Temperature-Dependent Rotation of Protonated Methyl Groups in Otherwise Deuterated Proteins Modulates DEER Distance Distributions.

Unlabelled: Temperature-dependent DEER effects are observed as a function of methyl rotation by either leucine- or nitroxide-specific protonated methyl groups in an otherwise deuterated background. Both species induce a site-specific enhancement in the apparent relaxation of the paramagnetic nitroxide label. The presence of a single protonated methyl group in close proximity (4-10 Å) to only one of the two nitroxide rotamer ensembles in AviTagged immunoglobulin-binding B domain of protein A results in a selective and substantial decrease in , manifested by differential decay of the peak intensities in the bimodal distance distribution as a function of the total dipolar evolution time, temperature, or both. The temperature-dependent differential decay of the individual distance components was globally analyzed by fitting the DEER dipolar time traces to a three-site jump model that is defined by the activation energy of leucine- or nitroxide-specific methyl rotation. Temperature-assisted T filtering will capture the DEER structural analysis of biomolecular systems heterogenic conformations, including complexes involving multimeric proteins.

Supplementary Information: The online version contains supplementary material available at 10.1007/s00723-024-01720-5.