat. Biotechnol. 20, 87– 90
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    14
    A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications
    Nagai, Takeharu; Ibata, Keiji; Park, Eun Sun; Kubota, Mie; Mikoshiba, Katsuhiko; Miyawaki, Atsushi
    Nature Biotechnology (2002), 20 (1), 87-90CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)
    The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biol. systems. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs. 1,2). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37° and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-). For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired. Here we describe the development of an improved version of YFP named "Venus". Venus contains a novel mutation, F46L, which at 37° greatly accelerates oxidn. of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl-. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labeling that were not possible before.
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    Shaner, N. C., Steinbach, P. A., and Tsien, R. Y. (2005) A guide to choosing fluorescent proteins Nat. Methods 2, 905– 909
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    A guide to choosing fluorescent proteins
    Shaner, Nathan C.; Steinbach, Paul A.; Tsien, Roger Y.
    Nature Methods (2005), 2 (12), 905-909CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    The recent explosion in the diversity of available fluorescent proteins (FPs) promises a wide variety of new tools for biol. imaging. With no unified std. for assessing these tools, however, a researcher is faced with difficult questions. Which FPs are best for general use. Which are the brightest. What addnl. factors det. which are best for a given expt.. Although in many cases, a trial-and-error approach may still be necessary in detg. the answers to these questions, a unified characterization of the best available FPs provides a useful guide in narrowing down the options.
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    Galarneau, A., Primeau, M., Trudeau, L. E., and Michnick, S. W. (2002) Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions Nat. Biotechnol. 20, 619– 622
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    β-Lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein-protein interactions
    Galarneau, Andre; Primeau, Martin; Trudeau, Louis-Eric; Michnick, Stephen W.
    Nature Biotechnology (2002), 20 (6), 619-622CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)
    We have previously described a strategy for detecting protein-protein interactions based on protein interaction-assisted folding of rationally designed fragments of enzymes. We call this strategy the protein fragment complementation assay (PCA). Here we describe PCAs based on the enzyme TEM-1 β-lactamase (EC: 3.5.2.6), which include simple colorimetric in vitro assays using the cephalosporin nitrocefin and assays in intact cells using the fluorescent substrate CCF2/AM (ref. 6). Constitutive protein-protein interactions of the GCN4 leucine zippers and of apoptotic proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an in vitro assay using cell lysates. With the same in vitro assay, we also demonstrate interactions of protein kinase PKB with substrate Bad. The in vitro assay is facile and amenable to high-throughput modes of screening with signal-to-background ratios in the range of 10:1 to 250:1, which is superior to other PCAs developed to date. Furthermore, we show that the in vitro assay can be used for quant. anal. of a small mol.-induced protein interaction, the rapamycin-induced interaction of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of rapamycin)). The assay reproduces the known dissocn. const. and no. of sites for this interaction. The combination of in vitro colorimetric and in vivo fluorescence assays of β-lactamase in mammalian cells suggests a wide variety of sensitive and high-throughput large-scale applications, including in vitro protein array anal. of protein-protein or enzyme-protein interactions and in vivo applications such as clonal selection for cells expressing interacting protein partners.
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    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XktFOhsrY%253D&md5=284ef04b2969d864811bab12a6de3e4b
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    Zhang, J. H., Chung, T. D., and Oldenburg, K. R. (1999) A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays J. Biomol. Screening 4, 67– 73
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    A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays
    Zhang; Chung; Oldenburg
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    The ability to identify active compounds (3hits2) from large chemical libraries accurately and rapidly has been the ultimate goal in developing high-throughput screening (HTS) assays. The ability to identify hits from a particular HTS assay depends largely on the suitability or quality of the assay used in the screening. The criteria or parameters for evaluating the 3suitability2 of an HTS assay for hit identification are not well defined and hence it still remains difficult to compare the quality of assays directly. In this report, a screening window coefficient, called 3Z-factor,2 is defined. This coefficient is reflective of both the assay signal dynamic range and the data variation associated with the signal measurements, and therefore is suitable for assay quality assessment. The Z-factor is a dimensionless, simple statistical characteristic for each HTS assay. The Z-factor provides a useful tool for comparison and evaluation of the quality of assays, and can be utilized in assay optimization and validation.
