Determining the stoichiometry of protein heterocomplexes in living cells with fluorescence fluctuation spectroscopy

Chen et al. 10.1073/pnas.0606557104.

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SI Figure 5
SI Materials and Methods

SI Figure 5

Fig. 5. Both RXRLBD and SRC1NID are tagged with GFP and coexpressed in CV-1 cells. Based on the experiments with CFP- and YFP-labeled proteins we chose cotransfection concentrations of the plasmid that favor a 3:1 coexpression ratio between RXRLBD and SRC1NID. The brightness of transfected cells is measured at an excitation wavelength of 902 nm in the nucleus of CV-1 cells. The ligand 9cRA was present in all measurements conducted. Cells transfected only with RXRLBD-GFP were measured as a control. Their normalized brightness titration curve (open squares) is reaching a value of two, indicative of a dimer. The normalized brightness (filled circles) of cells coexpressing SRC1NID-GFP/RXRLBD-GFP in the presence of ligand increases with protein concentration and reaches a value of close to four, which is consistent with a 3:1 binding model between RXRLBD and SRC1NID.

SI Materials and Methods

Construction of Expression Vectors and Cell Measurements. All fluorescent proteins used are based on the vectors pEGFP-C1, pEYFP-C1, and pECFP-C1 (Clontech, Mountain View, CA) and are referred to as GFP, YFP, and CFP throughout the text. RXRLBD-CFP, RXRLBD-YFP, and RXRLBD-GFP were amplified from Mouse RXRβ (GenBank accession no. X66224) and have been described previously. Full-length human SRC-1 was a kind gift from Dr. O'Malley (Department of Molecular and Cellular Biology, Baylor College of Medicine). Full-length SRC1 and SRC1NID were PCR amplified with a 5¢ primer that encoded a BspEI restriction site and a 3¢ primer that encoded a XhoI site. The PCR products were digested by BspEI and XhoI and ligated into pEGFP-C1 and pEYFP-C1 within the multiple cloning region. SRC1NIDT (amino acid residues 570-737) is based on SRC1NID and is generated by deletion mutation using QuikChange II Site-Directed Mutagenesis Kits (Stratagene, La Jolla, CA). The sequences were verified by automatic sequencing.

CV-1 cells were obtained from ATCC (Manassas, VA) and maintained in 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and Eagle minimum essential medium. Transfections were carried out by using Transfectin (Bio-Rad, Hercules, CA) according to manufacturer's instructions. Cells were subcultured into 8-well coverglass chamber slides (Nalge Nunc International, Rochester, NY) 48 h before measurements. The growth medium was exchanged to Leibovitz's L-15 medium (no phenol red) with 10% fetal bovine serum (Invitrogen) before starting measurements.

Cells were transfected with a mixture of the two DNA plasmids encoding the proteins. Both proteins are present in the cytoplasm and the nucleus. All measurements were performed after focusing the two-photon spot into the nucleus of cells. We measured the protein coexpression ratio of transfected cells for different mixtures of plasmids. The average expression ratio varied as the plasmid concentration ratio was changed. We used this dependence to select conditions that provide favorable coexpression ratios for brightness analysis.

Fluorescence Lifetime Measurements. A time correlated single photon counting (TCSPC) module (TimeHarp 200; Picoquant, Germany) was used to measure the fluorescence lifetimes of CFP. A photodiode (DET210; Thorlabs, Newton, NJ) monitored the laser pulses and provided the synchronization signal. The fluorescence signal was measured under magic angle conditions with a PMT (H7421-40; Hamamatsu, Japan). A bandpass filter (FF495-EX01-25; Semrock, Rochester, NY) was placed in front of the PMT to block the light emitted from YFP. The fluorescence lifetime data were analyzed with Globals Unlimited (Urbana, IL).

Brightness Analysis. Molecular brightness was determined by analyzing the data using generalized Q-analysis (1). For each experiment we also measured the brightness of cells expressing CFP and YFP individually for calibration purposes. Furthermore, cells expressing a CFP-YFP fusion protein were measured in parallel experiments to ensure that the brightness of CFP and YFP are additive at an excitation wavelength of 902 nm. RXRLBD labeled with either CFP or YFP exhibited a concentration-dependent dimerization process (2) and served as a further control. The normalized brightness was established by dividing the measured brightness of the sample by the brightness of either CFP or YFP. Photobleaching of fluorophores was negligible in our experiments as tested by described methods (3). We calibrated the observation volume by measuring a GFP solution of known concentration, which we determined by absorption spectroscopy using an extinction coefficient of 53,000 M^-1cm^-1 at 489 nm (4). On average, one molecule within the excitation volume corresponds to a concentration of 23 nM. The concentration was therefore calculated by multiplying the occupation number N with 23 nM per molecule.

Protein Coexpression Ratio. The method has been described in detail elsewhere (2). We briefly summarize the approach. Assume that protein A is labeled with CFP, and protein B is labeled with YFP. We first established the brightness value (e_CFP and e_YFP) and the dual-color fluorescence intensity ratio (r_CFP and r_YFP) of each fluorescent protein for excitation at 902 nm. We also determined the fluorescence lifetime t_CFP of the donor A-CFP. Next, the dual-color intensity ratio r_I of cells cotransfected with A-CFP and B-YFP was measured together with the fluorescence lifetime of the donor t^*_CFP. The concentration ratio r_N between A-CFP and B-YFP is calculated for each cell from r_I, t_C^*, and the previously determined control parameters. The lifetime t_C^* is only needed if FRET is present to correct the bias in the intensity ratio r_I (2).