title: Tunable photoactivation of a post-translationally modified signaling protein and its unmodified counterpart in live cells.
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.
authors: Laboratory of Synthetic Protein Chemistry, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
authors: Michael E
authors: ME
authors: Hahn
authors: Jean-Philippe
authors: JP
authors: Pellois
authors: Miquel
authors: M
authors: Vila-Perelló
authors: Tom W
authors: TW
authors: Muir
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004952:Esters / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D010766:Phosphorylation; D010777:Photochemistry; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000187:drug effects*; D051899:Smad2 Protein / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D004952:Esters; D051899:Smad2 Protein; D013438:Sulfhydryl Compounds