title: An in vivo covalent TMP-tag based on proximity-induced reactivity.
abstract: Chemical tags for live cell imaging are emerging as viable alternatives to the fluorescent proteins for labeling proteins with small molecule probes. Among reported chemical tags, trimethoprim (TMP)-tag stands out for having sufficient cell permeability and selectivity to allow imaging of intracellular proteins. TMP-tag provides a noncovalent label in which the protein of interest is fused to E. coli dihydrofolate reductase (DHFR) and then labeled with a cell-permeable TMP-probe heterodimer. To complement the utility of the noncovalent TMP-tag, we sought to render the TMP-tag covalent for applications such as single-molecule tracking and pulse-chase labeling that would benefit from a more permanent modification. On the basis of the long-standing use of proximity-induced reactivity for irreversible inhibitor design and its more recent application to in vitro chemical biology tools, we designed an eDHFR variant with a unique cysteine residue positioned to react with an acrylamide electrophile installed on the TMP-probe label. In vitro experiments show that the eDHFR:L28C nucleophile reacts rapidly and quantitatively with the TMP-acrylamide-probe. Most significantly, the balance in reactivity provided by the acrylamide electrophile allows intracellular proteins tagged with eDHFR:L28C to be labeled with a TMP-acrylamide-fluorescein heterotrimer in live cells with minimal background. Thus, the TMP electrophile described here can be used immediately as a covalent chemical tag in live cells. Moreover, proximity-induced reactivity is shown to be sufficiently selective for use in a living cell, suggesting a general approach for the development of orthogonal covalent chemical tags from existing noncovalent ligand-protein pairs.
authors.affiliation: Department of Chemistry, Columbia University, New York, NY 10027, USA.
authors.forename: Sarah S
authors.initials: SS
authors.lastname: Gallagher
authors.forename: Julia E
authors.initials: JE
authors.lastname: Sable
authors.forename: Michael P
authors.initials: MP
authors.lastname: Sheetz
authors.forename: Virginia W
authors.initials: VW
authors.lastname: Cornish
mesh_terms: D000818:Animals; D002470:Cell Survival; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D041681:NIH 3T3 Cells; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D014295:Trimethoprim / Q000737:chemistry*
chemical_list: D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase