Simultaneous linear unmixing of excitation-emission spectra (ExEm-unmixing)-based fluorescence resonance energy transfer (FRET) two-hybrid assay method, named as ExEm-FRET two-hybrid assay, was developed for evaluating the stoichiometric ratio of macromolecular complexes in living cells. Linear unmixing of the excitation-emission spectra (SDA) of cells obtains the weight factors of donor (WD), acceptor (WA) and acceptor sensitization (WS), yielding ED and EA (donor- and acceptor-centric FRET efficiency) images. ExEm-FRET two-hybrid assay employs pixel-to-pixel titration curves of ED/EA versus the free acceptor (Ca)/donor (Cd) concentration deduced from the three weight factors to obtain EA,max and ED,max (the maximal EA and ED), thus yielding the stoichiometric ratio (EA,max/ED,max) of donor-tagged protein to acceptor-tagged protein.
© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
Fluorescence resonance energy transfer (FRET) two-hybrid assay is an invaluable method for resolving the stoichiometric information of macromolecular complexes within living cells [1–3]. In FRET two-hybrid assay, a single cell is considered to be a “biological cuvette” for titration experiments between molecules. Both ED and EA (donor- and acceptor-centric FRET efficiency) of the pairs of donor fluorophore-tagged and acceptor fluorophore-tagged molecules are systematically measured from a lot of single cells with varying concentrations of free donors (Dfree) and acceptors (Afree) to obtain the FRET binding curves of both ED-Afree and EA-Dfree, yielding EA,max and ED,max (the maximal EA and ED, assuming each acceptor/donor molecule binds to donor/acceptor) as well as the stoichiometric ratio (EA,max/ED,max) of the fluorophore-tagged molecules .
FRET two-hybrid assay is currently carried out by implementing two existing fluorescence intensity-based FRET approaches: 33-FRET and E-FRET [1,2]. Based on the measured ED and EA images by using E-FRET and 33-FRET methods, we recently obtained EA,max and ED,max by using ED, EA and the ratio of donor to acceptor concentration across pixel-to-pixel to determine the stoichiometric ratio of intracellular complexes . Although E-FRET method is a popular approach for quantitative biochemistry of dynamic signal transduction in living cells [1–4], the requirement that all measurements must be implemented under the same conditions partly impedes the application of live-cell quantitative FRET measurements.
Simultaneous unmixing of excitation-emission spectra (ExEm-unmixing) has become a potential technology to resolve FRET signal (ExEm-FRET) due to the inherent ability of overcoming spectral crosstalk (a key issue of quantitative FRET measurement) [5–11]. Recently, a spectral wide-field microscope was developed for microscopic live-cell spectral imaging, and we implemented ExEm-FRET measurement on the microscope in single living cells [6,7,10,11]. The instrument settings for ExEm-FRET measurement can be adjusted for measuring cells under optimal imaging conditions, thus ExEm-FRET measurement greatly reduces the failure rate of quantitative FRET measurements in living cells [10,11]. Moreover, ExEm-FRET measurement has superior stability and robustness and can obtain relatively accurate results even for cells with low signal-to-noise ratio [6,11].
