title: ReAsH as a Quantitative Probe of In-Cell Protein Dynamics.
abstract: The tetracysteine (tc) tag/biarsenical dye system (FlAsH or ReAsH) promises to combine the flexibility of fluorescent protein tags with the small size of dye labels, allowing in-cell study of target proteins that are perturbed by large protein tags. Quantitative thermodynamic and kinetic studies in-cell using FlAsH and ReAsH have been hampered by methodological complexities presented by the fluorescence properties of the tag-dye complex probed by either Förster resonance energy transfer (FRET) or direct excitation. We label the model protein phosphoglycerate kinase (PGK) with AcGFP1 and ReAsH for direct comparison with AcGFP1/mCherry-labeled PGK. We find that fast relaxation imaging (FReI), combining millisecond temperature jump kinetics with fluorescence microscopy detection, circumvents many of the difficulties encountered working with the ReAsH system, allowing us to obtain quantitative FRET measurements of protein stability and kinetics both in vitro and in cells. We also demonstrate the to us surprising result that fluorescence from directly excited, unburied ReAsH at the C-terminus of the model protein also reports on folding in vitro and in cells. Comparing the ReAsH-labeled protein to a construct labeled with two fluorescent protein tags allows us to evaluate how a bulkier protein tag affects protein dynamics in cells and in vitro. We find that the average folding rate in the cell is closer to the in vitro rate with the smaller tag, highlighting the effect of tags on quantitative in-cell measurements.
authors.affiliation: Department of Physics, ‡Department of Chemistry, and §Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Hannah
authors.initials: H
authors.lastname: Gelman
authors.affiliation: Department of Physics, ‡Department of Chemistry, and §Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Anna Jean
authors.initials: AJ
authors.lastname: Wirth
authors.affiliation: Department of Physics, ‡Department of Chemistry, and §Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Martin
authors.initials: M
authors.lastname: Gruebele
mesh_terms: D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D057054:Molecular Imaging; D008970:Molecular Weight; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D055550:Protein Stability; D058767:Protein Unfolding; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D050505:Mutant Proteins; D010735:Phosphoglycerate Kinase; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000097573:Red Fluorescent Protein