Direct observation and measurement of the average quantum methyl-tunneling frequency in a protein using Tm-edited double electron–electron resonance EPR spectroscopy

Abstract

Using a leucine methyl protonated, otherwise fully deuterated, {U[²H]; [Leu-[¹³CH₃]}, isotopically labeled model protein, spin-labeled at two sites with the rigid R1p nitroxide sidechain, one of which is present in two distinct conformers, we show that the oscillations in the ratio of integrated intensities of the two peaks in the double electron–electron resonance (DEER)-derived probability distance distribution as a function of the second echo period time T in a series of T_m-edited DEER experiments arise from methyl tunnelling spin echo envelope modulation. Quantitative analysis yields an average methyl tunneling rate of 0.12 MHz, indicative of a high rotational barrier. These data provide the first direct observation of methyl tunneling in a protein by electron paramagnetic resonance.