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    Reaching for high-hanging fruit in drug discovery at protein-protein interfaces
    Wells, James A.; McClendon, Christopher L.
    Nature (London, United Kingdom) (2007), 450 (7172), 1001-1009CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    A review. Targeting the interfaces between proteins has huge therapeutic potential, but discovering small-mol. drugs that disrupt protein-protein interactions is an enormous challenge. Several recent success stories, however, indicate that protein-protein interfaces might be more tractable than has been thought. These studies discovered small mols. that bind with drug-like potencies to 'hotspots' on the contact surfaces involved in protein-protein interactions. Remarkably, these small mols. bind deeper within the contact surface of the target protein, and bind with much higher efficiencies, than do the contact atoms of the natural protein partner. Some of these small mols. are now making their way through clin. trials, so this high-hanging fruit might not be far out of reach.
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    The bromodomain: From epigenome reader to druggable target
    Sanchez, Roberto; Meslamani, Jamel; Zhou, Ming-Ming
    Biochimica et Biophysica Acta, Gene Regulatory Mechanisms (2014), 1839 (8), 676-685CODEN: BBAGC6; ISSN:1874-9399. (Elsevier B.V.)
    A review. Lysine acetylation is a fundamental post-translational modification that plays an important role in the control of gene transcription in chromatin in an ordered fashion. The bromodomain, the conserved structural module present in transcription-assocd. proteins, functions exclusively to recognize acetyl-lysine on histones and non-histone proteins. The structural analyses of bromodomains' recognition of lysine-acetylated peptides derived from histones and cellular proteins provide detailed insights into the differences and unifying features of biol. ligand binding selectivity by the bromodomains. Newly developed small-mol. inhibitors targeting bromodomain proteins further highlight the functional importance of bromodomain/acetyl-lysine binding as a key mechanism in orchestrating mol. interactions and regulation in chromatin biol. and gene transcription. These new studies argue that modulating bromodomain/acetyl-lysine interactions with small-mol. chems. offer new opportunities to control gene expression in a wide array of human diseases including cancer and inflammation. This article is part of a Special Issue entitled: Mol. mechanisms of histone modification function.
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    Targeting epigenetic readers in cancer
    Dawson, Mark A.; Kouzarides, Tony; Huntly, Brian J. P.
    New England Journal of Medicine (2012), 367 (7), 647-657CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
    A review on assocn. between epigenetics and chromatin biol., BET bromodomain inhibitors, and role of BET inhibitors in cancer.
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    Filippakopoulos, P., Qi, J., Picaud, S., Shen, Y., Smith, W. B., Fedorov, O., Morse, E. M., Keates, T., Hickman, T. T., Felletar, I., Philpott, M., Munro, S., McKeown, M. R., Wang, Y., Christie, A. L., West, N., Cameron, M. J., Schwartz, B., Heightman, T. D., La Thangue, N., French, C. A., Wiest, O., Kung, A. L., Knapp, S., and Bradner, J. E. (2010) Selective inhibition of BET bromodomains Nature 468, 1067– 1073
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    Selective inhibition of BET bromodomains
    Filippakopoulos, Panagis; Qi, Jun; Picaud, Sarah; Shen, Yao; Smith, William B.; Fedorov, Oleg; Morse, Elizabeth M.; Keates, Tracey; Hickman, Tyler T.; Felletar, Ildiko; Philpott, Martin; Munro, Shongah; McKeown, Michael R.; Wang, Yuchuan; Christie, Amanda L.; West, Nathan; Cameron, Michael J.; Schwartz, Brian; Heightman, Tom D.; La Thangue, Nicholas; French, Christopher; Wiest, Olaf; Kung, Andrew L.; Knapp, Stefan; Bradner, James E.
    Nature (London, United Kingdom) (2010), 468 (7327), 1067-1073CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here the authors report a cell-permeable small mol. (I,JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is obsd. in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chem. scaffold for the development of chem. probes more broadly throughout the bromodomain family.
