Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 15, Issue 12, Pages 23836-23850Publisher
MDPI AG
DOI: 10.3390/ijms151223836
Keywords
time-resolved fluorescence; protein folding; Alexa Fluor; FRET; rise time of acceptor fluorescence
Funding
- Netherlands Organization for Scientific Research
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Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Forster 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 apoflavodoxin's complex folding pathway.
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