Revealing the sensitivity of methyl tunneling towards local environment changes with quantum-rotor EPR spectroscopy

Methyl rotors have potential as local environment probes because their rotation barrier is sensitive to hindering interactions with the nearby surrounding. Quantum-rotor electron paramagnetic resonance (EPR) measurements allow access to this local environment information of the methyl rotor if it is coupled to an electron spin. This is the case for commonly used nitroxide-based spin-labels, where electron spin echo envelope modulation (ESEEM) signals exhibit two contributions on different time scales at low temperatures. The slower decaying contribution is related to matrix-driven nuclear pair ESEEM while the faster contribution originates from methyl tunneling of the electron spin-coupled methyl rotors. The tunneling ESEEM contribution contains local environment information in terms of a distribution of the rotation barrier, which can be quantified using the methyl quantum rotor model. Here, we study the sensitivity of the tunneling behavior towards changes in the rotors' surrounding by systematically investigating the two-pulse ESEEM signal of nitroxide spin probes containing two pairs of geminal methyl groups in different biologically-relevant matrix compositions. We find that the nitroxide ring structure of these probes strongly impacts the rotation barrier of the observed methyl rotors whereas the matrix surrounding does not affect the underlying rotation barrier distribution. These insights are crucial for designing nitroxide-based spin-labels as local environments probes in combination with site-directed spin-labeling for protein structure elucidation.