Resonance energy transfer between green fluorescent protein variants: complexities revealed with myosin fusion proteins.
Green fluorescent protein and its variants are frequently used as Förster (fluorescence) resonance energy transfer (FRET) pairs to determine the proximity of protein domains. We prepared fusion proteins comprising yellow fluorescent protein-Dictyostelium myosin II motor domain-cyan fluorescent protein (YFP-myosin-CFP) and compared their FRET properties with an existing construct (GFP-myosin-BFP), containing a green fluorescent protein acceptor and blue fluorescent protein donor [Suzuki, Y., Yasunaga, T., Ohkura, R., Wakabayashi, T. and Sutoh, K. (1998) Nature 396, 380-383]. The latter construct showed an apparent 40% reduction in acceptor fluorescence on ATP addition, when excited via the donor, compared with the YFP-myosin-CFP constructs which showed a small increase (<or=5%). We propose that this disparity primarily arises from the differential response of GFP and YFP on intramolecular association with the donor probe. Studies with isolated GFP and YFP at high concentrations show that they dimerize with similar K(d) values but the spectrum shifts toward the protonated state only with GFP. On excitation at 380 nm, the protonated GFPH emits at 510 nm via excited-state proton transfer, giving the appearance of extensive FRET. These findings have important implications for FRET measurements using GFP-type probes because they give rise to changes in donor and acceptor emission ratios through processes other than FRET and complicate the extraction of the true degree of energy transfer from experimental data. Furthermore, the unknown orientation factor prevents the distance of the lever arm swing from being derived from these FRET changes.
Zeng
Wei
W
Department of Biochemistry, University of Leicester, UK.
Seward
Harriet E
HE
Málnási-Csizmadia
András
A
Wakelin
Stuart
S
Woolley
Robert J
RJ
Cheema
Gurpreet S
GS
Basran
Jaswir
J
Patel
Trushar R
TR
Rowe
Arthur J
AJ
Bagshaw
Clive R
CR
D000818:Animals; D004023:Dictyostelium / Q000737:chemistry* / Q000166:cytology; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006801:Humans; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D024462:Myosin Type II / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins
C485182:Cyan Fluorescent Protein; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D024462:Myosin Type II