Combining site-directed spin labeling in vivo and in-cell EPR distance determination. Issue 9 (19th February 2020)

• Record Type: Journal Article
• Authors: Widder, Pia; Schuck, Julian; Summerer, Daniel; Drescher, Malte
• Is Part Of: Physical chemistry chemical physics. Volume 22:Issue 9(2020)
• Page Start: 4875
• Page End: 4879
• Publication Date: 2020-02-19
• Subjects: Chemistry, Physical and theoretical -- Periodicals; 541.3
• DOI: 10.1039/c9cp05584c
• Languages: English
• ISSNs: 1463-9076

Abstract

Abstract : Non-canonical amino acid incorporation via amber stop codon suppression and in vivo site-directed spin labeling allow in-cell EPR distance determination in E. coli . 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.