2015
ACS nano

Super-resolution microscopy of clickable amino acids reveals the effects of fluorescent protein tagging on protein assemblies

Authors:

Vreja, I. C., Nikic, I., Göttfert, F., Bates, M., Kröhnert, K., Outeiro, T. F., ... & Rizzoli, S. O.

Keywords:

click chemistry, fluorescent protein, protein engineering, super-resolution microscopy, unnatural amino acid, GSDIM

Abstract:

The advent of super-resolution microscopy (nanoscopy) has set high standards for fluorescence tagging. Fluorescent proteins (FPs) are convenient tags in conventional imaging, but their use in nanoscopy has been questioned due to their relatively large size and propensity to form multimers. Here, we compared the nanoscale organization of proteins with or without FP tags by introducing the unnatural amino acid propargyl-l-lysine (PRK) in 26 proteins known to form multimolecular arrangements and into their FP-tagged variants. We revealed the proteins by coupling synthetic fluorophores to PRK via click chemistry and visualized them using ground-state depletion microscopy followed by individual molecule return, as well as stimulated emission depletion microscopy. The arrangements formed by the FP-tagged and nontagged proteins were similar. Mild, but statistically significant differences were observed for only six proteins (23% of all proteins tested). This suggests that FP-based nanoscopy is generally reliable. Unnatural amino acids should be a reliable alternative for the few proteins that are sensitive to FP tagging.