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Quantifying macromolecular interactions in living cells using FRET two-hybrid assays

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Abstract

Förster resonance energy transfer (FRET) is a versatile method for analyzing protein–protein interactions within living cells. This protocol describes a nondestructive live-cell FRET assay for robust quantification of relative binding affinities for protein–protein interactions. Unlike other approaches, our method correlates the measured FRET efficiencies to relative concentration of interacting proteins to determine binding isotherms while including collisional FRET corrections. We detail how to assemble and calibrate the equipment using experimental and theoretical procedures. A step-by-step protocol is given for sample preparation, data acquisition and analysis. The method uses relatively inexpensive and widely available equipment and can be performed with minimal training. Implementation of the imaging setup requires up to 1 week, and sample preparation takes 1–3 d. An individual FRET experiment, including control measurements, can be completed within 4–6 h, with data analysis requiring an additional 1–3 h.

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Figure 1: Equipment setup for FRET imaging.
Figure 2: Schematic of optical components of the FRET setup for a given filter cube and fluorophore.
Figure 3: Spectral properties of FRET.
Figure 4: Components of the fluorescence signal at 535-nm emission.
Figure 5: Calibration experiments using cells expressing CFP–YFP dimers of high, intermediate and low FRET efficiency.
Figure 6: Conceptual graph of FRET binding curves.
Figure 7: Overview of the calibration phase of the FRET rig.
Figure 8: Overview of a FRET two-hybrid binding experiment.
Figure 9: Stability of spectral correction factors and amplification factors.
Figure 10: Determination of the G factor and the excitation ratio using dimers.
Figure 11: Examples of spurious FRET and FRET two-hybrid binding curves.

Change history

  • 01 December 2016

    In the original version that appeared online, equal contribution of authors was incorrectly attributed; this has been corrected to indicate that Elisabeth Butz and Manu Ben Johny contributed equally to the work. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank R. Rötzer, A. Rößing, T. Kiwitt and W. Yang for excellent technical support. This work was supported by funding from the German Research Foundation (SFB/TRR 152 TP12 to M.B., SFB/TRR 152 TP06 to C.W.-S. and SFB 870 TP B05 to C.W.-S.) and the National Institute of Mental Health (R01 MH065531 to D.T.Y. and M.B.-J.). D.T.Y. passed away on December 23, 2014. He was a remarkable teacher, inspirational scientist and a generous colleague. He pioneered the development and the usage of FRET two-hybrid assays to elucidate the mechanistic underpinnings of voltage-gated Ca2+ channels. His scientific brilliance and personal charm are greatly missed in the scientific community.

Author information

Authors and Affiliations

Authors

Contributions

E.S.B., M.B.-J., D.T.Y. and C.W.-S. developed and/or designed the study. E.S.B. and M.B.-J. collected and analyzed the data. M.S. and L.S. furnished initial experimental data for dimer calibrations. M.B.-J. and D.T.Y. provided theoretical derivations. M.S., M.B.-J. and P.S.Y. constructed YFP- and CFP-tagged constructs and dimers. E.S.B., M.B.-J. and C.W.-S. wrote the manuscript and created the figures. M.B. discussed and commented on the manuscript.

Corresponding authors

Correspondence to Manu Ben-Johny or Christian Wahl-Schott.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Linearity of fluorescence detection subsystem

(a) Fluorescence intensity measured through the YFP cube is linearly proportional to the concentration of Alexa Fluor 514 dye. This result illustrates the linearity of the PMT detector enabling robust estimation of number of acceptor molecules. (b) Fluorescence intensity measured through the CFP cube is also linearly proportional to the concentration of Proflavin dye.

Supplementary Figure 2 Main graphical user interface of Felix GX

(a) The tab “Delta Ram Analog” (right upper corner) is clicked to open the “Hardware Configuration” window (Supplementary Fig. 2a). (b) The setup tab is selected (left lower corner) to open the “Setup” window (Supplementary Fig. 2b). (c) The “Action” tab opens the “Macro” window from which the “Macro Editor” window is opened (Supplementary Fig. 4).

Supplementary Figure 3 The “Hardware Configuration” window and the “Setup” window.

(a) Settings for the “Hardware Configuration” window. (b) Settings for the “Setup” window. From the 10 tabs in the “Setup” window select “Acquisition Type” and click the option “Timebased”.

Supplementary Figure 4 Parameter settings specific for Timebased acquisition in the “Setup window”.

(a) Acquisition settings; (b) PMTs; (c) Backgrounds; (d) Traces tab.

Supplementary Figure 5 Setup of Macros using the “Macro” and the “Macro Editor” window.

(a) Open the “Macro” window by clicking the “Action“ tab in the main graphical user interface (Supplemental Fig. 1). Click the green “+” button to open the “Macro Editor” window (b and c). In this window, different actions (listed on the left hand side) can be added to the macro (list on the black panel on the right hand side). In this example, a pause step (b) and a protocol (Run Acquisition; c) is added to the macro by clicking the Add tab.

Supplementary Figure 6 Data input field of the Background sheet.

(a) Raw data pasted into the respective columns from measurements using CFP- FRET- and YFP-cubes at a distinct HV gain. (b) The excel sheet contains 9 individual sub sheets (labeled from left to right: Background, RD-value (CFP), RA-value (YFP), Dimer sheet, Dimer Analysis, Spurious FRET, Samples (FRET sample data), BC Analysis and HV lookup.

Supplementary Figure 7 Data input field of the RD-value (CFP) and the RA-value (YFP) sheets.

Raw data are pasted into the respective columns on the left hand side from measurements in (a) CFP-only or (b) YFP-only expressing cells using CFP- FRET- and YFP-cubes at a distinct HV gain. Background subtraction and RA value calculation is performed automatically. (c) The mean RD and RA value, resp., is calculated on the right hand side (gray panel). Statistical values (Standard deviation of the mean (SEM), Minimum (Min) and Maximum (Max)) provide a useful tool to assess the variability of the values. Note that only the mean RD1 and RA1 values are used for subsequent analysis.

Supplementary Figure 8 Data input field of the Dimer sheet.

(a) Raw data are pasted into the respective columns on the left hand side from measurements in CFP-YFP dimer expressing cells using CFP- FRET- and YFP-cubes at a distinct HV gain. Raw data for individual cells are collected separately for a given dimer as indicated. (b) Background subtracted values are shown on the right. RA1 and RD1 values are automatically picked from RD-value (CFP) and the RA-value (YFP) sheets. Statistics are shown on the right hand side (Mean, SEM).

Supplementary Figure 9 Dimer analysis sheet.

(a) Columns D-G list background subtracted raw data from the “Dimer” sheet. Data for individual cells are collected separately for a given dimer as indicated in Column B. RA1 and RD1 values are automatically transferred from “RD-value (CFP)” and the “RA-value (YFP)” sheets (grey panel on the top, left). Columns H-P comprise calculations as outlined in Procedure Step 97. (b) The graph (column K plotted against column L) depicts data points for individual cells. For each dimer, cells cluster together. In the table below, the mean values of EA and ED for each dimer as well as mean values of the x- and y- coordinates (from columns M and N) are indicated. (c) The latter can be plotted in a new graph and analyzed by a linear regression line. In this template, the regression line is automatically computed and the resulting equation displayed in the graph. Note, that the y-intercept and the slope of this line is copied to cell T56 and T57 respectively for further analysis.

Supplementary Figure 10 Data input field of the “Spurious FRET” sheet.

(a) Raw data from cells expressing untagged eCFP and eYFP are transferred to respective cells in Columns C-G. (b) Background-subtraction as well as subsequent calculation of Fc/(RA1*SYFP), CFPEST and EA (columns K-M) is automatically performed referring to the constants in the gray panel at the upper left. (c) The graph at the right hand side, illustrates CFPEST as a function of EA. The slope of the regression line is used for spurious FRET correction in the “BC analysis sheet”. The equation of the regression line is automatically displayed in the graph. The slope is subsequently entered into cell “P7” linked to the “BC analysis” sheet.

Supplementary Figure 11 Data input field of the binding curve analysis (“BC analysis”) sheet.

(a) Background subtracted raw data (columns D-H) are automatically adopted from the “Samples” sheet. The principal calculation steps required in order to solve binding curves are automatically performed in columns I-W for each (biological) cell according Steps 117-130 outlined in the Procedure section. (b) The table in the upper left of the worksheet contains all parameters (columns F and I) necessary to correct for spectral properties and spurious FRET. Thereby, all values except the excitation ratio ((ƐYFP(440)/ƐCFP(440)) are automatically transferred from previously analyzed sheets. Values for KdEff, EAmax and EDmax (Column K) are place holders that are adjusted during the fitting procedure. (c) Graphs shown displays data points for individual biological cells (blue dots) and the fitted binding isotherme (black lines). The graphs represent data for 33-FRET (Dfree plotted against EA) and E-FRET (Afree plotted against ED) and allow a control of the fitting procedure.

Supplementary Figure 12 Setting up Excel Solver for optimizing 33-FRET and E-FRET binding parameters.

(a) Least squares optimization of Cell K8 (Sum_Err_3Cube) by changing both KD_eff (KD,EFF, relative affinity) and EAmax (EA,max, maximum 33-FRET efficiency). In this scenario, two constraints are EAmax>0, and Kd_eff>0 in accordance with physical principles. (b) Least squares optimization of Cell K10 (Total Error) by changing both KD_eff (KD,EFF, relative affinity), EAmax (EA,max, maximum 33-FRET efficiency), and EDmax (ED,max, maximum E-FRET efficiency). Three constraints are imposed: EDmax>0, EAmax>0, and Kd_eff>0.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Notes 1–4 (PDF 4424 kb)

Supplementary Data 1

MS Excel analysis spreadsheet for theoretical calculation of MD and MA containing sample data. (XLSX 270 kb)

Supplementary Data 2

MATLAB import/export script source file (M). (XLSX 46 kb)

Supplementary Data 3

MS Excel analysis spreadsheet containing sample data. (XLSX 133 kb)

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Butz, E., Ben-Johny, M., Shen, M. et al. Quantifying macromolecular interactions in living cells using FRET two-hybrid assays. Nat Protoc 11, 2470–2498 (2016). https://doi.org/10.1038/nprot.2016.128

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