Dataset Information

Anomalous negative fluorescence anisotropy in yellow fluorescent protein (YFP 10C): quantitative analysis of FRET in YFP dimers.

ABSTRACT: Yellow fluorescent protein (YFP) is widely used as a genetically encoded fluorescent marker in biology. In the course of a comprehensive study of this protein, we observed an unusual, negative fluorescence anisotropy at pH 6.0 (McAnaney, T. B., Zeng, W., Doe, C. F. E., Bhanji, N., Wakelin, S., Pearson, D. S., Abbyad, P., Shi, X., Boxer, S. G., and Bagshaw, C. R. (2005) Biochemistry 44, 5510-5524). Here we report that the fluorescence anisotropy of YFP 10C depends on protein concentration in the low micromolar range that was not expected. We propose that the negative anisotropy is a result of unidirectional Förster resonance energy transfer (FRET) in a dimer of YFP, with the donor chromophore in the neutral form and the acceptor chromophore in the anionic form. This unusual mechanism is supported by studies of a monomeric YFP (A206K YFP) and transient-absorption spectroscopy of YFP 10C. A detailed analysis of the chromophore transition dipole moment direction is presented. The anisotropy and rate constant of this energy transfer are consistent with values produced by an analysis of the dimer structure observed in crystals.

SUBMITTER: Shi X

PROVIDER: S-EPMC2570256 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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Anomalous negative fluorescence anisotropy in yellow fluorescent protein (YFP 10C): quantitative analysis of FRET in YFP dimers.

Shi Xinghua X Basran Jaswir J Seward Harriet E HE Childs William W Bagshaw Clive R CR Boxer Steven G SG

Biochemistry 20071121 50

Yellow fluorescent protein (YFP) is widely used as a genetically encoded fluorescent marker in biology. In the course of a comprehensive study of this protein, we observed an unusual, negative fluorescence anisotropy at pH 6.0 (McAnaney, T. B., Zeng, W., Doe, C. F. E., Bhanji, N., Wakelin, S., Pearson, D. S., Abbyad, P., Shi, X., Boxer, S. G., and Bagshaw, C. R. (2005) Biochemistry 44, 5510-5524). Here we report that the fluorescence anisotropy of YFP 10C depends on protein concentration in the ...[more]

PMID: 18027983

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Project description:Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. However, these lifetimes may originate from interacting and noninteracting molecules, which hampers quantitative interpretation of FRET data. We describe a methodology for the detection of FRET that monitors the rise time of acceptor fluorescence on donor excitation thereby detecting only those molecules undergoing FRET. The large advantage of this method, as compared to donor fluorescence quenching method used more commonly, is that the transfer rate of FRET can be determined accurately even in cases where the FRET efficiencies approach 100% yielding highly quenched donor fluorescence. Subsequently, the relative orientation between donor and acceptor chromophores is obtained from time-dependent fluorescence anisotropy measurements carried out under identical conditions of donor excitation and acceptor detection. The FRET based calcium sensor Yellow Cameleon 3.60 (YC3.60) was used because it changes its conformation on calcium binding, thereby increasing the FRET efficiency. After mapping distances and orientation angles between the FRET moieties in YC3.60, cartoon models of this FRET sensor with and without calcium could be created. Independent support for these representations came from experiments where the hydrodynamic properties of YC3.60 under ensemble and single-molecule conditions on selective excitation of the acceptor were determined. From rotational diffusion times as found by fluorescence correlation spectroscopy and consistently by fluorescence anisotropy decay analysis it could be concluded that the open structure (without calcium) is flexible as opposed to the rather rigid closed conformation. The combination of two independent methods gives consistent results and presents a rapid and specific methodology to analyze structural and dynamical changes in a protein on ligand binding.

Dataset Information

Anomalous negative fluorescence anisotropy in yellow fluorescent protein (YFP 10C): quantitative analysis of FRET in YFP dimers.

Publications

Anomalous negative fluorescence anisotropy in yellow fluorescent protein (YFP 10C): quantitative analysis of FRET in YFP dimers.

Similar Datasets

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