Volume 9 Issue 3
Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding
Simon Lindhoud, Adrie H. Westphal, Carlo P. M. Van Mierlo, Antonie J. W. G. Visser and Jan Willem Borst
1Laboratory of Biochemistry, Wageningen University, Wageningen 6703HA, The Netherlands
2Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft 2628CJ, The Netherlands
3Microspectroscopy Centre, Wageningen University, Wageningen 6703HA, The Netherlands
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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.
Keywords:time-resolved fluorescence; protein folding; Alexa Fluor; FRET; rise time of acceptor fluorescence