Browse by author
Lookup NU author(s): Professor Tiago OuteiroORCiD
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
© 2015 American Chemical Society.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.
Author(s): Vreja IC, Nikic I, Gottfert F, Bates M, Krohnert K, Outeiro TF, Hell SW, Lemke EA, Rizzoli SO
Publication type: Article
Publication status: Published
Journal: ACS Nano
Online publication date: 25/10/2015
Acceptance date: 01/01/1900
Date deposited: 19/12/2017
ISSN (print): 1936-0851
ISSN (electronic): 1936-086X
Publisher: American Chemical Society
PubMed id: 26498474
Altmetrics provided by Altmetric