TEVprotease-based specific N-terminal cysteine labeling for imaging in living cells.

ELIZABETH JARES-ERIJMAN; E. PAPOUCHEVA; CARLA SPAGNUOLO; M. ROSSNER; M. WEHR; THOMAS M. JOVIN; GERTRUDE BUNT

Heidelberg, Alemania

Simposio; 4th International Symposium of the Volkswagen Foundation; 2005

European Molecular Biology Laboratory/ Volkswagen Stiftung

We report the synthesis of specially designed small membrane-permeable compounds for the implementation of a new methodology that combines chemoselective chemistry with native chemical ligation, resulting in the specific labeling of proteins in living eukaryotic cells. The method requires a transthioesterification step between a thioester in a small molecule bearing a fluorophore and the thiol group in a protein modified to expose an N-terminal cysteine. The ligated thioester intermediate undergoes spontaneous S -> N acyl rearrangement, thereby covalently linking the protein to the fluorophore of interest. The introduction of N-terminal cysteine constitutes a key aspect of the work. To optimize the methodology for eukaryotic cells, a GFP construct that carries a modified tobacco etch virus (TEV) protease recognition site at its N-terminus was expressed in mammalian cells together with a TEVprotease. The introduction of a cysteine immediately downstream of the cleavage position (P´) in the recognition sequence results in the exposure of a N-terminal cysteine upon TEV protease cleavage. Cells transfected with the GFP fusion protein were selectively labeled in vivo with rhodamine- and biotin-containing benzyl-based thioesters, the latter being visualized by Cy3-labeled avidin or Qdots. Modification of the emission properties of Qdots by binding to molecules such as photochromic dithieilylethens enables specific identification of selected nanoparticles. Therefore. the cleavage of modified TEV sites proenabling in vivo labeling with thioester-based probes with new sensing characteristics for studving cellular behavior.