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  22. 22
    Dawson, M. A., Prinjha, R. K., Dittmann, A., Giotopoulos, G., Bantscheff, M., Chan, W. I., Robson, S. C., Chung, C. W., Hopf, C., Savitski, M. M., Huthmacher, C., Gudgin, E., Lugo, D., Beinke, S., Chapman, T. D., Roberts, E. J., Soden, P. E., Auger, K. R., Mirguet, O., Doehner, K., Delwel, R., Burnett, A. K., Jeffrey, P., Drewes, G., Lee, K., Huntly, B. J., and Kouzarides, T. (2011) Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia Nature 478, 529– 533
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    Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia
    Dawson, Mark A.; Prinjha, Rab K.; Dittman, Antje; Giotopoulos, George; Bantscheff, Marcus; Chan, Wai-In; Robson, Samuel C.; Chung, Chun-wa; Hopf, Carsten; Savitski, Mikhail M.; Huthmacher, Carola; Gudgin, Emma; Lugo, Dave; Beinke, Soren; Chapman, Trevor D.; Roberts, Emma J.; Soden, Peter E.; Auger, Kurt R.; Mirguet, Olivier; Doehner, Konstanze; Delwel, Ruud; Burnett, Alan K.; Jeffrey, Phillip; Drewes, Gerard; Lee, Kevin; Huntly, Brian J. P.; Kouzarides, Tony
    Nature (London, United Kingdom) (2011), 478 (7370), 529-533CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Recurrent chromosomal translocations involving the mixed lineage leukemia (MLL) gene initiate aggressive forms of leukemia, which are often refractory to conventional therapies. Many MLL-fusion partners are members of the super elongation complex (SEC), a crit. regulator of transcriptional elongation, suggesting that aberrant control of this process has an important role in leukemia induction. Here we use a global proteomic strategy to demonstrate that MLL fusions, as part of SEC and the polymerase-assocd. factor complex (PAFc), are assocd. with the BET family of acetyl-lysine recognizing, chromatin adaptor' proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukemia, via the displacement of the BET family of proteins from chromatin. We show that a novel small mol. inhibitor of the BET family, GSK1210151A (I-BET151), has profound efficacy against human and murine MLL-fusion leukemic cell lines, through the induction of early cell cycle arrest and apoptosis. I-BET151 treatment in two human leukemia cell lines with different MLL fusions alters the expression of a common set of genes whose function may account for these phenotypic changes. The mode of action of I-BET151 is, at least in part, due to the inhibition of transcription at key genes (BCL2, C-MYC and CDK6) through the displacement of BRD3/4, PAFc and SEC components from chromatin. In vivo studies indicate that I-BET151 has significant therapeutic value, providing survival benefit in two distinct mouse models of murine MLL-AF9 and human MLL-AF4 leukemia. Finally, the efficacy of I-BET151 against human leukemia stem cells is demonstrated, providing further evidence of its potent therapeutic potential. These findings establish the displacement of BET proteins from chromatin as a promising epigenetic therapy for these aggressive leukemias.
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    Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., and Caron, M. G. (1999) Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization J. Biol. Chem. 274, 32248– 32257
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    23
    Association of β-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization
    Oakley, Robert H.; Laporte, Stephane A.; Holt, Jason A.; Barak, Larry S.; Caron, Marc G.
    Journal of Biological Chemistry (1999), 274 (45), 32248-32257CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Resensitization of G protein-coupled receptors (GPCRs) following agonist-mediated desensitization is a necessary step for maintaining physiol. responsiveness. However, the mol. mechanisms governing the nature of GPCR resensitization are poorly understood. Here, we examine the role of β-arrestin in the resensitization of the β2 adrenergic receptor (β2AR), known to recycle and resensitize rapidly, and the vasopressin V2 receptor (V2R), known to recycle and resensitize slowly. Upon agonist activation, both receptors recruit β-arrestin to the plasma membrane and internalize in a β-arrestin- and clathrin-dependent manner. However, whereas β-arrestin dissocs. from the β2AR at the plasma membrane, it internalizes with the V2R into endosomes. The differential trafficking of β-arrestin and the ability of these two receptors to dephosphorylate, recycle, and resensitize is completely reversed when the carboxyl-terminal tails of these two receptors are switched. Moreover, the ability of β-arrestin to remain assocd. with desensitized GPCRs during clathrin-mediated endocytosis is mediated by a specific cluster of phosphorylated serine residues in the receptor carboxyl-terminal tail. These results demonstrate that the interaction of β-arrestin with a specific motif in the GPCR carboxyl-terminal tail dictates the rate of receptor dephosphorylation, recycling, and resensitization, and thus provide direct evidence for a novel mechanism by which β-arrestins regulate the reestablishment of GPCR responsiveness.
