Isoindoline‐Based Nitroxides as Bioresistant Spin Labels for Protein Labeling through Cysteines and Alkyne‐Bearing Noncanonical Amino Acids. (6th December 2019)

• Record Type: Journal Article
• Authors: Braun, Theresa Sophie; Widder, Pia; Osswald, Uwe; Groß, Lina; Williams, Lara; Schmidt, Moritz; Helmle, Irina; Summerer, Daniel; Drescher, Malte
• Is Part Of: Chembiochem. Volume 21:Number 7(2020)
• Page Start: 958
• Page End: 962
• Publication Date: 2019-12-06
• Subjects: bioresistance -- EPR spectroscopy -- macromolecular dynamics -- protein conformation analysis -- site-directed spin labeling; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201900537
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: Electron paramagnetic resonance (EPR) spectroscopy in combination with site‐directed spin labeling (SDSL) is a powerful tool in protein structural research. Nitroxides are highly suitable spin labeling reagents, but suffer from limited stability, particularly in the cellular environment. Herein we present the synthesis of a maleimide‐ and an azide‐modified tetraethyl‐shielded isoindoline‐based nitroxide (M‐ and Az‐TEIO) for labeling of cysteines or the noncanonical amino acid para ‐ethynyl‐l ‐phenylalanine ( p ENF). We demonstrate the high stability of TEIO site‐specifically attached to the protein thioredoxin (TRX) against reduction in prokaryotic and eukaryotic environments, and conduct double electron–electron resonance (DEER) measurements. We further generate a rotamer library for the new residue p ENF‐Az‐TEIO that affords a distance distribution that is in agreement with the measured distribution. Abstract : DEER in the distance : Isoindoline‐based nitroxides for site‐directed spin labeling of proteins show a good half‐life in bacterial and eukaryotic cell extracts. Distance distributions revealed from double electron–electron resonance (DEER) measurements between two labeling sites in thioredoxin are in good agreement with the simulated distribution using a rotamer library.