This report aims to develop an ExEm-unmixing-based FRET two-hybrid assay method, named as ExEm-FRET two-hybrid assay. Linear unmixing of the excitation-emission spectra (SDA) of cells obtains the weight factors of donor (WD), acceptor (WA) and donor-acceptor sensitization (WS), yielding ED and EA images. ExEm-FRET two-hybrid assay employs pixel-to-pixel titration curves (the titration curves are obtained by analyzing each pixel of images) of ED/EA versus the free acceptor (Ca)/donor(Cd) concentration deduced from the three weight factors for obtaining EA,max and ED,max, thus yielding the stoichiometric ratio (EA,max/ED,max) of donor-tagged protein binding to acceptor-tagged protein. ExEm-FRET two-hybrid assay requires only a few cells, which greatly reduces the workload of quantitative FRET measurements. Furthermore, due to the superior robustness and sensitivity of ExEm-unmixing, ExEm-FRET two-hybrid assay is especially applicable for cells with low fluorescence signal, thus provides a powerful tool for exploring the molecular mechanism of intracellular signal transduction in living cells. We performed the ExEm-FRET two-hybrid assay on a self-developed automated FRET microscope for evaluating the binding of Bcl-XL to Bad. ExEm-FRET two-hybrid assay for living Hela cells co-expressing Bad-YFP and CFP-Bcl-XL showed 1.345 (in healthy cells) and 1.716 (in apoptotic cells) of the stoichiometric ratio of Bcl-XL binding to Bad, indicating coexistence of both Bad-Bcl-XL dimer and Bad-Bcl-XL2 trimer in living cells.
2.1 ExEm-FRET two-hybrid assay
The net excitation-emission spectrum (SDA) of a FRET sample can be linearly resolved into three excitation-emission components as follow [7,10],7,10], and QX is quantum yield of X; SD, SA and SS are the unit-area-normalized excitation-emission spectral signatures of donor, acceptor and donor-acceptor sensitization, respectively; WD, WA and WS are the weights of SD, SA and SS, respectively . According to Eq. (1) and the definitions of WD, WA and WS, we also obtain10],1,2]1,2,12]Eqs. (5) and (6), we obtain following equation,Eq. (6), Ca is determined as follow,1,2]
2.2 Determination of collisional FRET
As the spurious EA and ED due to the collision between donor and acceptor molecules are linearly proportional to the concentration of donor and acceptor, respectively , the magnitude of spurious FRET efficiency is related to CtD and CtA by the following,
2.3 Determination of FRET binding curves
The experimentally measured EA and ED must relate to the Cd and Ca, respectively:1], where the condition for finding the optimal solution is that the difference between EA/ED and EA,pred/ED/pred (Eqs. (3) and 12) is the smallest, and the FRET binding curves of both ED-Ca and EA-Cd are obtained as shown in Fig. 1 .
2.4 Five steps of implementing ExEm-FRET two-hybrid assay
Step 1: Predetermination of excitation–emission spectral signatures.
Excitation–emission spectral signatures (SD, SA and SS) are determined by using living cells separately expressing donor and acceptor fluorophores, respectively [7,10]. The excitation spectrum of donor/acceptor is obtained by fixing one emission channel and switching different excitation wavelengths. Fixing one excitation wavelength and collecting the fluorescence of different emission channels, the emission spectrum of donor/acceptor is obtained. SD/SA is the outer product of the excitation spectrum and the emission spectrum measured from donor-only/acceptor-only sample. SS is obtained by the outer product of donor excitation spectrum and acceptor emission spectrum.
Step 2: Determination of the system calibration factors (QA/QD and KA/KD).
Both QA/QD and KA/KD values are simultaneously measured by linearly unmixing the excitation–emission spectra of more than one FRET pairs (reference samples) just as described previously . SDA of the reference samples are linearly unmixed into WD, WA and WS according to Eq. (1). Plot of WS/WD versus WA/WD for the reference samples indicates a linear relationship with the absolute intercept (QA/QD) and the reciprocal of slope (KA/KD).
Step 3: FRET efficiency calculation of FRET sample.
After obtaining the three contributions (weights: WD, WS and WA) from the SDA of FRET sample, both EA and ED values are calculated according to Eq. (3). The EA and ED values still need to be further corrected (as shown in Step 4 below).
Step 4: Correction of spurious FRET.
When fluorophores exist at high concentration, the distance between a donor and an acceptor may be less than the Förster distance by random chance, and the donor and acceptor molecules would undergo FRET even there is no real potential binding interaction . Therefore, to obtain reliable live-cell titration curves of ED/EA versus Ca/Cd, the contribution of spurious FRET due to the collision between donor and acceptor molecules must be subtracted (Eq. (11). As shown in Eq. (2), the measured CtA and CtD are related to 1/KAQA and 1/KDQD, respectively. KAQA value can be assumed to be 1, and the 1/(KDQD) value can be determined according to the values of measured KA/KD and QA/QD.
