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.
Highlights
Over the past few decades, Förster resonance energy transfer (FRET) has become a popular tool to probe distances between fluorescent donor and acceptor molecules
FRET is the transfer of electronic excitation energy from an excited donor fluorophore to an acceptor chromophore in the ground state through non-radiative dipole-dipole coupling [5,6]
Protein molecules that contain only donor fluorophores and, emit more donor photons compared to molecules containing both labels cause an underestimation of the actual FRET efficiency [11]
Summary
Over the past few decades, Förster resonance energy transfer (FRET) has become a popular tool to probe distances between fluorescent donor and acceptor molecules. We have performed time-resolved measurements of sensitized acceptor fluorescence to study the denaturant-dependent folding of apoflavodoxin from Azotobacter vinelandii. This protein is labeled with donor fluorophore. A488 is often used as a donor fluorophore in single-molecule FRET experiments (see, e.g., [18,19,20,21]) and in protein ensemble measurements (see, e.g., [22,23]) Both equilibrium and kinetic (un)folding of apoflavodoxin have been characterized using guanidine hydrochloride (GuHCl) as the denaturant Equilibrium folding of apoflavodoxin can be described by the three-state model N↔U↔Ioff, in which N and U are native and unfolded protein, respectively, and Ioff is an intermediate that is kinetically off-pathway [24]. We show that measurement of the rise time of acceptor fluorescence reveals folding-induced conformational changes of apoflavodoxin and, tracks protein folding
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
