Rapid Quantitative Measurements of Paramagnetic Relaxation Enhancements in Cu(II)-Tagged Proteins by Proton-Detected Solid-State NMR Spectroscopy.

We demonstrate rapid quantitative measurements of site-resolved paramagnetic relaxation enhancements (PREs), which are a source of valuable structural restraints corresponding to electron-nucleus distances in the ∼10-20 Å regime, in solid-state nuclear magnetic resonance (NMR) spectra of proteins containing covalent Cu ²⁺ -binding tags. Specifically, using protein GB1 K28C-EDTA-Cu ²⁺ mutant as a model, we show the determination of backbone amide ¹⁵ N longitudinal and ¹ H transverse PREs within a few hours of experiment time based on proton-detected 2D or 3D correlation spectra recorded with magic-angle spinning frequencies ≥ ∼ 60 kHz for samples containing ∼10-50 nanomoles of ² H, ¹³ C, ¹⁵ N-labeled protein back-exchanged in H ₂ O. Additionally, we show that the electron relaxation time for the Cu ²⁺ center, needed to convert PREs into distances, can be estimated directly from the experimental data. Altogether, these results are important for establishing solid-state NMR based on paramagnetic-tagging as a routine tool for structure determination of natively diamagnetic proteins.