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    Laporte, S. A., Oakley, R. H., Zhang, J., Holt, J. A., Ferguson, S. S., Caron, M. G., and Barak, L. S. (1999) The beta2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis Proc. Natl. Acad. Sci. U.S.A. 96, 3712– 3717
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    Mahen, R., Koch, B., Wachsmuth, M., Politi, A. Z., Perez-Gonzalez, A., Mergenthaler, J., Cai, Y., and Ellenberg, J. (2014) Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells Mol. Biol. Cell 25, 3610
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    Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells
    Mahen, Robert; Koch, Birgit; Wachsmuth, Malte; Politi, Antonio Z.; Perez-Gonzalez, Alexis; Mergenthaler, Julia; Cai, Yin; Ellenberg, Jan
    Molecular Biology of the Cell (2014), 25 (22), 3610-3618, 9 pp.CODEN: MBCEEV; ISSN:1939-4586. (American Society for Cell Biology)
    Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quant. live-cell imaging and single-mol. spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quant. imaging studies in mammalian cells.
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  • Abstract

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    Figure 1

    Figure 1. Comparison of different acceptors for NanoBRET. (a) BRET spectra for Nluc-HT (black) and Nluc-HT conjugated to HaloTag Oregon Green (green), HaloTag TMR (red), or HaloTag NCT (blue) ligands. (b) Correlation of BRET with fractional occupancy. Differing degrees of occupancy were created by combining Nluc-HT-NCT (fully acceptor occupied) with Nluc-HT-PEG-biotin (unoccupied donor) in defined ratios at a constant donor concentration. Fractional occupancy (%) indicates the amount of acceptor-occupied Nluc-HT relative to the total amount of Nluc-HT in the sample. Shown are the means ± SD (n = 7) of a representative experiment. The results were plotted as mBRET units.

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    Figure 2

    Figure 2. Schematic of NanoBRET for detecting protein–protein interactions.

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    Figure 3

    Figure 3. Comparative analysis of BRET1 and NanoBRET using the Frb/FKBP model system. (a) Optimization of donor and acceptor orientation for the Frb/FKBP interaction. BRET ratios were determined on HEK293 cells transiently transfected with the indicated Frb and FKBP fusion proteins at a donor/acceptor ratio of 1:4, each measured using untreated and rapamycin treated (1 μM, 15 min) samples. The optimal configuration for either BRET1 or NanoBRET is indicated by the hatch pattern. (b) HEK293 cells were transiently transfected with either Frb-Nluc/FKBP-HT or Frb-RLuc8/FKBP-turboYFP at a donor/acceptor ratio of 1:4. The cells were plated in a 384-well plate and left untreated (n = 3) or treated with the indicated concentration of rapamycin (n = 3) for 15 min before determining the BRET ratio.

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    Figure 4

    Figure 4. Effect of expression level on assay sensitivity for detecting the interaction of FKBP with Frb. HeLa cells were transfected with a serial dilution of expression constructs for (a) Frb-Nluc and FKBP-HT or (b) Frb-Rluc8 and FKBP-TurboYFP (1:1 ratio) and plated in thre 96-well plates. BRET ratios were determined for untreated or rapamycin treated samples (1 μM rapamycin, 15 min). Shown is a representative experiment with all individual data points (n = 12) as well as mean ± SD plotted against DNA dilution used for transfection.

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    Figure 5

    Figure 5. NanoBRET assays for bromodomain protein interactions with chromatin. NanoBRET assays were developed to measure the binding of Nluc-BRD4 (a) or Nluc-CBP (b) to Histone H3.3-HT. HEK293 cells were transfected with the indicated combination of constructs. The BRET ratio for each sample was determined following treatment for 18 h with the indicated concentrations of I-BET151. Shown are the means ± SEM of three independent experiments performed in quadruplicate.