Step 5: Determination of FRET binding curves.
Pixel-to-pixel titration curves of ED/EA versus Ca/Cd deduced from the three weight factors (WD, WS, and WA) are employed for obtaining EA,max and ED,max (Fig. 1), thus yielding the stoichiometric ratio (EA,max/ED,max) of donor-tagged protein binding to acceptor-tagged protein.
3. Materials and methods
3.1 ExEm-FRET experiments
ExEm-FRET measurements were performed on a self-developed automated FRET microscope. Figure 2(a) depicts the diagram of the automated FRET microscope. A wide-field microscope (IX73, Olympus, Hamburg, Japan) with an objective (60 × /1.42 NA oil, Olympus), a mercury lamp (HGLGPS, Olympus) and a CMOS camera (Flash 4.0, Hamamatsu, Japan) was refitted by utilizing a motorized cube wheel (IX3-RFACA, Olympus) with transition time as few as 500 ms and a motorized emission filter wheel (FW103, Throlabs, Newton, NJ, USA) with transition time as few as 55 ms. Excitation of two wavelengths was changed by switching two band-pass excitation filters (Chroma, USA): 435/20 nm (Ex 1) and 470/20 nm (Ex 2). Five band-pass emission filters (Chroma, USA) were placed in a motorized emission filter wheel for emission spectra measurement: 470/20 nm (Em 1), 490/20 nm (Em 2), 510/20 nm (Em 3), 530/20 nm (Em 4) and 550/20 nm (Em 5). In this report, the exposure time of each image was 500 ms, and a complete ExEm-FRET measurement took about 5.5 s.
Image-stack (SDA) was used to denote the excitation-emission spectral images of FRET sample [7,10]. An SDA contains eight images, including five emission spectral images with 435 nm excitation and three emission spectral images with 470 nm excitation, respectively (Fig. 2b).
3.2 Cell culture, transfection and plasmids
Hela cells, cell culture, transfection, plasmids (CFP (C), YFP (Y), C80Y, C40Y, C10Y and C4Y) and Staurosporine (STS) have been described previously in our publications [13,14]. CFP-Bcl-XL was kindly supplied by Dr. Gilmore . To generate a plasmid encoding YFP fused to Bad, the coding region of Bad was prepared by PCR (the Bad cDNA from mCherry-Bad), and was ligated into pcDNA3-YFP. Plasmid of mCherry-Bad was kindly provided by Dr. Andrews .
Hela cells were washed three times in phosphate buffer solution (PBS) after 24 h transfection and Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum was added before ExEm-FRET imaging in healthy cells. For the samples needed for ExEm-FRET imaging in apoptotic cells, 1 μM STS was added for 7 h after 24 h transfection. Finally, the cells were washed three times in PBS and DMEM containing 10% fetal calf serum was added before ExEm-FRET imaging in apoptotic cells.
4. Result and discussion
4.1 Predetermination of excitation–emission spectral signatures and system calibration factors
Firstly, living Hela cells separately expressing untagged CFP (C) and untagged YFP (Y) were used to predetermine excitation–emission spectral signatures (SD and SA as well as SS) [7,10]. Figure 3(a) shows the emission-spectral images of CFP and YFP with 435 nm and 470 nm excitation, respectively (Left). BG (the intensity of non-fluorescent area) was subtracted from the intensities of the pixels of cells. The measured fluorescence intensities were calibrated by using the emission-spectral responses of the imaging system, and then were normalized to unit area as the emission spectrum of CFP and YFP, respectively  (Fig. 3a, Right). Figure 3(b) shows the fluorescence images at 510 nm of cells expressing CFP and fluorescence images at 530 nm of cells expressing YFP with 435 nm excitation and 470 nm excitation, respectively (Top). After BG subtraction and normalization of measured fluorescence intensities, the average excitation spectra of the cells separately expressing CFP and YFP were obtained (Fig. 3b, Bottom). SD/SA was the outer product of the excitation spectrum and the emission spectrum measured from donor-only/acceptor-only sample, and SS was obtained by the outer product of donor excitation spectrum and acceptor emission spectrum (Fig. 3c) .
