Anal. Chem. 2010, 82, 432–435
Technical Notes Experimental Determination of the Fo¨rster Distance for Two Commonly Used Bioluminescent Resonance Energy Transfer Pairs H. Dacres,* J. Wang, M. M. Dumancic, and S. C. Trowell CSIRO Food Futures Flagship & Division of Entomology, GPO Box 1700, Canberra, Australia, ACT 2601 Fo ¨rster resonance energy transfer (RET) is the nonradiative transfer of energy from a donor to an acceptor fluorophore. The Fo ¨rster distance (R0), being the fluorophore separation corresponding to 50% of the maximum RET efficiency (ERET), is a critical parameter for optimization of RET biosensors. Sensitive RET-based monitoring of molecular rearrangements requires that the separation of the donor and acceptor RET pair is matched to their Fo ¨rster distance. Here, for the first time, we experimentally determine the Fo ¨rster distance for BRET1, R0 ) 4.4 nm, and for BRET2, R0 ) 7.5 nm. The latter is the largest reported value for a genetically encoded RET pair. Fo¨rster resonance energy transfer1 (RET) is non-radiative transmission of energy from a donor to an adjacent acceptor fluorophore. Use of protein fluorophores enables sensitive RETbased measurement of inter- and intra-polypeptide rearrangements.2 A critical parameter for all forms of RET is the Fo¨rster distance (R0), the interfluorophore distance generating 50% of the maximum possible energy transfer. Because the efficiency of energy transfer declines as the sixth power of the donor-acceptor distance, R0 typically falls in the nanometer range. RET sensitivity is optimal when the experimental separation of a RET pair is well matched to its Fo ¨rster distance. For example, Gonzalez and Tsien tailored the spectral characteristics of a new fluorescence resonance energy transfer (FRET) donor so as to match its R0 to the movement of the acceptor upon plasma membrane depolarization, thereby optimizing sensitivity.3 To achieve this outcome for a range of conditions, researchers need to be able to choose from a repertoire of different RET pairs with known R0. Recent advances in GFP technology have increased the number of FRET pairs suitable for in vivo studies.4,5 FRET pairs * Corresponding author. E-mail:
[email protected]. Phone: 0011 61 (0)2 62464398. (1) Fo ¨rster, T. Discuss. Faraday Soc. 1959, 27, 7–17. (2) Miyawaki, A.; Tsien, R. Y. Methods Enzymol. 2000, 327, 472–500. (3) Gonzalez, J. E.; Tsien, R. Y. Chem. Biol. 1997, 4, 269–277. (4) Piston, D. W.; Kremers, G. J. Trends Biochem. Sci. 2007, 32, 407–414. (5) Rizzo, M. A.; Springer, G.; Segawa, K.; Zipfel, W. R.; Piston, D. W. Microsc. Microanal. 2006, 12, 238–254.
