Ultrafast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids.

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 p

SUBMITTER: Jana S 

PROVIDER: S-EPMC10440187 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature