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

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.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0024-408F-3 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-80EA-4
Genre: Journal Article

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 Creators:
Liu , D. l. S., Author
Nivón, L. G., Author
Richter, F., Author
Goldman, P. J., Author
Deerinck, T. J., Author
Yao, J. Z., Author
Richardson, D.1, Author              
Phipps, W. S., Author
Ye, A. Z., Author
Ellisman, M. H., Author
Drennan, C. L., Author
Baker, D., Author
Ting, A. Y., Author
Affiliations:
1Department of NanoBiophotonics, MPI for Biophysical Chemistry, Max Planck Society, ou_578627              

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Free keywords: fluorescence microscopy; enzyme redesign; LplA; PRIME; chemical probe targeting
 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.

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Language(s): eng - English
 Dates: 2014-10-132014-10-28
 Publication Status: Published in print
 Pages: -
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 Table of Contents: -
 Rev. Method: Peer
 Identifiers: DOI: 10.1073/pnas.1404736111
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Title: Proceedings of the National Academy of Sciences of the United States of America
Source Genre: Journal
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Pages: - Volume / Issue: 111 (43) Sequence Number: - Start / End Page: E4551 - E4559 Identifier: -