432
Analytical Chemistry, Vol. 82, No. 1, January 1, 2010
generally exhibit “cross-talk”, i.e., direct optical excitation of the acceptor, and “bleed-through” caused by the tail of the donor’s light emission overlapping the acceptor’s emission spectrum.6 Bioluminesence resonance energy transfer (BRET), in which the donor fluorophore is replaced by a luciferase and the system is energized by oxidation of a substrate, avoids “cross-talk” and enhances sensitivity. We recently demonstrated that two distinct BRET systems (Table S-1 in the Supporting Information) each give better limits of detection and sensitivity in a protease assay than the most commonly used FRET system.7,8 However, BRET and FRET have not been directly compared in terms of their Fo¨rster distances. R0 has been calculated for a variety of fluorescent protein derived FRET pairs with donor and acceptor emissions ranging from blue to red. Calculated R0 values ranged from 3.17 nm (BFP-DsRED)9 to 5.84 nm by combining a monomeric GFP variant (mEGFP) with a monomeric YFP variant (phiYFPm).5 Experimental validation of R0 is important because of the sensitivity of the calculations to minor errors in a number of experimentally determined parameters. R0 for FRET was recently tested experimentally by inserting flexible peptide linker sequences of varying lengths between CFP and YFP10 with observations closely matching expectations for R0 ) 4.8 nm.9,10 In contrast, R0 has never been determined for any BRET pair, potentially limiting the utility of BRET. To remedy this gap, we took a similar experimental approach to that used by Evers et al.10 EXPERIMENTAL SECTION Assembly of RET Constructs. The RET fusion pairs were amplified and restriction cloned into the EcoRI and BsrGI sites of the pRSET vector. A series of sequences encoding 1-9 units of the peptide (Gly)2Ser(Gly)2Ser were cloned between the FRET (6) Takanishi, C. L.; Bykova, E. A.; Cheng, W.; Zheng, J. Brain Res. 2006, 1091, 132–139. (7) Dacres, H.; Dumancic, M. M.; Horne, I.; Trowell, S. C. Anal. Biochem. 2009, 385, 194–202. (8) Dacres, H.; Dumancic, M. M.; Horne, I.; Trowell, S. C. Biosens. Bioelectron. 2009, 24, 1164–1170. (9) Patterson, G. H.; Piston, D. W.; Barisas, B. G. Anal. Biochem. 2000, 284, 438–440. (10) Evers, T. H.; van Dongen, E. M.; Faesen, A. C.; Meijer, E. W.; Merkx, M. Biochemistry 2006, 45, 13183–13192. 10.1021/ac9022956 2010 American Chemical Society Published on Web 12/03/2009
Table 1. RET Constructs Incorporating x (1-9) (Gly)2Ser(Gly)2Ser (FL) Repeats RET system
construct name
FRET
CFP-FLx-YFP YFP-FLx-CFP RLuc-FLx-YFP YFP-FLx-RLuc RLuc-FLx-GFP2 GFP2-FLx-RLuc
1
BRET
BRET2
and BRET pairs using a PstI site. The GFP2-BSA-RLuc sequence was synthesized by Genscript and restriction cloned into the EcoRI and XhoI sites of the pRSET vector. Standard molecular biology techniques were used, and every clone was sequenced to confirm its integrity and orientation. Expression and Purification of RET Proteins. Constructs were transformed into chemically competent BL21 (DE3) cells (Novagen) by heat-shock. Identity and integrity of inserts was confirmed by plasmid digestion and DNA sequencing. At least three independent colonies were selected for each construct and used to perform biological replicates. Cultures were grown up and lysed using a French press. The BRET and FRET constructs were affinity-purified over TALON Superflow Metal Affinity Resin (Clontech Laboratories, Inc.), and their purity was confirmed using SDS-polyacrylamide electrophoresis. Purified protein (1 µM) was used for all RET assays unless otherwise stated. Simultaneous Dual Emission Detection. Simultaneous dual emission RET measurements were carried out with a POLARstar OPTIMA microplate reader (BMG LabTech). BRET measurements used either the BRET2 emission filter set comprising RLuc/ClZ400A emission filter (410 nm bandpass, 80 nm) and the GFP2 emission filter (515 nm bandpass, 30 nm) or the BRET1 filter set consisting of a RLuc/CLZ emission filter (475 nm bandpass, 30 nm) and YFP (535 nm bandpass, 30 nm) emission filter. The FRET filter set consisted of a CFP excitation filter (450 nm bandpass, 10 nm) and the respective CFP (500 nm bandpass, 10 nm) and YFP (530 nm bandpass, 10 nm) emission filters. For full experimental details, see Supplementary Methods in the Supporting Information. RESULTS AND DISCUSSION Orientation Effect. Between one and nine (Gly)2Ser(Gly)2Ser flexible oligopeptide linker (FLx) repeats were interposed between the donor and acceptor of each of the three RET pairs. Nomenclature is given in Table 1. A His6 tag was incorporated at the N-termini of the fusion proteins to facilitate their affinity purification. Initial studies demonstrated that, for both BRET systems, orientations with the fluorescent acceptor at the N-terminus of the fusion protein generated a higher RET ratio (integrated acceptor emission intensity relative to the integrated donor intensity) than with the reverse orientation (Figure 1). This effect has been observed previously and attributed to enhancement when the N-terminus of Renilla GFP is unconstrained.7,11 In contrast, (11) Molinari, P.; Casella, I.; Costa, T. Biochem. J. 2008, 409, 251–261. (12) Vinkenborg, J. L.; Evers, T. H.; Reulen, S. W.; Meijer, E. W.; Merkx, M. ChemBioChem 2007, 8, 1119–1121. (13) Felber, L. M.; Cloutier, S. M.; Kundig, C.; Kishi, T.; Brossard, V.; Jichlinski, P.; Leisinger, H. J.; Deperthes, D. BioTechniques 2004, 36, 878–885.
