Resonance Energy Transfer between Green Fluorescent Protein Variants: Complexities Revealed with Myosin Fusion Proteins
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- 作者：Wei Zeng ; Harriet E. Seward ; Andrá ; s Má ; lná ; si-Csizmadia ; Stuart Wakelin ; Robert J. Woolley ; Gurpreet S. Cheema ; Jaswir Basran ; Trushar R. Patel ; Arthur J. Rowe ; Clive R. Bagshaw
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：September 5, 2006
- 年：2006
- 卷：45
- 期：35
- 页码：10482 - 10491
- 全文大小：427K
- 年卷期：v.45,no.35(September 5, 2006)
- ISSN：1520-4995
文摘
Green fluorescent protein and its variants are frequently used as Förster (fluorescence) resonanceenergy transfer (FRET) pairs to determine the proximity of protein domains. We prepared fusion proteinscomprising 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 latterconstruct showed an apparent 40% reduction in acceptor fluorescence on ATP addition, when excited viathe donor, compared with the YFP-myosin-CFP constructs which showed a small increase (
5%). Wepropose that this disparity primarily arises from the differential response of GFP and YFP on intramolecularassociation with the donor probe. Studies with isolated GFP and YFP at high concentrations show thatthey dimerize with similar Kd 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, givingthe appearance of extensive FRET. These findings have important implications for FRET measurementsusing GFP-type probes because they give rise to changes in donor and acceptor emission ratios throughprocesses other than FRET and complicate the extraction of the true degree of energy transfer fromexperimental data. Furthermore, the unknown orientation factor prevents the distance of the lever armswing from being derived from these FRET changes.
5%). Wepropose that this disparity primarily arises from the differential response of GFP and YFP on intramolecularassociation with the donor probe. Studies with isolated GFP and YFP at high concentrations show thatthey dimerize with similar Kd 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, givingthe appearance of extensive FRET. These findings have important implications for FRET measurementsusing GFP-type probes because they give rise to changes in donor and acceptor emission ratios throughprocesses other than FRET and complicate the extraction of the true degree of energy transfer fromexperimental data. Furthermore, the unknown orientation factor prevents the distance of the lever armswing from being derived from these FRET changes.