Site-Specific Labeling of Proteins in Live Mammalian Cells using Click Chemistry

An ideal fluorescent tag to study protein structure and dynamics inside living cells using super-resolution microscopy (SRM) needs to be bright, photostable and should not interfere with the functionality of the protein. Since synthetic fluorophores have exceptional photophysical properties and small size, techniques have been developed in order to tag proteins with these fluorophores in a specific and straightforward manner. Among those, the most versatile way of tagging a fluorescent probe site-specifically to the protein of interest (POI) is to use genetic code expansion technology to incorporate noncanonical amino acids (ncAAs). These ncAAs contain reactive functional groups such as strained alkene/alkyne, which can be exploited to label the POI through rapid and biocompatible click chemistry reactions, namely, strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC). In here, we utilize this technology in order to develop an improved labeling method to perform multicolor labeling of large protein complexes. Moreover, we fully explore the potential of the labeling method by testing newly available ncAAs and fluorophores to find the most efficient and specific pair, thus minimizing background labeling in live mammalian cells. Subsequent analysis with SRM provided us with profound insights into fundamental biological processes.