Figure 1. Effect of RET pair orientation on the RET efficiency of 1 µM of RET fusion proteins incorporating either 1 or 9 flexible linker repeats between the donor and acceptor (mean ( SD, n ) 3).
Figure 2. RET ratios of 1 µM of the nominated RET pairs incorporating up to nine flexible linker repeats inserted between the donor and acceptor. All points represent calculated means ( SD. For BRET, n ) 3. For FRET, n ) 4.
it is standard practice to construct FRET probes with the CFP donor at the N-terminus of the fusion protein12-17 because this gives the higher RET ratio. Furthermore, both FRET and BRET fusions with optimal orientations, i.e., CFP-FLx-YFP, YFP-FLxRLuc, and GFP2-FLx-RLuc, were more sensitive than those with inverted orientations to increasing linker length (Figure S-1 in the Supporting Information). All further studies were carried out using only fusion proteins with the optimal orientations. These 27 FLx constructs were each expressed in E. coli and purified in a single step by Co2+-affinity chromatography (Figure S-2 in the Supporting Information). Ratiometric Measurements and RET Efficiencies. The RET ratios for all three series of purified fusion proteins decreased with increasing linker length (Figure 2). For determination of R0 values, RET ratios were converted into energy transfer efficiencies (ERET) (eq 1) and flexible linker numbers into distances (rRET) allowing application of the Fo ¨rster equation (eq 2). ERET ) 1 -
( ) IDA ID
(1)
(14) Zhang, B. Biochem. Biophys. Res. Commun. 2004, 323, 674–678. (15) Tyas, L.; Brophy, V. A.; Pope, A.; Rivett, A. J.; Tavare, J. M. EMBO Rep. 2000, 1, 266–270. (16) Mahajan, N. P.; Harrison-Shostak, D. C.; Michaux, J.; Herman, B. Chem Biol 1999, 6, 401–409. (17) Pollok, B. A.; Heim, R. Trends Cell. Biol. 1999, 9, 57–60.
