Imaging Protein–Protein Interactions inside Living Cells via Interaction-Dependent Fluorophore Ligation
We report a new method, Interaction-Dependent PRobe Incorporation Mediated by Enzymes, or ID-PRIME, for imaging protein–protein interactions (PPIs) inside living cells. ID-PRIME utilizes a mutant of Escherichia coli lipoic acid ligase, LplA[superscript W37V], which can catalyze the covalent ligation...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | en_US |
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American Chemical Society (ACS)
2013
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| Online Access: | http://hdl.handle.net/1721.1/82558 https://orcid.org/0000-0002-8277-5226 |
| _version_ | 1811081157631016960 |
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| author | Slavoff, Sarah A. Liu, Daniel S. Cohen, Justin D. Ting, Alice Y. |
| author2 | Massachusetts Institute of Technology. Department of Chemistry |
| author_facet | Massachusetts Institute of Technology. Department of Chemistry Slavoff, Sarah A. Liu, Daniel S. Cohen, Justin D. Ting, Alice Y. |
| author_sort | Slavoff, Sarah A. |
| collection | MIT |
| description | We report a new method, Interaction-Dependent PRobe Incorporation Mediated by Enzymes, or ID-PRIME, for imaging protein–protein interactions (PPIs) inside living cells. ID-PRIME utilizes a mutant of Escherichia coli lipoic acid ligase, LplA[superscript W37V], which can catalyze the covalent ligation of a coumarin fluorophore onto a peptide recognition sequence called LAP1. The affinity between the ligase and LAP1 is tuned such that, when each is fused to a protein partner of interest, LplA[superscript W37V] labels LAP1 with coumarin only when the protein partners to which they are fused bring them together. Coumarin labeling in the absence of such interaction is low or undetectable. Characterization of ID-PRIME in living mammalian cells shows that multiple protein–protein interactions can be imaged (FRB–FKBP, Fos–Jun, and neuroligin–PSD-95), with as little as 10 min of coumarin treatment. The signal intensity and detection sensitivity are similar to those of the widely used fluorescent protein complementation technique (BiFC) for PPI detection, without the disadvantage of irreversible complex trapping. ID-PRIME provides a powerful and complementary approach to existing methods for visualization of PPIs in living cells with spatial and temporal resolution. |
| first_indexed | 2024-09-23T11:42:23Z |
| format | Article |
| id | mit-1721.1/82558 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T11:42:23Z |
| publishDate | 2013 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/825582022-09-27T21:20:56Z Imaging Protein–Protein Interactions inside Living Cells via Interaction-Dependent Fluorophore Ligation Slavoff, Sarah A. Liu, Daniel S. Cohen, Justin D. Ting, Alice Y. Massachusetts Institute of Technology. Department of Chemistry Slavoff, Sarah A. Liu, Daniel S. Cohen, Justin D. Ting, Alice Y. We report a new method, Interaction-Dependent PRobe Incorporation Mediated by Enzymes, or ID-PRIME, for imaging protein–protein interactions (PPIs) inside living cells. ID-PRIME utilizes a mutant of Escherichia coli lipoic acid ligase, LplA[superscript W37V], which can catalyze the covalent ligation of a coumarin fluorophore onto a peptide recognition sequence called LAP1. The affinity between the ligase and LAP1 is tuned such that, when each is fused to a protein partner of interest, LplA[superscript W37V] labels LAP1 with coumarin only when the protein partners to which they are fused bring them together. Coumarin labeling in the absence of such interaction is low or undetectable. Characterization of ID-PRIME in living mammalian cells shows that multiple protein–protein interactions can be imaged (FRB–FKBP, Fos–Jun, and neuroligin–PSD-95), with as little as 10 min of coumarin treatment. The signal intensity and detection sensitivity are similar to those of the widely used fluorescent protein complementation technique (BiFC) for PPI detection, without the disadvantage of irreversible complex trapping. ID-PRIME provides a powerful and complementary approach to existing methods for visualization of PPIs in living cells with spatial and temporal resolution. National Institutes of Health (U.S.) (R01 GM072670) Camille & Henry Dreyfus Foundation David A. Johnson Summer Fellowship 2013-11-22T19:46:40Z 2013-11-22T19:46:40Z 2011-11 2011-07 Article http://purl.org/eprint/type/JournalArticle 0002-7863 1520-5126 http://hdl.handle.net/1721.1/82558 Slavoff, Sarah A., Daniel S. Liu, Justin D. Cohen, and Alice Y. Ting. “Imaging Protein–Protein Interactions inside Living Cells via Interaction-Dependent Fluorophore Ligation.” Journal of the American Chemical Society 133, no. 49 (December 14, 2011): 19769-19776. https://orcid.org/0000-0002-8277-5226 en_US http://dx.doi.org/10.1021/ja206435e Journal of the American Chemical Society 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 American Chemical Society (ACS) PMC |
| spellingShingle | Slavoff, Sarah A. Liu, Daniel S. Cohen, Justin D. Ting, Alice Y. Imaging Protein–Protein Interactions inside Living Cells via Interaction-Dependent Fluorophore Ligation |
| title | Imaging Protein–Protein Interactions inside Living Cells via Interaction-Dependent Fluorophore Ligation |
| title_full | Imaging Protein–Protein Interactions inside Living Cells via Interaction-Dependent Fluorophore Ligation |
| title_fullStr | Imaging Protein–Protein Interactions inside Living Cells via Interaction-Dependent Fluorophore Ligation |
| title_full_unstemmed | Imaging Protein–Protein Interactions inside Living Cells via Interaction-Dependent Fluorophore Ligation |
| title_short | Imaging Protein–Protein Interactions inside Living Cells via Interaction-Dependent Fluorophore Ligation |
| title_sort | imaging protein protein interactions inside living cells via interaction dependent fluorophore ligation |
| url | http://hdl.handle.net/1721.1/82558 https://orcid.org/0000-0002-8277-5226 |
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