Four C-Y FRET pairs (C4Y, C10Y, C40Y and C80Y) were used to simultaneously measure KA/KD and QA/QD values as described previously [1,10]. In this report, KAQA value was assumed to be 1, and the measured KA/KD and QA/QD values were 0.237 and 1.667, respectively. We obtained 0.394 of 1/(KDQD) value. The excitation–emission spectral signatures (SD, SA and SS) and KA/KD and QA/QD values for ExEm-FRET imaging just need to be measured periodically due to the stability of our system [10,13].
In order to reduce the effect of pixel mismatch on measurement results, two ways were adopted for reducing the effect of pixel mismatch on our results. Firstly, the pixel mismatches among five detection channels were calibrated by imaging standard ruler sample and corrected in post-processing. Secondly, the cell edges with mismatch were filtered by using a binary mask just as described in our previous report . According to the emission spectra of donor (CFP) and acceptor (YFP), we chose 470/20 and 530/20 nm channels to fabricate a binary mask for filtering the mismatched pixels from FRET images as following steps: (1) Set 1.5*IBG (IBG, the intensity of background) as the thresholds of 470/20 and 530/20 nm images, respectively; (2) Generate binary masks of 470/20 and 530/20 nm images by assigning pixels whose gray intensity are less than 1.5*IBG with 0 and pixels whose gray intensity are not less than 1.5*IBG with 1; (3) Get the final binary mask through a logical AND of 470/20 and 530/20 nm binary masks.
4.2 Correction of spurious FRET
To obtain reliable titration curves of EA-Cd and ED-Ca, the contribution of spurious FRET due to collisions must be subtracted [1,17]. After BG subtraction and calibration by using the system responses, the SDA of cells co-expressing CFP and YFP (Fig. 4a) was linearly unmixed for obtaining WA, WS, and WD factors according to Eq. (1) (Fig. 4b, Top). According to Eqs. (2) and (3), we obtained the images of CtD, EA, CtA, and ED (Fig. 4b, Bottom) and the corresponding pixel-to-pixel EA- CtD plot (Fig. 4c) and ED- CtA (Fig. 4d). The spurious FRET efficiency due to collisions is linearly proportional to the concentration of fluorescent proteins with 3.4 × 10−6 of slope in the EA- CtD plot, which can also be obtained from the ED- CtA plot. In our experimental system, we found that the spurious FRET efficiency caused by the collision between untagged CFP and untagged YFP was very small, which can be ignored in our system. However, the spurious FRET efficiency may be prohibitively high when molecules are placed restrictions on membrane, which can be estimated and corrected by localizing donor and acceptor fluorescent proteins on the membrane.
4.3 Evaluation of the binding of Bad to Bcl-XL
We next used ExEm-FRET two-hybrid assay to evaluate the interaction of Bad, an apoptotic BH-3 only protein, with Bcl-XL, an anti-apoptotic protein, in healthy cells [16,18]. Figure 5(a) shows an SDA of living cells co-expressing Bad-YFP and CFP-Bcl-XL (Top). Linear unmixing of the SDA according to Eq. (1) obtained the corresponding WA, WS and WD images (Fig. 5a, Bottom). The CtD and CtA mages were obtained by using Eq. (2). The EA and ED images were obtained by using Eq. (3) and corrected by Eq. (11) (Fig. 5b). According to Eq. (8a), EA was plotted against estimated Cd (Fig. 5c, Left), where each black symbol corresponds to the data from a single pixel, and the EA,max value was about 0.364. Similarly, according to Eq. (8d), ED was plotted as a function of estimated Ca, and the ED,max value was about 0.274 (Fig. 5c, Right). 1.282 (0.364/0.274) of the stoichiometric ratio of Bcl-XL binding to Bad was obtained. We analyzed about 50 cells from 10 frames and obtained 1.345 (0.394/0.293) of the stoichiometric ratio of Bcl-XL binding to Bad, indicating that the complexes exist mainly in Bad-Bcl-XL dimer and there may be a small amount of Bad-Bcl-XL2 trimer in healthy cells.
