title: Rise-time of FRET-acceptor fluorescence tracks protein folding.
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 apoflavodoxin's complex folding pathway.
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Wageningen 6703HA, The Netherlands. s.lindhoud@tudelft.nl.
authors.forename: Simon
authors.initials: S
authors.lastname: Lindhoud
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Wageningen 6703HA, The Netherlands. adrie.westphal@wur.nl.
authors.forename: Adrie H
authors.initials: AH
authors.lastname: Westphal
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Wageningen 6703HA, The Netherlands. carlo.vanmierlo@wur.nl.
authors.forename: Carlo P M
authors.initials: CP
authors.lastname: van Mierlo
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Wageningen 6703HA, The Netherlands. antoniejvisser@gmail.com.
authors.forename: Antonie J W G
authors.initials: AJ
authors.lastname: Visser
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Wageningen 6703HA, The Netherlands. janwillem.borst@wur.nl.
authors.forename: Jan Willem
authors.initials: JW
authors.lastname: Borst
mesh_terms: D001059:Apoproteins / Q000737:chemistry; D005418:Flavodoxin / Q000737:chemistry; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D013194:Staining and Labeling; D013997:Time Factors
chemical_list: D001059:Apoproteins; D005418:Flavodoxin; D005456:Fluorescent Dyes; D011506:Proteins; C026932:apoflavodoxin