Physical Chemistry

Protein Spin Labeling with a Photocaged Nitroxide Using Diels-Alder Chemistry


EPR spectroscopy of diamagnetic bio-macromolecules is based on site-directed spin labeling (SDSL). Here, we present a novel labeling strategy for proteins. We developed and synthesized a nitroxide-based spin label that can be ligated to proteins by an inverse-electron-demand Diels-Alder (DAinv) cycloaddition to genetically encoded non-canonical amino acids (ncAA). The nitroxide moiety is shielded by a photoremovable protecting group (PPG) with an attached tetraethylene glycol unit to achieve water solubility. We demonstrate SDSL of two model proteins with the PaNDA (Photoactivatable Nitroxide for DAinv reaction) label. Our strategy features high reaction rates combined with high selectivity, as well as high stability of the nitroxide in E. coli lysate.


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Supplementary material

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SI Kugele