Analytical Chemistry, Vol. 82, No. 1, January 1, 2010
433
rRET ) R0((1/ERET) - 1)1/6
(2)
where ΙDA is the ratio of integrated donor emission intensity to acceptor fluorescence intensity in the presence of RET and ΙD is the ratio of donor emission intensity to acceptor fluorescence intensity in the absence of RET18 (see Supplementary Methods in the Supporting Information). A ratiometric method (eq 1) was employed to calculate energy transfer efficiencies to compensate for the observed decrease in quantum yield of the BRET2 donor as the energy transfer efficiency decreases.7,8 In contrast, decreases in BRET1 and FRET energy transfer efficiencies are accompanied by increasing donor emission intensities.7,8 This difference would invalidate the use of traditional donor intensity parameters for directly comparing the energy transfer efficiencies of the three RET systems.19 FRET Calculations. A Fo¨rster distance of 4.8 nm, as calculated by Evers et al.,10 was applied to the Fo¨rster equation (eq 2) to translate calculated EFRET values into distances (rFRET) between the two FRET fluorophores. Fluorophore separations ranged from 3.97 to 4.62 nm (Table S-2 in the Supporting Information) as the number of flexible linker repeats was increased from one to nine. Evers et al.10 estimated the separations of the same fluorophores using the same series of linkers as 4.1-5 nm for FL1 to FL9, respectively. However, in Evers et al.’s experiment, an additional 17 amino acid residues were inserted between the fluorophores. This would increase the separation of the fluorophores and may also have relaxed the constraints on their relative orientation. The relatively small increments in fluorophore separation as the number of linkers is increased is attributed to the (Gly)2Ser(Gly)2Ser sequence adopting a compact random coil structure. Evers et al. have previously shown that the experimentally determined energy transfer efficiencies are consistent with distance estimates for such a random coil.10 Experimental Determination of BRET Fo ¨rster Distances. Distances between the BRET RLuc donor and fluorescent protein acceptor are expected to be larger than for the FRET fusion proteins as RLuc (36 kDa) is larger than GFP and its derivatives (27 kDa). The radii (Rd) of RLuc and GFP were calculated, assuming globularity and a specific volume of ∼0.74 cm3/kg, by substitution into the following equation:20 3
Rd ) 0.676√MW
(3)
The radius of RLuc was calculated to be 2.23 nm compared to 2.03 nm for CFP, YFP, and GFP2, and the difference was used to estimate rBRET values, from our rFRET values (Table S-2 in the Supporting Information), for both BRET systems (Table S-3 in the Supporting Information). (18) Adams, D. S. Lab Maths: A Handbook of Measurements, Calculations and Other Quantitative Skills for Use at the Bench; Cold Spring Laboratory Press: Cold Spring Harbor, NY, 2003. (19) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic/Plenum Publishers: New York, 1999. (20) Harpur, A. G.; Bastiaens, P. I. H. In Molecular Cloning; Sambrook, J., Russell, D. W., Eds.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2001; pp 18.69-18.95. (21) Ohashi, T.; Galiacy, S. D.; Briscoe, G.; Erickson, H. P. Protein Sci. 2007, 16, 1429–1438. (22) Levi, V.; Gonza´lez Flecha, F. L. Biochim. Biophys. Acta 2002, 1599, 141– 148. (23) Slayter, E. M. J. Mol. Biol. 1965, 14, 443–452.
434
Analytical Chemistry, Vol. 82, No. 1, January 1, 2010
Figure 3. RET efficiency (ERET) of FRET, BRET1, and BRET2 as a function of donor/acceptor separation (in nanometers). - - -, distances (rFRET (mean ( standard deviation (SD), n ) 4)) for the FRET system calculated using EFRET (mean ( SD, n ) 4) and a Fo¨rster distance of 4.8 nm. s, BRET1 (rBRET1) and BRET2 (rBRET2) distances (mean ( SD, n ) 4). BRET2 distances were plotted versus calculated EBRET values (mean ( SD, n ) 3). The calculated EBRET value (mean ( SD, n ) 5) for GFP2-BSA-RLuc was plotted against the distance of 9.81 nm.
Energy transfer efficiencies were calculated (eq 1) for BRET1 (EBRET1) and BRET2 (EBRET2) and fitted to the Fo ¨rster equation (eq 4) by nonlinear regression (Figure 3).
