Tm filtering by 1H-methyl labeling in a deuterated protein for pulsed double electron–electron resonance EPR

Modulating the phase-memory relaxation time (Tm) of a spin label by introducing 1H-methyl groups in a perdeuterated protein background is used in DEER experiments to assign interactions in multimodal P(r) distributions. Proof of principle is demonstrated using Protein A where one nitroxide label occupies two distinct regions of conformational space. The presence of a single protonated methyl group in close proximity (4-8 Å) to only one of the two nitroxide rotamer ensembles results in a selective and substantial decrease in Tm, manifested by differential decay of the peak intensities in the bimodal P(r) distance distribution as a function of the total dipolar evolution time. This form of Tm filtering will facilitate DEER structural analysis of biomolecular systems with three spin labels, including complexes involving multimeric proteins.