ExEm-FRET two-hybrid assay was also used to evaluate the binding of Bad with Bcl-XL in staurosporine(STS)-induced apoptotic cells . Treatment with 1 μM STS for 7 h induced cell shrinkage (Fig. 6) and apoptosis (data not shown). Figure 6(a) shows an SDA of apoptotic cells co-expressing Bad-YFP and CFP-Bcl-XL (Top). The corresponding WA, WS and WD images were obtained from the SDA (Fig. 6a, Bottom), and the corresponding EA, CtD, CtA and ED images were shown in Fig. 6(b). The EA,max value was about 0.377 from the corresponding EA-Cd plot, and the ED,max value was about 0.202 from the corresponding ED-Ca plot. Statistical result from 50 cells in 10 frames was 1.716 (0.398/0.232) stoichiometric ratio of Bcl-XL binding to Bad, indicating that the complexes exist mainly in Bad-Bcl-XL2 trimers in apoptotic cells.
Interactions between pro-apoptotic and anti-apoptotic proteins play an important role in the regulation of apoptosis [20–23]. Bad has been demonstrated to not only abrogate the protective capacity of Bcl-XL but also accelerate the apoptotic response of cells to a death signal [20,23]. It was reported that Bad inhibits the survival functions of Bcl-XL by direct interaction with Bcl-XL, forming hetero-dimer [22,23].
However, our results that the stoichiometric ratio of Bcl-XL binding to Bad is always between 1 and 2 in both healthy and STS-induced apoptotic cells (Figs. 5 and 6) further suggest the existence of Bad-Bcl-XL2 trimer . In vitro studies have confirmed the existence of Bad-Bcl-XL dimer, but have not observed Bad-Bcl-XL2 trimer [21–23]. We speculate that the affinity of Bad-Bcl-XL2 trimer may be not as strong as that of Bad-Bcl-XL dimer, thus Bad-Bcl-XL2 trimer has not been detected by in vitro studies.
In order to exclude the possibility that dimer and trimer exist separately in different cells, we also analyzed one of the cells in Fig. 5 and obtained 1.240 (0.381/0.307) of the stoichiometric ratio of Bcl-XL binding to Bad (Fig. 7). The results shown in Fig. 7 further indicate the coexistence of both Bad-Bcl-XL dimer and Bad-Bcl-XL2 trimer in living cells. However, in practical experiments, it is difficult for one cell to satisfy the conditions that both Cd and Ca have a wide concentration range. Therefore, in reality, simultaneous analysis of a few cells was adopted for obtaining more desirable titration curves of ED-Ca and EA-Cd.
The 1.345 and 1.716 stoichiometric ratios of Bcl-XL-Bad complexes also indicate that Bad has two binding sites with Bcl-XL. Bcl-XL may bind preferentially to one of the sites and then bind to the other one when Bcl-XL concentration is relative higher than Bad concentration. Our studies and other reports have demonstrated that Bcl-XL has strong anti-apoptotic ability and can prevent STS-induced apoptosis [2,23]. After STS treatment for 7 h, only the cells with relative high expression of Bcl-XL may be used for analysis, which increased the proportion of Bad-Bcl-XL2 trimer. Therefore, the stoichiometric ratio of Bcl-XL to Bad from STS-induced cells was higher than that from healthy cells.
