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Isoindoline‐Based Nitroxides as Bioresistant Spin Labels for Protein Labeling through Cysteines and Alkyne‐Bearing Noncanonical Amino Acids

2019; Wiley; Volume: 21; Issue: 7 Linguagem: Inglês

10.1002/cbic.201900537

1439-7633

Theresa Braun, Pia Widder, Uwe Osswald, Lina Groß, Lara Williams, Moritz J. Schmidt, Irina Helmle, Daniel Summerer, Malte Drescher,

Magnetism in coordination complexes

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.