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Isoindoline‐Based Nitroxides as Bioresistant Spin Labels for Protein Labeling through Cysteines and Alkyne‐Bearing Noncanonical Amino Acids
Author(s) -
Braun Theresa Sophie,
Widder Pia,
Osswald Uwe,
Groß Lina,
Williams Lara,
Schmidt Moritz,
Helmle Irina,
Summerer Daniel,
Drescher Malte
Publication year - 2020
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201900537
Subject(s) - site directed spin labeling , chemistry , electron paramagnetic resonance , isoindoline , nitroxide mediated radical polymerization , azide , spin label , bioorthogonal chemistry , conformational isomerism , stereochemistry , combinatorial chemistry , click chemistry , biochemistry , nuclear magnetic resonance , organic chemistry , polymerization , molecule , physics , radical polymerization , membrane , polymer
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.