ERET )
R06 R06 + rRET6
(4)
The Fo¨rster distance was determined to be 4.40 ± 0.02 nm (mean ± SEM; n ) 3) for BRET1 and 7.50 ± 0.03 nm (mean ± SEM; n ) 3) for BRET2 (Figure 3). Both fits were highly correlated, with R2 values of 0.92 and 0.99, respectively, for the BRET1 and BRET2 curves. To validate the shape of the BRET2 curve independently, bovine serum albumin (BSA) was inserted between the BRET2 donor and acceptor (GFP2-BSA-RLuc). In order to avoid artifactually increasing the RET efficiency21 by dimerization of BSA spacers, we avoided using BSA in concentrations greater than one tenth of its equilibrium disassociation constant (∼10 µM).22 BSA is a 69 kDa globular protein with a calculated diameter of 5.55 nm (eq 3), consistent with previous determinations.23 The chromophore-to-chromophore distance for GFP2-BSA-RLuc was calculated (eq 3) to be 9.81 nm. Plotting the calculated ERET (Table S-2 in the Supporting Information) for this fusion protein against a chromophore separation of 9.81 nm produced an excellent fit (R2 ) 0.999) with the previously constructed BRET2 curve confirming that R0 ) 7.5 nm. Comparison of Different Energy Transfer Systems. Since the first application of the BRET1 system by Xu et al.,24 it has been assumed that the BRET1 Fo ¨rster distance would be similar to that of the CFP/YFP FRET system investigated here. This assumption was based on the similarities in the spectral overlap of RLuc emission and YFP absorption with CFP emission and YFP absorption. The integrated overlap of the BRET1 and FRET systems (Figure S-3 in the Supporting Information) illustrate that the two RET systems are very similar albeit the overlap area (24) Xu, Y.; Piston, D. W.; Johnson, C. H. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 151–156.
of the BRET1 peaks is slightly smaller. This is consistent with R0(BRET1) being similar to but smaller than R0(FRET). However, comparison of spectral overlap areas is not of itself definitive because Fo ¨rster distance also depends upon quantum yield of the donor and the relative dipole orientations. The overlap area of the BRET2 peaks as presented by Pfleger and Eidne25 appears to be much larger than for either BRET1 or FRET, which is consistent with our findings. For these systems, the degree of spectral overlap between the donor and acceptor appears to be the principal practical determinant of R0. The large difference between the determined R0(BRET2), compared to the BRET1 and FRET systems, is apparent from the RET curves (Figure 3). Previous studies describing the cloning of tandem repeats of the influenza Hemagglutinin epitope demonstrated that a BRET2 signal was still detectable from fusion proteins incorporating six copies of the epitope. According to Gehret et al.,26 this epitope tag has an N to C terminal distance of ∼2 nm so that linkers incorporating six epitope tags would have an interchromophore distance greater than 10 nm. This is consistent with the measured ratio (Table S-2 in the Supporting Information) for the GFP2-BSA-RLuc fusion protein presented here. The Fo¨rster distance for BRET2 is the largest reported to date for a genetically encodable RET pair. The longer working distance range of the BRET2 system lends itself to the study of larger proteins and multicomponent systems such as G(25) Pfleger, K. D. G.; Eidne, K. A. Nat. Methods 2006, 3, 165–174. (26) Gehret, A. U.; Bajaj, A.; Naider, F.; Dumont, M. E. J. Biol. Chem. 2006, 281, 20698–20714.
protein receptor complexes, ribosomes, and nucleo-protein complexes. Our findings also illustrate the possibility of tailoring novel BRET systems with R0 < 4.8 nm or > 7.5 nm or between 4.8-7.5 nm for optimum reporting of possible molecular movements over the widest range of distances. CONCLUSIONS This technical note presents the first determination of the Fo¨rster distance for any BRET system and compares the experimentally determined values to those of a standard FRET pair. Since the first use of BRET in 1999, over 350 studies have been reported without knowledge of the Fo¨rster distance or whether the best RET system was being used for the particular application. The data reported here will enable better-informed decisions on choice of BRET systems. Comparison with a classical FRET system demonstrates that BRET2 has a larger working distance range with a calculated Fo ¨rster distance of 7.5 nm compared to 4.4 and 4.8 nm for BRET and FRET, respectively. This information also permits rational substitution of BRET1 for FRET in a range of research and diagnostic applications. The longer working distance range of the BRET2 system lends itself to the study of larger macromolecular assemblies. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review May 11, 2009. Accepted November 9, 2009. AC9022956
Analytical Chemistry, Vol. 82, No. 1, January 1, 2010
435
