Site-directed spin labeling (SDSL) is a valuable and established tool to elucidate structure, function, and dynamics of proteins and protein complexes. Site-directed spin labelling onto the proteins in their native environment has suffered due to the slow conjugation reaction kinetics, poor biorthogonality and biocompatibility of the conjugation reaction with paramagnetic center. For live cells labelling studies it is critical to balance the label size, stability and fast reactivity of the reagents together. Here we developed a new tiny tetrazine amino acid (Tet-v4.0) and evolved PylRS/tRNACUA pair for genetic incorporation. The Tet-v4.0 amino acids inside the protein offers the combination of desired features including small, ultra-fast kinetics (&gt;106 M-1s-1), quantitative labeling and stable linkage with sTCO reagents. We demonstrate the Tet-v4.0 can be incorporated at different single and double, user-defined sites of a maltose binding protein (MBP) and generate a shortest linkage sTCO-spin labels to attach a EPR label as close as possible to the protein backbone. We directly compared size and flexibility of our Tet-v4.0 spin labelling with the gold standard for cysteine reactive methanethiosulfonate (MTSSL) spin labeling by double-electron electron resonance (DEER) measurements. This approach provides an elegant and potential system for advance SDSL–EPR studies in living cells. Therefore, we anticipate that the structural novelty and characteristic diversity of genetically encoded Tet-v4.0 will have broad impact on advanced biophysical study in complex biological system.

Genetically encoded tiny tetrazine amino acids because size and speed does matter

ACS Spring 2022

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