Dimerization between aequorea fluorescent proteins does not affect interaction between tagged estrogen receptors in living cells

Abstract

Forster resonance energy transfer (FRET) detection of protein interaction in living cells is commonly measured following the expression of interacting proteins genetically fused to the cyan (CFP) and yellow (YFP) derivatives of the Aequorea victoria fluorescent protein (FP). These FPs can dimerize at mM concentrations, which may introduce artifacts into the measurement of interaction between proteins that are fused with the FPs. Here, FRET analysis of the interaction between estrogen receptors (alpha isoform, ERalpha) labeled with "wild-type" CFP and YFP is compared with that of ERalpha labeled with "monomeric" A206K mutants of CFP and YFP. The intracellular equilibrium dissociation constant for the hormone-induced ERalpha-ERalpha interaction is similar for ERalpha labeled with wild-type or monomeric FPs. However, the measurement of energy transfer measured for ERalpha-ERalpha interaction in each cell is less consistent with the monomeric FPs. Thus, dimerization of the FPs does not affect the kinetics of ERalpha-ERalpha interaction but, when brought close together via ERalpha-ERalpha interaction, FP dimerization modestly improves FRET measurement.

Structure, Affinity, and Availability of Estrogen Receptor Complexes in the Cellular Environment

“…Images were collected between 30 and 40 min post-treatment on a previously described microscope using a 20ϫ/0.75 NA objective (22,23). Initial studies conducted on cells treated with estradiol for 2 hours showed similar results.…”

“…The binding at 30 min, therefore, had reached steady state, which is a prerequisite for equilibrium biochemical analysis. The Acceptor, Donor and FRET images were collected in rapid succession, with the Acceptor images collected at two integration times to capture large variations in YFP-SRC expression levels, as previously described (22,23). Image collection was followed by previously described semi-automated procedures for background-subtraction, nuclei identification, and debris identification/elimination (22).…”

“…Instrument-calibrated bleed-through corrections were implemented to determine raw background-subtracted CFP, YFP, and FRET fluorescence values within each cell nucleus. Further instrument calibrations (22,24) were used to convert those values to the percent of CFP fluorescence lost to energy transfer (E). The CFP fluorescence intensities then were corrected for that loss of CFP fluorescence and for the calibrated ability of the equipment to detect CFP relative to YFP (3,22,24).…”

“…Further instrument calibrations (22,24) were used to convert those values to the percent of CFP fluorescence lost to energy transfer (E). The CFP fluorescence intensities then were corrected for that loss of CFP fluorescence and for the calibrated ability of the equipment to detect CFP relative to YFP (3,22,24). Fluorescence intensities were converted to ER␣ and SRC-RID concentrations (in nM) after instrument calibrations that used a stable cell line expressing a consistent, measured amount of a dual-labeled CFP-AR-YFP (where AR is the androgen receptor) expressed in HeLa cells.…”

“…As with all FRET measurements of interactions between factors, a large cell-to-cell scatter in the FRET measurement of ER⅐SRC-RID interaction has made hormone response difficult to accurately quantify (18 -20). Here, we introduce calibrated FRET procedures that complete prior progress toward interpreting the scatter typical of FRET measurements as a function of the kinetic model of biochemical interaction (3,13,21,22). The new analytical and calibration procedures allowed direct measurement in the cell of traditional biochemical parameters (such as the K d and Bmax, measured in nM) for each of the three ER⅐SRC-RID interactions.…”