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    Figure 6

    Figure 6. Real time imaging of ligand induced interaction of AVPR2-HT with Nluc-β-arrestin2 interaction. HeLa cells were transfected with expression constructs for AVPR2-HT and Nluc-β-arrestin2. Sequential acquisition of images was initiated under unstimulated conditions. After establishing a baseline for seven acquisition cycles, 1 μM arginine vasopressin was added to the sample by injection followed by acquisition cycles (13.4 s per cycle) covering approximately 20 min. (a) The panel shows pseudocolored images of the donor (cyan) and acceptor (yellow) channel at indicated time points following treatment with AVP. The images are representative of the results obtained in five independent experiments (scale bar = 10 μm). (b) Kinetic of Nluc-β-arrestin2 recruitment following stimulation with AVP measured by imaging or in plate reader. The imaging data represent five individual regions of interest (ROI), each representing a single cell. The data shown for the plate based assay format are the results of a representative experiment performed in triplicate. In order to compare results obtained from different analytical platforms, the data were plotted as relative change in BRET normalized to the maximum BRET ratio obtained for each data set.

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  • References

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      Merzlyak, Ekaterina M.; Goedhart, Joachim; Shcherbo, Dmitry; Bulina, Mariya E.; Shcheglov, Aleksandr S.; Fradkov, Arkady F.; Gaintzeva, Anna; Lukyanov, Konstantin A.; Lukyanov, Sergey; Gadella, Theodorus W. J.; Chudakov, Dmitriy M.
      Nature Methods (2007), 4 (7), 555-557CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Fluorescent proteins have become extremely popular tools for in vivo imaging and esp. for the study of localization, motility and interaction of proteins in living cells. Here the authors report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.
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    14. 14
      Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., and Miyawaki, A. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications Nat. Biotechnol. 20, 87– 90
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      14
      A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications
      Nagai, Takeharu; Ibata, Keiji; Park, Eun Sun; Kubota, Mie; Mikoshiba, Katsuhiko; Miyawaki, Atsushi
      Nature Biotechnology (2002), 20 (1), 87-90CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)
      The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biol. systems. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs. 1,2). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37° and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-). For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired. Here we describe the development of an improved version of YFP named "Venus". Venus contains a novel mutation, F46L, which at 37° greatly accelerates oxidn. of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl-. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labeling that were not possible before.
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    15. 15
      Shaner, N. C., Steinbach, P. A., and Tsien, R. Y. (2005) A guide to choosing fluorescent proteins Nat. Methods 2, 905– 909
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      15
      A guide to choosing fluorescent proteins
      Shaner, Nathan C.; Steinbach, Paul A.; Tsien, Roger Y.
      Nature Methods (2005), 2 (12), 905-909CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      The recent explosion in the diversity of available fluorescent proteins (FPs) promises a wide variety of new tools for biol. imaging. With no unified std. for assessing these tools, however, a researcher is faced with difficult questions. Which FPs are best for general use. Which are the brightest. What addnl. factors det. which are best for a given expt.. Although in many cases, a trial-and-error approach may still be necessary in detg. the answers to these questions, a unified characterization of the best available FPs provides a useful guide in narrowing down the options.
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    16. 16
      Galarneau, A., Primeau, M., Trudeau, L. E., and Michnick, S. W. (2002) Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions Nat. Biotechnol. 20, 619– 622
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      16
      β-Lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein-protein interactions
      Galarneau, Andre; Primeau, Martin; Trudeau, Louis-Eric; Michnick, Stephen W.
      Nature Biotechnology (2002), 20 (6), 619-622CODEN: NABIF9; ISSN:1087-0156. (Nature America Inc.)
