Super-resolution Microscopy of Clickable Amino Acids Reveals the Effects of Fluorescent Protein Tagging on Protein Assemblies.
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.
International Max Planck Research School for Molecular Biology , Göttingen, Germany.
Ingrid C
IC
Vreja
Structural and Computational Biology Unit, EMBL , 69117 Heidelberg, Germany.
Ivana
I
Nikić
Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry , 37077 Göttingen, Germany.
Fabian
F
Göttfert
Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry , 37077 Göttingen, Germany.
Mark
M
Bates
Katharina
K
Kröhnert
Tiago F
TF
Outeiro
Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry , 37077 Göttingen, Germany.
Stefan W
SW
Hell
Structural and Computational Biology Unit, EMBL , 69117 Heidelberg, Germany.
Edward A
EA
Lemke
Silvio O
SO
Rizzoli
D000596:Amino Acids / Q000737:chemistry*; D057930:Click Chemistry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D014162:Transfection
D000596:Amino Acids; D005456:Fluorescent Dyes; D008164:Luminescent Proteins
GSDIM; STED; click chemistry; fluorescent protein; protein engineering; super-resolution microscopy; unnatural amino acid