Cargando…

Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

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 correspon...

Descripción completa

Detalles Bibliográficos
Autores principales: Lindhoud, Simon, Westphal, Adrie H., van Mierlo, Carlo P. M., Visser, Antonie J. W. G., Borst, Jan Willem
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4284793/
https://www.ncbi.nlm.nih.gov/pubmed/25535076
http://dx.doi.org/10.3390/ijms151223836
_version_ 1782351465909583872
author Lindhoud, Simon
Westphal, Adrie H.
van Mierlo, Carlo P. M.
Visser, Antonie J. W. G.
Borst, Jan Willem
author_facet Lindhoud, Simon
Westphal, Adrie H.
van Mierlo, Carlo P. M.
Visser, Antonie J. W. G.
Borst, Jan Willem
author_sort Lindhoud, Simon
collection PubMed
description 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.
format Online
Article
Text
id pubmed-4284793
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-42847932015-01-21 Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding Lindhoud, Simon Westphal, Adrie H. van Mierlo, Carlo P. M. Visser, Antonie J. W. G. Borst, Jan Willem Int J Mol Sci Article 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. MDPI 2014-12-19 /pmc/articles/PMC4284793/ /pubmed/25535076 http://dx.doi.org/10.3390/ijms151223836 Text en © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Lindhoud, Simon
Westphal, Adrie H.
van Mierlo, Carlo P. M.
Visser, Antonie J. W. G.
Borst, Jan Willem
Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding
title Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding
title_full Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding
title_fullStr Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding
title_full_unstemmed Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding
title_short Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding
title_sort rise-time of fret-acceptor fluorescence tracks protein folding
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4284793/
https://www.ncbi.nlm.nih.gov/pubmed/25535076
http://dx.doi.org/10.3390/ijms151223836
work_keys_str_mv AT lindhoudsimon risetimeoffretacceptorfluorescencetracksproteinfolding
AT westphaladrieh risetimeoffretacceptorfluorescencetracksproteinfolding
AT vanmierlocarlopm risetimeoffretacceptorfluorescencetracksproteinfolding
AT visserantoniejwg risetimeoffretacceptorfluorescencetracksproteinfolding
AT borstjanwillem risetimeoffretacceptorfluorescencetracksproteinfolding