      We have previously described a strategy for detecting protein-protein interactions based on protein interaction-assisted folding of rationally designed fragments of enzymes. We call this strategy the protein fragment complementation assay (PCA). Here we describe PCAs based on the enzyme TEM-1 β-lactamase (EC: 3.5.2.6), which include simple colorimetric in vitro assays using the cephalosporin nitrocefin and assays in intact cells using the fluorescent substrate CCF2/AM (ref. 6). Constitutive protein-protein interactions of the GCN4 leucine zippers and of apoptotic proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an in vitro assay using cell lysates. With the same in vitro assay, we also demonstrate interactions of protein kinase PKB with substrate Bad. The in vitro assay is facile and amenable to high-throughput modes of screening with signal-to-background ratios in the range of 10:1 to 250:1, which is superior to other PCAs developed to date. Furthermore, we show that the in vitro assay can be used for quant. anal. of a small mol.-induced protein interaction, the rapamycin-induced interaction of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of rapamycin)). The assay reproduces the known dissocn. const. and no. of sites for this interaction. The combination of in vitro colorimetric and in vivo fluorescence assays of β-lactamase in mammalian cells suggests a wide variety of sensitive and high-throughput large-scale applications, including in vitro protein array anal. of protein-protein or enzyme-protein interactions and in vivo applications such as clonal selection for cells expressing interacting protein partners.
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    17. 17
      Zhang, J. H., Chung, T. D., and Oldenburg, K. R. (1999) A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays J. Biomol. Screening 4, 67– 73
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      17
      A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays
      Zhang; Chung; Oldenburg
      Journal of biomolecular screening (1999), 4 (2), 67-73 ISSN:.
      The ability to identify active compounds (3hits2) from large chemical libraries accurately and rapidly has been the ultimate goal in developing high-throughput screening (HTS) assays. The ability to identify hits from a particular HTS assay depends largely on the suitability or quality of the assay used in the screening. The criteria or parameters for evaluating the 3suitability2 of an HTS assay for hit identification are not well defined and hence it still remains difficult to compare the quality of assays directly. In this report, a screening window coefficient, called 3Z-factor,2 is defined. This coefficient is reflective of both the assay signal dynamic range and the data variation associated with the signal measurements, and therefore is suitable for assay quality assessment. The Z-factor is a dimensionless, simple statistical characteristic for each HTS assay. The Z-factor provides a useful tool for comparison and evaluation of the quality of assays, and can be utilized in assay optimization and validation.
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    18. 18
      Wells, J. A. and McClendon, C. L. (2007) Reaching for high-hanging fruit in drug discovery at protein-protein interfaces Nature 450, 1001– 1009
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      18
      Reaching for high-hanging fruit in drug discovery at protein-protein interfaces
      Wells, James A.; McClendon, Christopher L.
      Nature (London, United Kingdom) (2007), 450 (7172), 1001-1009CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      A review. Targeting the interfaces between proteins has huge therapeutic potential, but discovering small-mol. drugs that disrupt protein-protein interactions is an enormous challenge. Several recent success stories, however, indicate that protein-protein interfaces might be more tractable than has been thought. These studies discovered small mols. that bind with drug-like potencies to 'hotspots' on the contact surfaces involved in protein-protein interactions. Remarkably, these small mols. bind deeper within the contact surface of the target protein, and bind with much higher efficiencies, than do the contact atoms of the natural protein partner. Some of these small mols. are now making their way through clin. trials, so this high-hanging fruit might not be far out of reach.
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    19. 19
      Sanchez, R., Meslamani, J., and Zhou, M. M. (2014) The bromodomain: from epigenome reader to druggable target Biochim. Biophys. Acta 1839, 676– 685
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      19
      The bromodomain: From epigenome reader to druggable target
      Sanchez, Roberto; Meslamani, Jamel; Zhou, Ming-Ming
      Biochimica et Biophysica Acta, Gene Regulatory Mechanisms (2014), 1839 (8), 676-685CODEN: BBAGC6; ISSN:1874-9399. (Elsevier B.V.)
      A review. Lysine acetylation is a fundamental post-translational modification that plays an important role in the control of gene transcription in chromatin in an ordered fashion. The bromodomain, the conserved structural module present in transcription-assocd. proteins, functions exclusively to recognize acetyl-lysine on histones and non-histone proteins. The structural analyses of bromodomains' recognition of lysine-acetylated peptides derived from histones and cellular proteins provide detailed insights into the differences and unifying features of biol. ligand binding selectivity by the bromodomains. Newly developed small-mol. inhibitors targeting bromodomain proteins further highlight the functional importance of bromodomain/acetyl-lysine binding as a key mechanism in orchestrating mol. interactions and regulation in chromatin biol. and gene transcription. These new studies argue that modulating bromodomain/acetyl-lysine interactions with small-mol. chems. offer new opportunities to control gene expression in a wide array of human diseases including cancer and inflammation. This article is part of a Special Issue entitled: Mol. mechanisms of histone modification function.
