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Access to Faster Eukaryotic Cell Labeling with Encoded Tetrazine Amino Acids.

ABSTRACT: Labeling of biomolecules in live eukaryotic cells has been limited by low component stability and slow reaction rates. We show that genetically encoded tetrazine amino acids in proteins reach reaction rates of 8 × 10⁴ M^-1 s^-1 with sTCO reagents, making them the fastest site-specific bioorthogonal labels in eukaryotic systems. We demonstrate that tetrazine amino acids are stable on proteins and are capable of quantitative labeling with sTCO reagents. The exceptionally high reaction rate of this ligation minimizes label concentration, allowing for substoichiometric in vivo eukaryotic protein labeling where the concentration of the label is less than the concentration of the protein. This approach offers unprecedented control over the composition and stability of the protein tag. We anticipate that this system will have a broad impact on labeling and imaging studies because it can be used where all generally orthogonal PylRS/tRNA pairs are employed.

SUBMITTER: Jang HS

PROVIDER: S-EPMC7771912 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Access to Faster Eukaryotic Cell Labeling with Encoded Tetrazine Amino Acids.

Jang Hyo Sang HS Jana Subhashis S Blizzard Robert J RJ Meeuwsen Joseph C JC Mehl Ryan A RA

Journal of the American Chemical Society 20200410 16

Labeling of biomolecules in live eukaryotic cells has been limited by low component stability and slow reaction rates. We show that genetically encoded tetrazine amino acids in proteins reach reaction rates of 8 × 104 M-1 s-1 with sTCO reagents, making them the fastest site-specific bioorthogonal labels in eukaryotic systems. We demonstrate that tetrazine amino acids are stable on proteins and are capable of quantitative labeling with sTCO reagents. The exception ...[more]

PMID: 32251579

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Project description:Studying protein structures and dynamics directly in the cellular environments in which they function is essential to fully understand the molecular mechanisms underlying cellular processes. Site-directed spin-labeling (SDSL)-in combination with double electron-electron resonance (DEER) spectroscopy-has emerged as a powerful technique for determining both the structural states and the conformational equilibria of biomacromolecules. In-cell DEER spectroscopy on proteins in mammalian cells has thus far not been possible due to the notable challenges of spin-labeling in live cells. In-cell SDSL requires exquisite biorthogonality, high labeling reaction rates and low background signal from unreacted residual spin label. While the bioorthogonal reaction must be highly specific and proceed under physiological conditions, many spin labels display time-dependent instability in the reducing cellular environment. Additionally, high concentrations of spin label can be toxic. Thus, an exceptionally fast bioorthogonal reaction is required that can allow for complete labeling with low concentrations of spin-label prior to loss of signal. Here we utilized genetic code expansion to site-specifically encode a novel family of small, tetrazine-bearing non-canonical amino acids (Tet-v4.0) at multiple sites in green fluorescent protein (GFP) and maltose binding protein (MBP) expressed both in E. coli and in human HEK293T cells. We achieved specific and quantitative spin-labeling of Tet-v4.0-containing proteins by developing a series of strained trans -cyclooctene (sTCO)-functionalized nitroxides-including a gem -diethyl-substituted nitroxide with enhanced stability in cells-with rate constants that can exceed 10 6 M -1 s -1 . The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live HEK293T cells within minutes, requiring only sub-micromolar concentrations of sTCO-nitroxide added directly to the culture medium. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro . Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and successfully discerned the conformational state of MBP within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purification.Toc

Dataset Information

Access to Faster Eukaryotic Cell Labeling with Encoded Tetrazine Amino Acids.

Publications

Access to Faster Eukaryotic Cell Labeling with Encoded Tetrazine Amino Acids.

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