Next, the relaxation profile of the new spin label was investigated by determining the phase memory time Tm for the spin label 12 in a protonated medium and the doubly-labelled protein in a solution containing deuterated solvents by pulsed EPR spectroscopy (Figure ). Tm reflects the intensity decay of an electron spin echo during pulsed EPR experiments, and this factor contributes significantly to the resolution and accuracy of DEER distance measurements. Spin echo decay curves were recorded at Q-band at various temperatures (Figure and), and numerical values for Tm were extracted (see the Supplementary Information). Spin label 12 has a Tm of 4.26 µs at 50 K, and remains stable up to 160 K (Tm,160K = 3.28 µs, Figure ). Tm at 180 K (just above the glass melting temperature) is 2.35 µs, which corresponds to a 45% decay and is in agreement with our previous results performed on a similar nitroxide scaffold. The same pattern is evident for the labelled protein, but with phase memory times of approximately double duration (8.56 µs at 50 K, Figure ) due to the protein now being measured in a deuterated solvent mixture. A comparison of the logarithm of the phase memory decay rates (i.e. the reciprocal of Tm) across temperatures is shown in Figure . It is evident that the absence of rotatable methyl groups and relative rigidity of the nitroxide scaffold 12 results in excellent relaxation behaviour both in the free label and when attached to the protein. These findings validate the utility of spin label 12 for the investigation of proteins at elevated temperatures.