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Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells

Author(s)
Cohen, Justin D.; Thompson, Samuel M.; Ting, Alice Y.
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Abstract
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.
Date issued
2011-08
URI
http://hdl.handle.net/1721.1/73988
Department
Massachusetts Institute of Technology. Department of Chemistry
Journal
Biochemistry
Publisher
American Chemical Society (ACS)
Citation
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.
Version: Author's final manuscript
ISSN
0006-2960
1520-4995

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