We for the first time employed ExEm-FRET method to measure the stoichiometric information of macromolecular complexes within living cells. Live-cell ExEm-FRET two-hybrid assay for evaluating the binding of Bad to Bcl-XL in Hela cells yielded similar results to the previous report that revealed the coexistence of both Bad-Bcl-XL dimer and Bad-Bcl-XL2 trimer in living cells . Recently, our studies have confirmed that ExEm-FRET measurement has superior stability and robustness and can obtain relatively accurate results even for cells with low signal-to-noise ratio [6,10,11]. Due to the superior robustness and sensitivity of ExEm-FRET [6,11], ExEm-FRET two-hybrid assay is very applicable to evaluating stoichiometry of macromolecular complexes in living cells. On our self-developed automated FRET microscope, one switch of the motorized cube wheel and six-time switch of the motorized emission filter wheel are required for a complete ExEm-FRET measurement. Although ExEm-FRET measurement requires about eight spectral images, a complete measurement takes about only 3 s on our self-developed automated FRET microscope when the expression level of fluorescent protein is relatively high.
We here develop an ExEm-FRET two-hybrid assay to determine the stoichiometric information of intracellular complexes by linearly unmixing the excitation-emission spectra of cells. Because of using pixel-to-pixel titration curves of donor-centric FRET efficiency (ED) versus the free acceptor (Ca) and acceptor-centric FRET efficiency (EA) versus the free donor (Cd) to obtain the ED,max and EA,max (the maximal EA and ED) values, respectively, ExEm-FRET two-hybrid assay requires only a few cells, which greatly reduces the workload of quantitative FRET measurements. Furthermore, due to the superior robustness and sensitivity of ExEm-unmixing, ExEm-FRET two-hybrid assay is especially applicable for cells with low fluorescence signal, thus provides a powerful tool for exploring the molecular mechanism of intracellular signal transduction in living cells.
National Natural Science Foundation of China (NSFC) (Grant No. 61875056 and 61527825).
1. E. S. Butz, M. Ben-Johny, M. Shen, P. S. Yang, L. Sang, M. Biel, D. T. Yue, and C. Wahl-Schott, “Quantifying macromolecular interactions in living cells using FRET two-hybrid assays,” Nat. Protoc. 11(12), 2470–2498 (2016). [CrossRef] [PubMed]
2. M. Y. Du, F. F. Yang, Z. H. Mai, W. F. Qu, F. R. Lin, L. C. Wei, and T. S. Chen, “FRET two-hybrid assay linearly fitting FRET efficiency to concentration ratio between acceptor and donor,” Appl. Phys. Lett. 112(15), 153702 (2018). [CrossRef]
3. M. G. Erickson, B. A. Alseikhan, B. Z. Peterson, and D. T. Yue, “Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells,” Neuron 31(6), 973–985 (2001). [CrossRef] [PubMed]
5. A. D. Hoppe, B. L. Scott, T. P. Welliver, S. W. Straight, and J. A. Swanson, “N-way FRET microscopy of multiple protein-protein interactions in live cells,” PLoS One 8(6), e64760 (2013). [CrossRef] [PubMed]
6. F. Lin, C. Zhang, M. Du, L. Wang, Z. Mai, and T. Chen, “Superior robustness of ExEm-spFRET to IIem-spFRET method in live-cell FRET measurement,” J. Microsc. 272(2), 145–150 (2018). [CrossRef] [PubMed]
7. M. Du, L. Zhang, S. Xie, and T. Chen, “Wide-field microscopic FRET imaging using simultaneous spectral unmixing of excitation and emission spectra,” Opt. Express 24(14), 16037–16051 (2016). [CrossRef] [PubMed]
