A simple "proximity" correction for Förster resonance energy transfer efficiency determination in membranes using lifetime measurements.
Accurate measurements of oligomerization in membranes by Förster resonance energy transfer (FRET) are always compromised by a substantial contribution from random chance colocalization of donors and acceptors. Recently, Li and coworkers demonstrated the use of computer simulation in estimating the contribution of this "proximity" component to correct the FRET efficiency and estimate the free energy of dimer formation of the G380R mutants of fibroblast growth factor receptor 3 (FGFR3) transmembrane domain immersed into lipid bilayer. Because tight dimerization will result in complete energy transfer from donor to acceptor, we have used the same experimental system of fluorescein- and rhodamine-labeled G380R mutants of FGFR3 for the experimental assessment of the proximity FRET corrections using fluorescence lifetime measurements. The experimental proximity FRET correction, based on time-resolved fluorescence measurements, is expected to have general advantages over theoretical correction, especially in the case of nonrandomly distributed monomers.
Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, KS 66160, USA.
Yevgen O
YO
Posokhov
Mikhail
M
Merzlyakov
Kalina
K
Hristova
Alexey S
AS
Ladokhin
D002462:Cell Membrane / Q000378:metabolism*; D019281:Dimerization; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008051:Lipid Bilayers / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors
D008051:Lipid Bilayers; D051498:Receptor, Fibroblast Growth Factor, Type 3