dHis-troying Barriers: Deuteration Provides a Pathway to Increase Sensitivity and Accessible Distances for Cu²⁺ Labels

Recently, site-directed Cu²⁺ labeling has emerged as an incisive biophysical tool to directly report on distance constraints that pertain to the structure, conformational transitions, and dynamics of proteins and nucleic acids. However, short phase memory times inherent to the Cu²⁺ labels limit measurable distances to 4–5 nm. In this work we systematically examine different methods to dampen electron–nuclear and electron–electron coupled interactions to decrease rapid relaxation. We show that using Cu²⁺ spin concentrations up to ca. 800 μM has an invariant effect on relaxation and that increasing the cryoprotectant concentration reduces contributions of solvent protons to relaxation. On the other hand, the deuteration of protein and solvent dramatically increases the duration of the dipolar modulated signal by over 6-fold to 32 μs. Based on this increase in signal longevity, distances up to 9 nm and beyond can potentially be measured with Cu²⁺ labels.