Tunable photoactivation of a post-translationally modified signaling protein and its unmodified counterpart in live cells

Michael E. Hahn, Jean Philippe Pellois, Miquel Vila-Perelló, Tom W. Muir

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

An ideal technology for direct imaging of post-translationally modified proteins would be one in which the appearance of a fluorescent signal is linked to a modification dependent protein-activation event. Herein, we utilize the protein semisynthesis technique, expressed protein ligation (EPL), to prepare caged analogues of the signaling protein Smad2; the function and fluorescence of the analogues were then photocontrolled in a correlated fashion. We show that this strategy permits titration of the cellular levels of active phosphorylated Smad2 in its biologically relevant, full-length form. We also prepared a nonphosphorylated, caged full-length Smad2 analogue labeled with an orthogonal fluorophore, and simultaneously imaged the phosphorylated and nonphosphorylated forms of the protein in the same cell. This strategy should enable the dissection of the cellular consequences of post-translational modifications (PTMs) by direct comparison of the behavior of the modified and unmodified forms of the protein following uncaging.

Original languageEnglish (US)
Pages (from-to)2100-2105
Number of pages6
JournalChemBioChem
Volume8
Issue number17
DOIs
StatePublished - Nov 23 2007
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Organic Chemistry

Keywords

  • Caged proteins
  • Fluorescent probes
  • Protein engineering
  • Protein modifications
  • Protein semisynthesis

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