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

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 (pENF). 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 pENF-Az-TEIO that affords a distance distribution that is in agreement with the measured distribution.

SUBMITTER: Braun TS 

PROVIDER: S-EPMC7187341 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature