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Monitoring protein misfolding by site-specific labeling of proteins in vivo

Incorporating fluorescent amino acids by suppression of the TAG amber codon is a useful tool for site-specific labeling of proteins and visualizing their localization in living cells. Here we use a plasmid encoded orthogonal tRNA/aminoacyl-tRNA synthetase pair to site-specifically label firefly luci...

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Main Authors: Hsieh, Tzung-yang (Author) , Nillegoda, Nadinath B. (Author) , Tyedmers, Jens (Author) , Bukau, Bernd (Author) , Mogk, Axel (Author) , Kramer, Günter (Author)
Format: Article (Journal)
Language:English
Published: June 10, 2014
In: PLOS ONE
Year: 2014, Volume: 9, Issue: 6
ISSN:1932-6203
DOI:10.1371/journal.pone.0099395
Online Access:Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1371/journal.pone.0099395
Verlag, lizenzpflichtig, Volltext: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099395
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Author Notes:Tzung-yang Hsieh, Nadinath B. Nillegoda, Jens Tyedmers, Bernd Bukau, Axel Mogk, Günter Kramer
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Summary:Incorporating fluorescent amino acids by suppression of the TAG amber codon is a useful tool for site-specific labeling of proteins and visualizing their localization in living cells. Here we use a plasmid encoded orthogonal tRNA/aminoacyl-tRNA synthetase pair to site-specifically label firefly luciferase with the environmentally sensitive fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2- aminopropanoic acid (ANAP) and explore the detectability of conformational changes in labeled luciferase in the yeast cytoplasm. We find that ANAP labeling efficiency is greatly increased in [PSI+] cells and show that analysis of the ANAP fluorescence emission by confocal imaging allows for tracking the thermal unfolding and aggregation of luciferase in vivo. Furthermore we demonstrate that flow cytometry can be used to study conformational changes in luciferase and chaperone-mediated refolding in quantitative terms and at the level of single cells. This experimental setup for the first time allows for the direct analysis of the folding state of a protein in living cells and may serve as valuable new tool for examining mechanisms of protein folding, misfolding and aggregation.
Item Description:Gesehen am 12.08.2020
Physical Description:Online Resource
ISSN:1932-6203
DOI:10.1371/journal.pone.0099395

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