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Issue 9, 2020
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Combining site-directed spin labeling in vivo and in-cell EPR distance determination

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Abstract

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.

Graphical abstract: Combining site-directed spin labeling in vivo and in-cell EPR distance determination

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Supplementary files

Article information


Submitted
14 Oct 2019
Accepted
02 Jan 2020
First published
18 Feb 2020

This article is Open Access

Phys. Chem. Chem. Phys., 2020,22, 4875-4879
Article type
Communication

Combining site-directed spin labeling in vivo and in-cell EPR distance determination

P. Widder, J. Schuck, D. Summerer and M. Drescher, Phys. Chem. Chem. Phys., 2020, 22, 4875
DOI: 10.1039/C9CP05584C

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