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Journal Article

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells.

MPS-Authors
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Richardson,  D.
Department of NanoBiophotonics, MPI for Biophysical Chemistry, Max Planck Society;

Fulltext (public)

2073009.pdf
(Publisher version), 2MB

Supplementary Material (public)

2073009_Suppl.pdf
(Supplementary material), 3MB

Citation

Liu, D. l. S., Nivón, L. G., Richter, F., Goldman, P. J., Deerinck, T. J., Yao, J. Z., et al. (2014). Computational design of a red fluorophore ligase for site-specific protein labeling in living cells. Proceedings of the National Academy of Sciences of the United States of America, 111(43), E4551-E4559. doi:10.1073/pnas.1404736111.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0024-408F-3
Abstract
Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.