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    20. 20
      Dawson, M. A., Kouzarides, T., and Huntly, B. J. (2012) Targeting epigenetic readers in cancer N. Engl. J. Med. 367, 647– 657
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      20
      Targeting epigenetic readers in cancer
      Dawson, Mark A.; Kouzarides, Tony; Huntly, Brian J. P.
      New England Journal of Medicine (2012), 367 (7), 647-657CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
      A review on assocn. between epigenetics and chromatin biol., BET bromodomain inhibitors, and role of BET inhibitors in cancer.
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    21. 21
      Filippakopoulos, P., Qi, J., Picaud, S., Shen, Y., Smith, W. B., Fedorov, O., Morse, E. M., Keates, T., Hickman, T. T., Felletar, I., Philpott, M., Munro, S., McKeown, M. R., Wang, Y., Christie, A. L., West, N., Cameron, M. J., Schwartz, B., Heightman, T. D., La Thangue, N., French, C. A., Wiest, O., Kung, A. L., Knapp, S., and Bradner, J. E. (2010) Selective inhibition of BET bromodomains Nature 468, 1067– 1073
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      21
      Selective inhibition of BET bromodomains
      Filippakopoulos, Panagis; Qi, Jun; Picaud, Sarah; Shen, Yao; Smith, William B.; Fedorov, Oleg; Morse, Elizabeth M.; Keates, Tracey; Hickman, Tyler T.; Felletar, Ildiko; Philpott, Martin; Munro, Shongah; McKeown, Michael R.; Wang, Yuchuan; Christie, Amanda L.; West, Nathan; Cameron, Michael J.; Schwartz, Brian; Heightman, Tom D.; La Thangue, Nicholas; French, Christopher; Wiest, Olaf; Kung, Andrew L.; Knapp, Stefan; Bradner, James E.
      Nature (London, United Kingdom) (2010), 468 (7327), 1067-1073CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here the authors report a cell-permeable small mol. (I,JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is obsd. in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chem. scaffold for the development of chem. probes more broadly throughout the bromodomain family.
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    22. 22
      Dawson, M. A., Prinjha, R. K., Dittmann, A., Giotopoulos, G., Bantscheff, M., Chan, W. I., Robson, S. C., Chung, C. W., Hopf, C., Savitski, M. M., Huthmacher, C., Gudgin, E., Lugo, D., Beinke, S., Chapman, T. D., Roberts, E. J., Soden, P. E., Auger, K. R., Mirguet, O., Doehner, K., Delwel, R., Burnett, A. K., Jeffrey, P., Drewes, G., Lee, K., Huntly, B. J., and Kouzarides, T. (2011) Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia Nature 478, 529– 533
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      22
      Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia
      Dawson, Mark A.; Prinjha, Rab K.; Dittman, Antje; Giotopoulos, George; Bantscheff, Marcus; Chan, Wai-In; Robson, Samuel C.; Chung, Chun-wa; Hopf, Carsten; Savitski, Mikhail M.; Huthmacher, Carola; Gudgin, Emma; Lugo, Dave; Beinke, Soren; Chapman, Trevor D.; Roberts, Emma J.; Soden, Peter E.; Auger, Kurt R.; Mirguet, Olivier; Doehner, Konstanze; Delwel, Ruud; Burnett, Alan K.; Jeffrey, Phillip; Drewes, Gerard; Lee, Kevin; Huntly, Brian J. P.; Kouzarides, Tony
      Nature (London, United Kingdom) (2011), 478 (7370), 529-533CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Recurrent chromosomal translocations involving the mixed lineage leukemia (MLL) gene initiate aggressive forms of leukemia, which are often refractory to conventional therapies. Many MLL-fusion partners are members of the super elongation complex (SEC), a crit. regulator of transcriptional elongation, suggesting that aberrant control of this process has an important role in leukemia induction. Here we use a global proteomic strategy to demonstrate that MLL fusions, as part of SEC and the polymerase-assocd. factor complex (PAFc), are assocd. with the BET family of acetyl-lysine recognizing, chromatin adaptor' proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukemia, via the displacement of the BET family of proteins from chromatin. We show that a novel small mol. inhibitor of the BET family, GSK1210151A (I-BET151), has profound efficacy against human and murine MLL-fusion leukemic cell lines, through the induction of early cell cycle arrest and