Ultrafast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids
Year: 2023
Type: article
Abstract: 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. DEER combined with in situ SDSL in live cells is challenging since current bioorthogonal labeling approaches are too slow to allow for complete labeling with low concentrations of spin label prior to loss of signal from cellular reduction. Here, we overcome this limitation by genetically encoding a novel family of small, tetrazine-bearing noncanonical amino acids (Tet-v4.0) at multiple sites in proteins expressed in Escherichia 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 cells within minutes, requiring only sub-micromolar concentrations of sTCO-nitroxide. 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 support assignment of the conformational state of an MBP mutant 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.
DOI: 10.1021/jacs.3c00967
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
Awards: High-sensitivity upgrade of DEER/EPR spectrometer, Analysis tools for quantifying protein conformational landscapes using DEER spectroscopy, Development of an improved core technology for efficient genetic code expansion in biomedical research, Structural mechanisms for gating of bacterial cyclic nucleotide-gated ion channels, G2589913962, Ideal eukaryotic tetrazine ligations for imaging protein dynamics in live cells, New Nitroxide Spin Labels for Distance Measurements in Biological Systems, G4126275445, Nitrogen Centered Radicals, Decoherence of Molecular Spin Qubits, Gating Mechanisms of Retinal Cyclic Nucleotide-Regulated Ion Channels, Ion Mobility Time-of-flight Mass Spectrometry System, G8646758398, G9000014155, Conformational Energetics and Heterogeneity to Reveal Gating Mechanisms of TRPV and TRPM Ion Channels, Vision Training Grant, The GCE4All Center: Unleashing the Potential of Genetic Code Expansion for Biomedical Research
OpenAlex ID: W4382047977