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.
Date Issued
August 2011
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
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.
MIT Department
Massachusetts Institute of Technology. Department of Chemistry
Terms of Use
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DOI of Published Version
https://doi.org/10.1021/bi201037r