apoptosis. I-BET151 treatment in two human leukemia cell lines with different MLL fusions alters the expression of a common set of genes whose function may account for these phenotypic changes. The mode of action of I-BET151 is, at least in part, due to the inhibition of transcription at key genes (BCL2, C-MYC and CDK6) through the displacement of BRD3/4, PAFc and SEC components from chromatin. In vivo studies indicate that I-BET151 has significant therapeutic value, providing survival benefit in two distinct mouse models of murine MLL-AF9 and human MLL-AF4 leukemia. Finally, the efficacy of I-BET151 against human leukemia stem cells is demonstrated, providing further evidence of its potent therapeutic potential. These findings establish the displacement of BET proteins from chromatin as a promising epigenetic therapy for these aggressive leukemias.
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    23. 23
      Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., and Caron, M. G. (1999) Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization J. Biol. Chem. 274, 32248– 32257
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      23
      Association of β-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization
      Oakley, Robert H.; Laporte, Stephane A.; Holt, Jason A.; Barak, Larry S.; Caron, Marc G.
      Journal of Biological Chemistry (1999), 274 (45), 32248-32257CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Resensitization of G protein-coupled receptors (GPCRs) following agonist-mediated desensitization is a necessary step for maintaining physiol. responsiveness. However, the mol. mechanisms governing the nature of GPCR resensitization are poorly understood. Here, we examine the role of β-arrestin in the resensitization of the β2 adrenergic receptor (β2AR), known to recycle and resensitize rapidly, and the vasopressin V2 receptor (V2R), known to recycle and resensitize slowly. Upon agonist activation, both receptors recruit β-arrestin to the plasma membrane and internalize in a β-arrestin- and clathrin-dependent manner. However, whereas β-arrestin dissocs. from the β2AR at the plasma membrane, it internalizes with the V2R into endosomes. The differential trafficking of β-arrestin and the ability of these two receptors to dephosphorylate, recycle, and resensitize is completely reversed when the carboxyl-terminal tails of these two receptors are switched. Moreover, the ability of β-arrestin to remain assocd. with desensitized GPCRs during clathrin-mediated endocytosis is mediated by a specific cluster of phosphorylated serine residues in the receptor carboxyl-terminal tail. These results demonstrate that the interaction of β-arrestin with a specific motif in the GPCR carboxyl-terminal tail dictates the rate of receptor dephosphorylation, recycling, and resensitization, and thus provide direct evidence for a novel mechanism by which β-arrestins regulate the reestablishment of GPCR responsiveness.
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    24. 24
      Laporte, S. A., Oakley, R. H., Zhang, J., Holt, J. A., Ferguson, S. S., Caron, M. G., and Barak, L. S. (1999) The beta2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis Proc. Natl. Acad. Sci. U.S.A. 96, 3712– 3717
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      There is no corresponding record for this reference.
    25. 25
      Mahen, R., Koch, B., Wachsmuth, M., Politi, A. Z., Perez-Gonzalez, A., Mergenthaler, J., Cai, Y., and Ellenberg, J. (2014) Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells Mol. Biol. Cell 25, 3610
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      25
      Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells
      Mahen, Robert; Koch, Birgit; Wachsmuth, Malte; Politi, Antonio Z.; Perez-Gonzalez, Alexis; Mergenthaler, Julia; Cai, Yin; Ellenberg, Jan
      Molecular Biology of the Cell (2014), 25 (22), 3610-3618, 9 pp.CODEN: MBCEEV; ISSN:1939-4586. (American Society for Cell Biology)
      Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quant. live-cell imaging and single-mol. spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quant. imaging studies in mammalian cells.
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