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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2014
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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 |
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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 |
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