EGFP oligomers as natural fluorescence and hydrodynamic standards.
EGFP oligomers are convenient standards for experiments on fluorescent protein-tagged biomolecules. In this study, we characterized their hydrodynamic and fluorescence properties. Diffusion coefficients D of EGFP1-4 were determined by analytical ultracentrifugation with fluorescence detection and by fluorescence correlation spectroscopy (FCS), yielding 83.4…48.2 μm(2)/s and 97.3…54.8 μm(2)/s from monomer to tetramer. A "barrels standing in a row" model agreed best with the sedimentation data. Oligomerization red-shifted EGFP emission spectra without any shift in absorption. Fluorescence anisotropy decreased, indicating homoFRET between the subunits. Fluorescence lifetime decreased only slightly (4%) indicating insignificant quenching by FRET to subunits in non-emitting states. FCS-measured D, particle number and molecular brightness depended on dark states and light-induced processes in distinct subunits, resulting in a dependence on illumination power different for monomers and oligomers. Since subunits may be in "on" (bright) or "off" (dark) states, FCS-determined apparent brightness is not proportional to that of the monomer. From its dependence on the number of subunits, the probability of the "on" state for a subunit was determined to be 96% at pH 8 and 77% at pH 6.38, i.e., protonation increases the dark state. These fluorescence properties of EGFP oligomeric standards can assist interpreting results from oligomerized EGFP fusion proteins of biological interest.
Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Hungary.
György
G
Vámosi
DKFZ, Biophysics of Macromolecules, D-69120 Heidelberg, Germany.
Norbert
N
Mücke
DKFZ, Biophysics of Macromolecules, D-69120 Heidelberg, Germany.
Gabriele
G
Müller
DKFZ, Biophysics of Macromolecules, D-69120 Heidelberg, Germany.
Jan Wolfgang
JW
Krieger
Institute for Biophysical Chemistry, Hannover Medical School, D-30625 Hannover, Germany.
Ute
U
Curth
DKFZ, Biophysics of Macromolecules, D-69120 Heidelberg, Germany.
Jörg
J
Langowski
DKFZ, Biophysics of Macromolecules, D-69120 Heidelberg, Germany.
Katalin
K
Tóth
D005453:Fluorescence; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000592:standards; D057446:Hydrodynamics; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000592:standards; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins