10.1021/acs.jpclett.7b02709.s001 Dwaipayan Mukhopadhyay Dwaipayan Mukhopadhyay Philippe S. Nadaud Philippe S. Nadaud Matthew D. Shannon Matthew D. Shannon Christopher P. Jaroniec Christopher P. Jaroniec Rapid Quantitative Measurements of Paramagnetic Relaxation Enhancements in Cu(II)-Tagged Proteins by Proton-Detected Solid-State NMR Spectroscopy American Chemical Society 2017 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 2017-11-17 00:00:00 Journal contribution 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.