Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells
Mutation of a gatekeeper residue, tryptophan 37, in E. coli lipoic acid ligase (LplA), expands substrate specificity such that unnatural probes much larger than lipoic acid can be recognized. This approach, however, has not been successful for anionic substrates. An example is the blue fluorophore P...
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| Format: | Article |
| Language: | en_US |
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American Chemical Society (ACS)
2012
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| Online Access: | http://hdl.handle.net/1721.1/73988 https://orcid.org/0000-0002-8277-5226 |
| _version_ | 1826191475362234368 |
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| author | Cohen, Justin D. Thompson, Samuel M. Ting, Alice Y. |
| author2 | Massachusetts Institute of Technology. Department of Chemistry |
| author_facet | Massachusetts Institute of Technology. Department of Chemistry Cohen, Justin D. Thompson, Samuel M. Ting, Alice Y. |
| author_sort | Cohen, Justin D. |
| collection | MIT |
| description | Mutation of a gatekeeper residue, tryptophan 37, in E. coli lipoic acid ligase (LplA), expands substrate specificity such that unnatural probes much larger than lipoic acid can be recognized. This approach, however, has not been successful for anionic substrates. An example is the blue fluorophore Pacific Blue, which is isosteric to 7-hydroxycoumarin and yet not recognized by the latter’s ligase ([superscript W37V]LplA) or any tryptophan 37 point mutant. Here we report the results of a structure-guided, two-residue screening matrix to discover an LplA double mutant, [superscript E20G/W37T]LplA, that ligates Pacific Blue as efficiently as [superscript W37V]LplA ligates 7-hydroxycoumarin. The utility of this Pacific Blue ligase for specific labeling of recombinant proteins inside living cells, on the cell surface, and inside acidic endosomes is demonstrated. |
| first_indexed | 2024-09-23T08:56:27Z |
| format | Article |
| id | mit-1721.1/73988 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T08:56:27Z |
| publishDate | 2012 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/739882022-09-26T09:21:16Z Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells Cohen, Justin D. Thompson, Samuel M. Ting, Alice Y. Massachusetts Institute of Technology. Department of Chemistry Ting, Alice Y. Cohen, Justin D. Thompson, Samuel M. Ting, Alice Y. Mutation of a gatekeeper residue, tryptophan 37, in E. coli lipoic acid ligase (LplA), expands substrate specificity such that unnatural probes much larger than lipoic acid can be recognized. This approach, however, has not been successful for anionic substrates. An example is the blue fluorophore Pacific Blue, which is isosteric to 7-hydroxycoumarin and yet not recognized by the latter’s ligase ([superscript W37V]LplA) or any tryptophan 37 point mutant. Here we report the results of a structure-guided, two-residue screening matrix to discover an LplA double mutant, [superscript E20G/W37T]LplA, that ligates Pacific Blue as efficiently as [superscript W37V]LplA ligates 7-hydroxycoumarin. The utility of this Pacific Blue ligase for specific labeling of recombinant proteins inside living cells, on the cell surface, and inside acidic endosomes is demonstrated. National Institutes of Health (U.S.) (Grant R01 GM072670) Camille & Henry Dreyfus Foundation American Chemical Society Massachusetts Institute of Technology 2012-10-15T19:26:31Z 2012-10-15T19:26:31Z 2011-08 2011-08 Article http://purl.org/eprint/type/JournalArticle 0006-2960 1520-4995 http://hdl.handle.net/1721.1/73988 Cohen, Justin D., Samuel Thompson, and Alice Y. Ting. “Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells.” Biochemistry 50.38 (2011): 8221–8225. https://orcid.org/0000-0002-8277-5226 en_US http://dx.doi.org/10.1021/bi201037r Biochemistry 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) Prof. Ting via Erja Kajosalo |
| spellingShingle | Cohen, Justin D. Thompson, Samuel M. Ting, Alice Y. Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells |
| title | Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells |
| title_full | Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells |
| title_fullStr | Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells |
| title_full_unstemmed | Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells |
| title_short | Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells |
| title_sort | structure guided engineering of a pacific blue fluorophore ligase for specific protein imaging in living cells |
| url | http://hdl.handle.net/1721.1/73988 https://orcid.org/0000-0002-8277-5226 |
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