Issue 9, 2020

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

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

Supplementary files

Article information

Article type
Communication
Submitted
14 Okt 2019
Accepted
02 Jan 2020
First published
18 Feb 2020
This article is Open Access
Creative Commons BY license

Phys. Chem. Chem. Phys., 2020,22, 4875-4879

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

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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