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Int. J. Mol. Sci. 2014, 15(12), 23836-23850; doi:10.3390/ijms151223836

Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

1
Laboratory of Biochemistry, Wageningen University, Wageningen 6703HA, The Netherlands
2
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft 2628CJ, The Netherlands
3
Microspectroscopy Centre, Wageningen University, Wageningen 6703HA, The Netherlands
*
Author to whom correspondence should be addressed.
Received: 28 October 2014 / Revised: 26 November 2014 / Accepted: 28 November 2014 / Published: 19 December 2014
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Abstract

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway. View Full-Text
Keywords: time-resolved fluorescence; protein folding; Alexa Fluor; FRET; rise time of acceptor fluorescence time-resolved fluorescence; protein folding; Alexa Fluor; FRET; rise time of acceptor fluorescence
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Lindhoud, S.; Westphal, A.H.; van Mierlo, C.P.M.; Visser, A.J.W.G.; Borst, J.W. Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding. Int. J. Mol. Sci. 2014, 15, 23836-23850.

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