Second-Generation Covalent
TMP-Tag for Live Cell Imaging
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Abstract
Chemical tags are now viable alternatives to fluorescent
proteins
for labeling proteins in living cells with organic fluorophores that
have improved brightness and other specialized properties. Recently,
we successfully rendered our TMP-tag covalent with a proximity-induced
reaction between the protein tag and the ligand-fluorophore label.
This initial design, however, suffered from slow <i>in vitro</i> labeling kinetics and limited live cell protein labeling. Thus,
here we report a second-generation covalent TMP-tag that has a fast
labeling half-life and can readily label a variety of intracellular
proteins in living cells. Specifically, we designed an acrylamide-trimethoprim-fluorophore
(A-TMP-fluorophore v2.0) electrophile with an optimized linker for
fast reaction with a cysteine (Cys) nucleophile engineered just outside
the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chemical
synthesis for routine production of a variety of A-TMP-probe v2.0
labels. We then screened a panel of eDHFR:Cys variants and identified
eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin
v2.0 <i>in vitro</i>. Finally, we demonstrated live cell
imaging of various cellular protein targets with A-TMP-fluorescein,
A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation
covalent TMP-tag adds to the limited number of chemical tags that
can be used to covalently label intracellular proteins efficiently
in living cells. Moreover, the success of this second-generation design
further validates proximity-induced reactivity and organic chemistry
as tools not only for chemical tag engineering but also more broadly
for synthetic biology