ReAsH as a Quantitative Probe of In-Cell Protein Dynamics

Biochemistry. 2016 Apr 5;55(13):1968-76. doi: 10.1021/acs.biochem.5b01336. Epub 2016 Mar 18.

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cell Line, Tumor
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes / chemistry*
  • Green Fluorescent Proteins / chemistry*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Hot Temperature
  • Humans
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Fluorescence
  • Models, Molecular*
  • Molecular Imaging
  • Molecular Weight
  • Mutant Proteins / chemistry*
  • Mutant Proteins / metabolism
  • Phosphoglycerate Kinase / chemistry*
  • Phosphoglycerate Kinase / genetics
  • Phosphoglycerate Kinase / metabolism
  • Protein Conformation
  • Protein Engineering
  • Protein Folding
  • Protein Stability
  • Protein Unfolding
  • Recombinant Fusion Proteins
  • Red Fluorescent Protein
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism

Substances

  • Fluorescent Dyes
  • Luminescent Proteins
  • Mutant Proteins
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Green Fluorescent Proteins
  • Phosphoglycerate Kinase