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Title: Resonance energy transfer between green fluorescent protein variants: complexities revealed with myosin fusion proteins.
Authors: Zeng, W
Seward, HE
Málnási-Csizmadia, A
Wakelin, S
Woolley, RJ
Cheema, GS
Basran, J
Patel, TR
Rowe, AJ
Bagshaw, CR
First Published: 5-Sep-2006
Citation: BIOCHEMISTRY, 2006, 45 (35), pp. 10482-10491
Abstract: 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.
DOI Link: 10.1021/bi060943u
ISSN: 0006-2960
Type: Journal Article
Appears in Collections:Published Articles, Dept. of Biochemistry

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