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Combining Site-Directed Spin Labeling in Vivo and In-Cell EPR Distance Determination

preprint
submitted on 29.08.2019 and posted on 30.08.2019 by Pia Widder, Julian Schuck, Daniel Summerer, Malte Drescher
Structural studies on proteins directly in their native environment are required for a comprehensive understanding of their function. Electron paramagnetic resonance (EPR) spectroscopy and in particular double electron-electron resonance (DEER) distance determination are suited to investigate spin-labeled proteins directly in the cell. The combination of intracellular bioorthogonal labeling with in-cell DEER measurements does not require additional purification or delivery steps of spin-labeled protein to the cells. In this study, we express eGFP in E.coli and use copper-catalyzed azide-alkyne cycloaddition (CuAAC) for the site-directed spin labeling of the protein in vivo, followed by in-cell EPR distance determination. Inter-spin distance measurements of spin-labeled eGFP agree with in vitro measurements and calculations based on the rotamer library of the spin label.

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, Project C3)

History

Email Address of Submitting Author

pia.widder@uni-konstanz.de

Institution

Universität Konstanz

Country

Germany

ORCID For Submitting Author

0000-0002-5068-2658

Declaration of Conflict of Interest

There are no conflicts of interest to declare.

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