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A genetically encoded alkyne directs palladium-mediated protein labeling on live mammalian cell surface.

ABSTRACT: The merging of site-specific incorporation of small bioorthogonal functional groups into proteins via amber codon suppression with bioorthogonal chemistry has created exciting opportunities to extend the power of organic reactions to living systems. Here we show that a new alkyne amino acid can be site-selectively incorporated into mammalian proteins via a known orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and directs an unprecedented, palladium-mediated cross-coupling reaction-driven protein labeling on live mammalian cell surface. A comparison study with the alkyne-encoded proteins in vitro indicated that this terminal alkyne is better suited for the palladium-mediated cross-coupling reaction than the copper-catalyzed click chemistry.

SUBMITTER: Li N

PROVIDER: S-EPMC4340352 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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A genetically encoded alkyne directs palladium-mediated protein labeling on live mammalian cell surface.

Li Nan N Ramil Carlo P CP Lim Reyna K V RK Lin Qing Q

ACS chemical biology 20141105 2

The merging of site-specific incorporation of small bioorthogonal functional groups into proteins via amber codon suppression with bioorthogonal chemistry has created exciting opportunities to extend the power of organic reactions to living systems. Here we show that a new alkyne amino acid can be site-selectively incorporated into mammalian proteins via a known orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and directs an unprecedented, palladium-mediated cross-coupling reaction-driven prot ...[more]

PMID: 25347611

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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

A genetically encoded alkyne directs palladium-mediated protein labeling on live mammalian cell surface.

Publications

A genetically encoded alkyne directs palladium-mediated protein labeling on live mammalian cell surface.

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