Rapid Quantitative Measurements of Paramagnetic Relaxation Enhancements in Cu(II)-Tagged Proteins by Proton-Detected Solid-State NMR Spectroscopy Dwaipayan Mukhopadhyay Philippe S. Nadaud Matthew D. Shannon Christopher P. Jaroniec 10.1021/acs.jpclett.7b02709.s001 https://acs.figshare.com/articles/journal_contribution/Rapid_Quantitative_Measurements_of_Paramagnetic_Relaxation_Enhancements_in_Cu_II_-Tagged_Proteins_by_Proton-Detected_Solid-State_NMR_Spectroscopy/5616562 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<sup>2+</sup>-binding tags. Specifically, using protein GB1 K28C-EDTA-Cu<sup>2+</sup> mutant as a model, we show the determination of backbone amide <sup>15</sup>N longitudinal and <sup>1</sup>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 <sup>2</sup>H,<sup>13</sup>C,<sup>15</sup>N-labeled protein back-exchanged in H<sub>2</sub>O. Additionally, we show that the electron relaxation time for the Cu<sup>2+</sup> 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. 2017-11-17 00:00:00 PRE proton-detected 2 D 3 D correlation spectra 13 C 28C GB 2 H experiment time Proton-Detected Solid-State NMR Spectroscopy natively diamagnetic proteins binding tags Paramagnetic Relaxation Enhancements H 2 O electron relaxation time 1 H Cu backbone amide 15 N Rapid Quantitative Measurements relaxation enhancements structure determination 15 N-labeled protein back-exchanged