Rise-Time of FRET-Acceptor Fluorescence TracksProtein Folding

International Journal of Molecular Sciences

Antonie J. W. G. Visser¹  Carlo P. M. van Mierlo¹  Adrie H. Westphal¹  Jan Willem Borst¹  Simon Lindhoud¹ 

[1] Laboratory of Biochemistry, Wageningen University, Wageningen 6703HA, The Netherlands;

关键词: time-resolved fluorescence;    protein folding;    Alexa Fluor;    FRET;    rise time of acceptor fluorescence;   

DOI  :  10.3390/ijms151223836

来源: DOAJ

【 摘 要 】

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 containsdonor-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.

【 授权许可】

Unknown