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.
Highlights
Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool in protein structural research
Be studied by double electron–electron resonance (DEER) even in the context of their native, cellular environment,[4] as biological organisms own a limited amount of endogenous EPR-active species, for example, manganese, copper and iron ions
Owing to their small size, nitroxides are well tolerated by proteins, feature high sensitivity due to their narrow EPR spectrum, and enable monitoring dynamics of the labeled protein because their spectral shape depends on rotational diffusion
Summary
Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool in protein structural research. There are two key features for in cell applicability of nitroxide based spin labels: 1) the stability of the nitroxide head group against intracellular reduction, which is known to be a limiting factor in biological surroundings[4,9] and 2) the attachment strategy.
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