ChemRxiv
These are preliminary reports that have not been peer-reviewed. They should not be regarded as conclusive, guide clinical practice/health-related behavior, or be reported in news media as established information. For more information, please see our FAQs.
1/1
2 files

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

preprint
submitted on 01.04.2019 and posted on 02.04.2019 by Anandi Kugele, Bjarne Silkenath, Jakob Langer, Valentin Wittmann, Malte Drescher
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.

Funding

European Research Council under the European Union's Horizon 2020 research and innovation programme (Grant Agreement Number: 772027 - SPICE - ERC-2017-COG)

Deutsche Forschungsgemeinschaft (SFB 969, Projects B5 and C3)

History

Email Address of Submitting Author

anandi.kugele@uni-konstanz.de

Institution

University of Konstanz

Country

Germany

ORCID For Submitting Author

0000-0002-1016-8910

Declaration of Conflict of Interest

There are no conflicts of interest to declare.

Exports