title: Monitoring protein misfolding by site-specific labeling of proteins in vivo.
abstract: 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.
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Deutsches Krebsforschungszentrum (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany.
authors.forename: Tzung-yang
authors.initials: TY
authors.lastname: Hsieh
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Deutsches Krebsforschungszentrum (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany.
authors.forename: Nadinath B
authors.initials: NB
authors.lastname: Nillegoda
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Deutsches Krebsforschungszentrum (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Tyedmers
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Deutsches Krebsforschungszentrum (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Bukau
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Deutsches Krebsforschungszentrum (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Mogk
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Deutsches Krebsforschungszentrum (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany.
authors.forename: Günter
authors.initials: G
authors.lastname: Kramer
mesh_terms: D005434:Flow Cytometry; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006358:Hot Temperature; D057927:Hydrophobic and Hydrophilic Interactions; D049409:Luciferases, Firefly / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010454:Peptide Termination Factors / Q000378:metabolism; D066329:Protein Aggregates; D011489:Protein Denaturation; D017510:Protein Folding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D015091:beta-Alanine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: C546053:3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid; D006360:Heat-Shock Proteins; D050505:Mutant Proteins; D010454:Peptide Termination Factors; D066329:Protein Aggregates; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015091:beta-Alanine; C070972:HsP104 protein, S cerevisiae; D049409:Luciferases, Firefly