Computational design of a red fluorophore ligase for site-specific protein labeling in living cells
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 Escherich...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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National Academy of Sciences (U.S.)
2015
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| Online Access: | http://hdl.handle.net/1721.1/96304 https://orcid.org/0000-0001-5486-2755 https://orcid.org/0000-0002-8277-5226 |
| _version_ | 1811070526604443648 |
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| author | Baker, David Ting, Alice Y. Liu, Daniel S. Nivon, Lucas G. Richter, Florian Goldman, Peter J. Yao, Jennifer Z. Phipps, William S. Ye, Anne Z. Deerinck, Thomas J. Richardson, Douglas Ellisman, Mark H. Drennan, Catherine L |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Baker, David Ting, Alice Y. Liu, Daniel S. Nivon, Lucas G. Richter, Florian Goldman, Peter J. Yao, Jennifer Z. Phipps, William S. Ye, Anne Z. Deerinck, Thomas J. Richardson, Douglas Ellisman, Mark H. Drennan, Catherine L |
| author_sort | Baker, David |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T08:37:26Z |
| format | Article |
| id | mit-1721.1/96304 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T08:37:26Z |
| publishDate | 2015 |
| publisher | National Academy of Sciences (U.S.) |
| record_format | dspace |
| spelling | mit-1721.1/963042022-09-30T10:01:22Z Computational design of a red fluorophore ligase for site-specific protein labeling in living cells Baker, David Ting, Alice Y. Liu, Daniel S. Nivon, Lucas G. Richter, Florian Goldman, Peter J. Yao, Jennifer Z. Phipps, William S. Ye, Anne Z. Deerinck, Thomas J. Richardson, Douglas Ellisman, Mark H. Drennan, Catherine L Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Drennan, Catherine L. Ting, Alice Y. Liu, Daniel S. Yao, Jennifer Z. Phipps, William S. Ye, Anne Z. Goldman, Peter J. 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. National Institutes of Health (U.S.) (Grant DP1 OD003961) National Institutes of Health (U.S.) (R01 GM072670) American Chemical Society 2015-04-01T15:48:59Z 2015-04-01T15:48:59Z 2014-10 2014-03 Article http://purl.org/eprint/type/JournalArticle 0027-8424 1091-6490 http://hdl.handle.net/1721.1/96304 Liu, D. S., L. G. Nivon, F. Richter, P. J. Goldman, T. J. Deerinck, J. Z. Yao, D. Richardson, et al. “Computational Design of a Red Fluorophore Ligase for Site-Specific Protein Labeling in Living Cells.” Proceedings of the National Academy of Sciences 111, no. 43 (October 13, 2014): E4551–E4559. https://orcid.org/0000-0001-5486-2755 https://orcid.org/0000-0002-8277-5226 en_US http://dx.doi.org/10.1073/pnas.1404736111 Proceedings of the National Academy of Sciences of the United States of America Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf National Academy of Sciences (U.S.) National Academy of Sciences (U.S.) |
| spellingShingle | Baker, David Ting, Alice Y. Liu, Daniel S. Nivon, Lucas G. Richter, Florian Goldman, Peter J. Yao, Jennifer Z. Phipps, William S. Ye, Anne Z. Deerinck, Thomas J. Richardson, Douglas Ellisman, Mark H. Drennan, Catherine L Computational design of a red fluorophore ligase for site-specific protein labeling in living cells |
| title | Computational design of a red fluorophore ligase for site-specific protein labeling in living cells |
| title_full | Computational design of a red fluorophore ligase for site-specific protein labeling in living cells |
| title_fullStr | Computational design of a red fluorophore ligase for site-specific protein labeling in living cells |
| title_full_unstemmed | Computational design of a red fluorophore ligase for site-specific protein labeling in living cells |
| title_short | Computational design of a red fluorophore ligase for site-specific protein labeling in living cells |
| title_sort | computational design of a red fluorophore ligase for site specific protein labeling in living cells |
| url | http://hdl.handle.net/1721.1/96304 https://orcid.org/0000-0001-5486-2755 https://orcid.org/0000-0002-8277-5226 |
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