ja6b08733_si_001.avi (2.87 MB)
Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells
media
posted on 2016-10-21, 18:33 authored by Tao Peng, Howard C. HangOver the past years,
fluorescent proteins (e.g., green fluorescent
proteins) have been widely utilized to visualize recombinant protein
expression and localization in live cells. Although powerful, fluorescent
protein tags are limited by their relatively large sizes and potential
perturbation to protein function. Alternatively, site-specific labeling
of proteins with small-molecule organic fluorophores using bioorthogonal
chemistry may provide a more precise and less perturbing method. This
approach involves site-specific incorporation of unnatural amino acids
(UAAs) into proteins via genetic code expansion, followed by bioorthogonal
chemical labeling with small organic fluorophores in living cells.
While this approach has been used to label extracellular proteins
for live cell imaging studies, site-specific bioorthogonal labeling
and fluorescence imaging of intracellular proteins in live cells is
still challenging. Herein, we systematically evaluate site-specific
incorporation of diastereomerically pure bioorthogonal UAAs bearing
stained alkynes or alkenes into intracellular proteins for inverse-electron-demand
Diels–Alder cycloaddition reactions with tetrazine-functionalized
fluorophores for live cell labeling and imaging in mammalian cells.
Our studies show that site-specific incorporation of axial diastereomer
of trans-cyclooct-2-ene-lysine robustly affords highly
efficient and specific bioorthogonal labeling with monosubstituted
tetrazine fluorophores in live mammalian cells, which enabled us to
image the intracellular localization and real-time dynamic trafficking
of IFITM3, a small membrane-associated protein with only 137 amino
acids, for the first time. Our optimized UAA incorporation and bioorthogonal
labeling conditions also enabled efficient site-specific fluorescence
labeling of other intracellular proteins for live cell imaging studies
in mammalian cells.