PMID:22098454
| Citation |
Slavoff, SA, Liu, DS, Cohen, JD and Ting, AY (2011) Imaging protein-protein interactions inside living cells via interaction-dependent fluorophore ligation. J. Am. Chem. Soc. 133:19769-76 |
|---|---|
| Abstract |
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(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(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. |
| Links |
PubMed PMC3547671 Online version:10.1021/ja206435e |
| Keywords |
Animals; Cell Line; Coumarins/analysis; Coumarins/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Fluorescent Dyes/analysis; Fluorescent Dyes/metabolism; Humans; Ligases/genetics; Ligases/metabolism; Microscopy, Fluorescence/methods; Models, Molecular; Mutation; Protein Interaction Mapping/methods; Proteins/metabolism |
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