8. Y. Sun, H. Wallrabe, C. F. Booker, R. N. Day, and A. Periasamy, “Three-color spectral FRET microscopy localizes three interacting proteins in living cells,” Biophys. J. 99(4), 1274–1283 (2010). [CrossRef] [PubMed]
9. J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophys. J. 94(3), 986–1000 (2008). [CrossRef] [PubMed]
10. C. Zhang, F. Lin, M. Du, W. Qu, Z. Mai, J. Qu, and T. Chen, “Simultaneous measurement of quantum yield ratio and absorption ratio between acceptor and donor by linearly unmixing excitation-emission spectra,” J. Microsc. 270(3), 335–342 (2018). [CrossRef] [PubMed]
11. W. Su, M. Du, F. Lin, C. Zhang, and T. Chen, “Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation-emission spectra,” J. Biophotonics 12(4), e201800314 (2019). [CrossRef] [PubMed]
12. G. Heras-Martinez, J. Andrieu, B. Larijani, and J. Requejo-Isidro, “Quantifying intracellular equilibrium dissociation constants using single-channel time-resolved FRET,” J. Biophotonics 11(1), e201600272 (2018). [CrossRef]
14. F. R. Lin, M. Y. Du, F. F. Yang, L. C. Wei, and T. S. Chen, “Improved spectrometer-microscope for quantitative FRET measurement based on simultaneous spectral unmixing of excitation and emission spectra,” J. Biomed. Opt. 23(1), 1–10 (2018). [PubMed]
15. A. J. Valentijn, A. D. Metcalfe, J. Kott, C. H. Streuli, and A. P. Gilmore, “Spatial and temporal changes in Bax subcellular localization during anoikis,” J. Cell Biol. 162(4), 599–612 (2003). [PubMed]
16. A. Aranovich, Q. Liu, T. Collins, F. Geng, S. Dixit, B. Leber, and D. W. Andrews, “Differences in the mechanisms of proapoptotic BH3 proteins binding to Bcl-XL and Bcl-2 quantified in live MCF-7 cells,” Mol. Cell 45(6), 754–763 (2012). [CrossRef] [PubMed]
17. C. King, S. Sarabipour, P. Byrne, D. J. Leahy, and K. Hristova, “The FRET signatures of noninteracting proteins in membranes: simulations and experiments,” Biophys. J. 106(6), 1309–1317 (2014). [CrossRef] [PubMed]
18. F. Edlich, S. Banerjee, M. Suzuki, M. M. Cleland, D. Arnoult, C. Wang, A. Neutzner, N. Tjandra, and R. J. Youle, “Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol,” Cell 145(1), 104–116 (2011). [CrossRef] [PubMed]
19. H. Düssmann, M. Rehm, C. G. Concannon, S. Anguissola, M. Würstle, S. Kacmar, P. Völler, H. J. Huber, and J. H. Prehn, “Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation,” Cell Death Differ. 17(2), 278–290 (2010). [CrossRef] [PubMed]
20. A. Kelekar, B. S. Chang, J. E. Harlan, S. W. Fesik, and C. B. Thompson, “Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL,” Mol. Cell. Biol. 17(12), 7040–7046 (1997). [CrossRef] [PubMed]
21. J. Zha, H. Harada, K. Osipov, J. Jockel, G. Waksman, and S. J. Korsmeyer, “BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity,” J. Biol. Chem. 272(39), 24101–24104 (1997). [CrossRef] [PubMed]
22. E. Yang, J. Zha, J. Jockel, L. H. Boise, C. B. Thompson, and S. J. Korsmeyer, “Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death,” Cell 80(2), 285–291 (1995). [CrossRef] [PubMed]
23. A. M. Petros, D. G. Nettesheim, Y. Wang, E. T. Olejniczak, R. P. Meadows, J. Mack, K. Swift, E. D. Matayoshi, H. Zhang, C. B. Thompson, and S. W. Fesik, “Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies,” Protein Sci. 9(12), 2528–2534 (2000). [CrossRef] [PubMed]