Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins.
BACKGROUND
The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry.


METHODS
We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control.


RESULTS
We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (alpha) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing alpha. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins.


CONCLUSIONS
We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry.
Nagy
Peter
P
Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
Bene
László
L
Hyun
William C
WC
Vereb
György
G
Braun
Manuela
M
Antz
Christof
C
Paysan
Jacques
J
Damjanovich
Sándor
S
Park
John W
JW
Szöllősi
János
J
D002138:Calibration; D051357:Cyclin-Dependent Kinase 2 / Q000032:analysis / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000032:analysis / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis
D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D051357:Cyclin-Dependent Kinase 2